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C.D.C. Reviewing Cases of Heart Problem in Youngsters After Getting Vaccinated and AHA Reassures that Benefits Overwhelm the Risks of Vaccination
Reporter: Amandeep Kaur, B.Sc. , M.Sc.
The latest article in New York times reported by Apoorva Mandavilli outlines the statement of officials that C.D.C. agency is investigating few cases of young adults and teenagers who might have developed myocarditis after getting vaccinated. It is not confirmed by the agency that whether this condition is caused by vaccine or not.
According to the vaccine safety group of the Centers for Disease Control and Prevention, the reports of heart problems experienced by youngsters is relatively very small in number. The group stated that these cases could be unlinked to vaccination. The condition of inflammation of heart muscle which can occur due to certain infections is known as myocarditis.
Moreover, the agency still has to determine any evidence related to vaccines causing the heart issues. The C.D.C. has posted on its website the updated guidance for doctors and clinicians, urging them to be alert to uncommon symptoms related to heart cases among teenagers who are vaccine recipients.
In New York, Dr. Celine Gounder, an infectious disease specialist at Bellevue Hospital Center stated that “It may simply be a coincidence that some people are developing myocarditis after vaccination. It’s more likely for something like that to happen by chance, because so many people are getting vaccinated right now.”
The article reported that the cases appeared mainly in young adults after about four days of their second shot of mRNA vaccines, made by Moderna and Pfizer-BioNTech. Such cases are more prevalent in males as compared to females.
The vaccine safety group stated “Most cases appear to be mild, and follow-up of cases is ongoing.” It is strongly recommended by C.D.C. that American young adults from the age of 12 and above should get vaccinated against COVID-19.
Dr. Yvonne Maldonado, chair of the American Academy of Pediatrics’s Committee on Infectious Diseases stated “We look forward to seeing more data about these cases, so we can better understand if they are related to the vaccine or if they are coincidental. Meanwhile, it’s important for pediatricians and other clinicians to report any health concerns that arise after vaccination.”
Experts affirmed that the potentially uncommon side effects of myocarditis get insignificant compared to the potential risks of SARS-CoV-2 infection, including the continuous syndrome known as “long Covid.” It is reported in the article that acute Covid can lead to myocarditis.
According to the data collected by A.A.P, about 16 thousand children were hospitalized and more than 3.9 million children were infected by coronavirus till the second week of May. In the United States, about 300 children died of SARS-CoV-2 infection, which makes it among the top 10 death causes in children since the start of pandemic.
Dr. Jeremy Faust, an emergency medicine physician at Brigham and Women’s Hospital in Boston stated that “And that’s in the context of all the mitigation measures taken.”
According to researchers, about 10 to 20 of every 1 lakh people each year develop myocarditis in the general population, facing symptoms from fatigue and chest pain to arrhythmias and cardiac arrest, whereas some have mild symptoms which remain undiagnosed.
Currently, the number of reports of myocarditis after vaccination is less than that reported normally in young adults, confirmed by C.D.C. The article reported that the members of vaccine safety group felt to communicate the information about upcoming cases of myocarditis to the providers.
The C.D.C. has not yet specified the ages of the patients involved in reporting. Since December 2020, the Pfizer-BioNTech vaccine was authorized for young people of age 16 and above. The Food and Drug Administration extended the authorization to children of age 12 to 15 years, by the starting of this month.
On 14th May, the clinicians have been alerted by C.D.C. regarding the probable link between myocarditis and vaccination. Within three days, the team started reviewing data on myocarditis, reports filed with the Vaccine Adverse Event Reporting System and others from the Department of Defense.
A report on seven cases has been submitted to the journal Pediatrics for review and State health departments in Washington, Oregon and California have notified emergency providers and cardiologists about the potential problem.
In an interview, Dr. Liam Yore, past president of the Washington State chapter of the American College of Emergency Physicians detailed a case of teenager with myocarditis after vaccination. The patient was provided treatment for mild inflammation of the inner lining of the heart and was discharged afterwards. Later, the young adult returned for care due to decrease in the heart’s output. Dr. Yore reported that still he had come across worse cases in youngsters with Covid, including in a 9-year-old child who arrived at the hospital after a cardiac arrest last winter.
He stated that “The relative risk is a lot in favor of getting the vaccine, especially considering how coronavirus vaccine have been administered.”
In the United States, more than 161 million people have received their first shot of vaccine in which about 4.5 million people were between the age 12 to 18 years.
Benefits Overwhelm Risks of COVID Vaccination, AHA Reassures
The latest statement of American Heart Association (AHA)/ American Stroke Association (ASA) on May 23rd states that the benefits of COVID-19 vaccination enormously outweigh the rare risk for myocarditis cases, which followed the C.D.C. report that the agency is tracking the Vaccine Adverse Events Reporting System (VAERS) and the Vaccine Safety Datalink (VSD) for myocarditis cases linked with mRNA vaccines against coronavirus.
The myocarditis cases in young adults are more often observed after the second dose of vaccine rather than the first one, and have more cases of males than females. The CDC’s COVID-19 Vaccine Safety Technical Work Group (VaST) observed such heart complications after 4 days of vaccination.
CDC reported that “Within CDC safety monitoring systems, rates of myocarditis reports in the window following COVID-19 vaccination have not differed from expected baseline rates.”
The CDC team stated that “The evidence continues to indicate that the COVID-19 vaccines are nearly 100% effective at preventing death and hospitalization due to COVID-19 infection, and Strongly urged all young adults and children 12 years and above to get vaccinated as soon as possible.”
Even though the analysis of myocarditis reports related to coronavirus vaccine is in progress, the AHA/ASA stated that “myocarditis is typically the result of an actual viral infection, and it is yet to be determined if these cases have any correlation to receiving a COVID-19 vaccine.”
Richard Besser, MD, president and CEO of the Robert Wood Johnson Foundation (RWJF) and former acting director of the CDC stated on ABC’s Good Morning America “We’ve lost hundreds of children and there have been thousands who have been hospitalized, thousands who developed an inflammatory syndrome, and one of the pieces of that can be myocarditis.” He added “still, from my perspective, the risk of COVID is so much greater than any theoretical risk from the vaccine.”
After COVID-19 vaccination the symptoms that occur include tiredness, muscle pain, headaches, chills, nausea and fever. The AHA/ASA stated that “typically appear within 24 to 48 hours and usually pass within 36-48 hours after receiving the vaccine.”
All healthcare providers are suggested to be aware of the rare adverse symptoms such as myocarditis, low platelets, blood clots, and severe inflammation. The agency stated that “Healthcare professionals should strongly consider inquiring about the timing of any recent COVID vaccination among patients presenting with these conditions, as needed, in order to provide appropriate treatment quickly.”
President Mitchell S.V. Elkind, M.D., M.S., FAHA, FAAN, Immediate Past President Robert A. Harrington, M.D., FAHA, President-Elect Donald M. Lloyd-Jones, M.D., Sc.M., FAHA, Chief Science and Medical Officer Mariell Jessup, M.D., FAHA, and Chief Medical Officer for Prevention Eduardo Sanchez, M.D, M.P.H., FAAFP are science leaders of AHA/ASA and reflected their views in the following statements:
We strongly urge all adults and children ages 12 and older in the U.S. to receive a COVID vaccine as soon as they can receive it, as recently approved by the U.S. Food and Drug Administration and the CDC. The evidence continues to indicate that the COVID-19 vaccines are nearly 100% effective at preventing death and hospitalization due to COVID-19 infection. According to the CDC as of May 22, 2021, over 283 million doses of COVID-19 vaccines have been administered in the U.S. since December 14, 2020, and more than 129 million Americans are fully vaccinated (i.e., they have received either two doses of the Pfizer-BioNTech or Moderna COVID-19 vaccine, or the single-dose Johnson & Johnson/Janssen COVID-19 vaccine).
We remain confident that the benefits of vaccination far exceed the very small, rare risks. The risks of vaccination are also far smaller than the risks of COVID-19 infection itself, including its potentially fatal consequences and the potential long-term health effects that are still revealing themselves, including myocarditis. The recommendation for vaccination specifically includes people with cardiovascular risk factors such as high blood pressure, obesity and type 2 diabetes, those with heart disease, and heart attack and stroke survivors, because they are at much greater risk of an adverse outcome from the COVID-19 virus than they are from the vaccine.
We commend the CDC’s continual monitoring for adverse events related to the COVID-19 vaccines through VAERS and VSD, and the consistent meetings of ACIP’s VaST Work Group, demonstrating transparent and robust attention to any and all health events possibly related to a COVID-19 vaccine. The few cases of myocarditis that have been reported after COVID-19 vaccination are being investigated. However, myocarditis is usually the result of a viral infection, and it is yet to be determined if these cases have any correlation to receiving a COVID-19 vaccine, especially since the COVID-19 vaccines authorized in the U.S. do not contain any live virus.
We also encourage everyone to keep in touch with their primary care professionals and seek care immediately if they have any of these symptoms in the weeks after receiving the COVID-19 vaccine:chest pain including sudden, sharp, stabbing pains;difficulty breathing/shortness of breath;abnormal heartbeat;severe headache;blurry vision;fainting or loss of consciousness;weakness or sensory changes;confusion or trouble speaking;seizures;unexplained abdominal pain; ornew leg pain or swelling.
We will stay up to date with the CDC’s recommendations regarding all potential complications related to COVID-19 vaccines, including myocarditis, pericarditis, central venous sinus thrombosis (CVST) and other blood clotting events, thrombosis thrombocytopenia syndrome (TTS), and vaccine-induced immune thrombosis thrombocytopenia (VITT).
The American Heart Associationrecommends all health care professionals be aware of these very rare adverse events that may be related to a COVID-19 vaccine, including myocarditis, blood clots, low platelets, or symptoms of severe inflammation. Health care professionals should strongly consider inquiring about the timing of any recent COVID vaccination among patients presenting with these conditions, as needed, in order to provide appropriate treatment quickly. As detailed in last month’s AHA/ASA statement, all suspected CVST or blood clots associated with the COVID-19 vaccine should be treated initially using non-heparin anticoagulants. Heparin products should not be administered in any dose if TTS/VITT is suspected, until appropriate testing can be done to exclude heparin-induced antibodies. In addition, health care professionals are required to report suspected vaccine-related adverse events to the Vaccine Adverse Event Reporting System, in accordance with federal regulations.
Individuals should refer to their local and state health departments for specific information about when and where they can get vaccinated. We implore everyone ages 12 and older to get vaccinated so we can return to being together, in person – enjoying life with little to no risk of severe COVID-19 infection, hospitalization or death.
We also support the CDC recommendations last week that loosen restrictions on mask wearing and social distancing for people who are fully vaccinated. For those who are unable to be vaccinated, we reiterate the importance of handwashing, social distancing and wearing masks, particularly for people at high risk of infection and/or severe COVID-19. These simple precautions remain crucial to protecting people who are not vaccinated from the virus that causes COVID-19.
FDA chief Scott Gottlieb steps down, leaving pet projects behind
FDA Commissioner Scott Gottlieb was appointed by President Trump in 2017. (FDA)
Not quite two years into his stewardship of the FDA, Scott Gottlieb, M.D., is stepping down. His sudden departure will likely send shock waves through biopharma, because the commissioner was popular for his market-based policies to lower drug costs and work to speed new brands to approval.
Gottlieb won Senate confirmation as FDA commissioner back in May 2017, an uncertain time for the drug industry. During the selection process, his name had been favored by a majority of biopharma executives polled by an analyst. At the time, one anonymous executive said he’s the “least likely to rail for the obliteration of the current efficacy, safety, risk-benefit model that is a foundation for advancement of new treatments in the United States”—unlike at least one other candidate for the job.
That preference proved out after Gottlieb took over the agency, as his policies favored speed and competition. And when the news broke Tuesday afternoon, the industry trade group PhRMA praised his “exemplary leadership.”
“During his tenure, he focused on innovation in drug development and review, increased competition, and advanced the regulatory framework for approving novel technologies, including gene therapies,” the organization said in a statement. “His efforts have made a meaningful impact for patients in need of innovative medicines.”
Gottlieb tweeted Tuesday that he’s “immensely grateful” for the opportunity to lead the FDA. In a letter to colleagues, he wrote that “there’s nothing that could pull me away from this role other than the challenge of being apart from my family for these past two years and missing my wife and two young children.”
President Donald Trump tweeted that he’s done an “absolutely terrific job” and will be missed.
In his resignation letter, tweeted by STAT reporter Matthew Herper, Gottlieb reviewed his efforts at the agency over the past two years. It’s a long list, but here are a few examples:
The FDA worked to avoid drug shortages after a hurricane struck Puerto Rico,
led a global investigation into drug impurities,
advanced reviews of cell and gene therapies, and
bolstered approvals of complex generic drugs.
Also under his command, the FDA took quick and decisive action on drug costs. The commissioner worked to boost generic approvals and crack down on regulatory “gaming” that stifles competition. He additionally blamed branded drug companies for an “anemic” U.S. biosimilars market and recently blasted insulin pricing.
His sudden departure will likely leave many agency efforts to lower costs up in the air. After the news broke, many pharma watchers posted on Twitter that Gottlieb’s resignation is a loss for the industry.
During his tenure as FDA commissioner, Gottlieb’s name had been floated for HHS chief when former HHS secretary Tom Price resigned due to a travel scandal, but Gottlieb said he was best suited for the FDA commissioner job. Now, former Eli Lilly executive Alex Azar serves as HHS secretary, and on Tuesday afternoon, Azar praised Gottlieb for his work at the agency.
New FDA commissioner Gottlieb unveils price-fighting strategies
New FDA commissioner Scott Gottlieb laid out some approaches the agency will take to fight high prices.
During the campaign and since the U.S. presidential election, President Donald Trump has pledged to bring down drug costs and, in some cases, railed against the industry for its pricing. Now, his new FDA commissioner is laying out some approaches the agency will take to fight high prices.
Importantly, the FDA can’t regulate drug prices, but it can implement measures aimed at deterring the types of price hikes that have made so many headlines over more than a year.
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At a U.S. House of Representatives budget hearing on Thursday, new FDA commissioner Scott Gottlieb said his agency will publish and regularly update a list of medications that are off patent and have no competition, work to improve generic review times and seek to “curtail gaming” of regulations by the industry that allows companies to extend patent monopolies.
On the first point, Gottlieb said a regularly updated list of off-patent meds could “entice competitors into the market” and ultimately lower costs. One such old, off-patent med with no generic competition is infamous to industry watchers: Daraprim.
Back in 2015, after picking up the medication from Marathon Pharma, Martin Shkreli’s Turing Pharma raised Daraprim’s price exponentially, triggering intense backlash.
In his opening remarks at the budget hearing, Gottlieb said he’s working on a “drug competition action plan” that he will unveil soon. However, the new commissioner did offer a few strategies to lower costs.
The FDA will work to “improve processes that enable generic versions of complex drugs to be approved for marketing,” and simplify the overall generic review process, Gottlieb said. He wants the agency to achieve those goals while “completely eliminating” a backlog of generic medications waiting for a review, he told members of Congress.
Further, the FDA will seek to stop misuse of Risk Evaluation Mitigation Strategies that have hurt generic drug developers’ ability to get samples of branded medications needed to develop copycats.
The proposals will likely be welcomed by those who’ve asked for drug pricing reform but have seen little action from Congress despite more than a year of calls from its members to rein in drug prices.
In a Thursday statement, the pharmacy benefit manager industry group PCMA hailed the proposals.
“As the Trump administration seeks to combat high drug costs, we applaud FDA Commissioner Gottlieb’s decision to publish a ‘watch list’ of off-patent drugs,” PCMA CEO Mark Merritt said in a statement. “This move, which PCMA and others have long advocated, will help deter manufacturers from buying the rights to drugs like Daraprim for the explicit purpose of raising their price.”
UPDATED 3/19/2019
National Cancer Chief, Ned Sharpless, Named F.D.A.’s Acting Commissioner
Dr. Norman E. Sharpless was named acting commissioner of the Food and Drug Administration on Tuesday. For the last 18 months, he had been director of the National Cancer Institute.CreditTom Williams/CQ Roll Call, via Getty Images
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Dr. Norman E. Sharpless was named acting commissioner of the Food and Drug Administration on Tuesday. For the last 18 months, he had been director of the National Cancer Institute.CreditCreditTom Williams/CQ Roll Call, via Getty Images
WASHINGTON — Dr. Norman E. (Ned) Sharpless, director of the National Cancer Institute, will serve as acting commissioner of the Food and Drug Administration, Alex M. Azar III, secretary of health and human services, announced on Tuesday.
Dr. Sharpless temporarily will fill the post being vacated by Dr. Scott Gottlieb, who stunned public health experts, lawmakers and consumer groups last week when he abruptly announced that he was resigningfor personal reasons.
Dr. Sharpless has been director of the cancer center, part of the National Institutes of Health, since October 2017. He is also chief of the aging biology and cancer section in the National Institute on Aging’s Laboratory of Genetics and Genomics. His research focuses on the relationship between aging and cancer, and development of new treatments for melanoma, lung cancer and breast cancer.
“Dr. Sharpless’s deep scientific background and expertise will make him a strong leader for F.D.A.,” said Mr. Azar, in a statement. “There will be no let up in the agency’s focus, from ongoing efforts on drug approvals and combating the opioid crisis to modernizing food safety and addressing the rapid rise in youth use of e-cigarettes.”
Dr. Douglas Lowy, known for seminal research on the link between human papillomavirus and multiple cancer types including cervical, and ultimately leading to development of a vaccine, will be named head of the NCI to replace Dr. Sharpless. Dr. Lowy currently is Deputy Director of the NCI.
Other posts on the Food and Drug Administration and FDA Approvals during Dr. Gotlieb’s Tenure on this Open Access Journal Include:
DUBLIN, May 27, 2015 /PRNewswire/ — Actavis plc (NYSE: ACT) announced today that VIBERZI™ (eluxadoline) was approved by the Food and Drug Administration (FDA) as a twice-daily, oral treatment for adults suffering from irritable bowel syndrome with diarrhea (IBS-D). VIBERZI (eluxadoline) has mixed opioid receptor activity, it is a mu receptor agonist, a delta receptor antagonist, and a kappa receptor agonist.
“The FDA’s approval of VIBERZI is the first step to providing physicians with a new, evidence-based, treatment option for their adult patients with IBS-D,” said David Nicholson, Executive Vice President, Actavis Global Brands R&D. “At Actavis, we are dedicated to providing new treatment options, and the development of new agents that help address the most bothersome symptoms of IBS-D. We are very pleased to be working with the FDA to advance this IBS-D treatment and we eagerly await DEA scheduling determination later this year.”
IBS-D is a multifactorial disorder marked by recurrent abdominal pain or discomfort and altered bowel function that affects as many as 15 million adult Americans, impacting about twice as many women as men.i,ii,iii There are few treatment options available for IBS-D, particularly options that relieve both the diarrhea and abdominal pain associated with IBS-D.
“The unpredictable symptoms experienced by patients with IBS-D can have a significant impact on everyday life,” said William D. Chey, MD, Nostrant Professor of Gastroenterology at the University of Michigan Health System. “It’s exciting when physicians are able to add an additional treatment option like VIBERZI to their toolbox for patients with IBS-D.”
The FDA has recommended that VIBERZI be classified as a controlled substance. This recommendation has been submitted to the U.S. Drug Enforcement Administration (DEA). Once VIBERZI receives final scheduling designation, the updated label will be available. Pending final scheduling designation, product launch is anticipated in Q1 2016.
About VIBERZI
VIBERZI is an orally active compound indicated for the treatment of irritable bowel syndrome with diarrhea (IBS-D) in men and women. VIBERZI (eluxadoline) has mixed opioid receptor activity, it is a mu receptor agonist, a delta receptor antagonist, and a kappa receptor agonist.
Efficacy was established in two Phase III clinical studies, demonstrating significant superiority over placebo on the composite endpoint of simultaneous improvement in both abdominal pain and diarrhea at both 75 mg and 100 mg twice daily doses. The primary efficacy responder endpoint was evaluated over the duration of double-blind, placebo-controlled treatment. Response rates were compared based on patients who met the daily composite response criteria (improvement in both abdominal pain and stool consistency on the same day) for at least 50% of the days from weeks 1 to 12 (FDA endpoint) and weeks 1 to 26 (European Medicines Agency endpoint).
The most common adverse events in the two Phase III clinical trials were constipation (7% and 8% for eluxadoline 75 mg and 100 mg; 2% for placebo) and nausea (8% and 7% for eluxadoline 75 mg and 100 mg; 5% for placebo). Rates of severe constipation were less than 1% in patients receiving 75 mg and 100 mg eluxadoline. Rates of discontinuation due to constipation were low for both eluxadoline and placebo (≤2%) and similar rates of constipation occurred between the active and placebo arms beyond 3 months of treatment. A total of 2,426 subjects were enrolled across the two studies.
Irritable bowel syndrome with diarrhea (IBS-D) is a functional bowel disorder characterized by chronic abdominal pain and frequent diarrhea, which affects approximately 15 million patients in the U.S. Although the exact cause of IBS-D is not known, symptoms are thought to result from a disturbance in the way the gastrointestinal tract and nervous system interact.
IBS-D can be debilitating and there are limited therapeutic options for managing the chronic symptoms. IBS-D is associated with economic burden in direct medical costs and indirect social costs such as absenteeism and lost productivity, along with decreased quality of life.
About Actavis
Actavis plc (NYSE: ACT), headquartered in Dublin, Ireland, is a unique, global pharmaceutical company and a leader in a new industry model—Growth Pharma. Actavis is focused on developing, manufacturing and commercializing innovative branded pharmaceuticals, high-quality generic and over-the-counter medicines and biologic products for patients around the world.
Actavis markets a portfolio of best-in-class products that provide valuable treatments for the central nervous system, eye care, medical aesthetics, gastroenterology, women’s health, urology, cardiovascular and anti-infective therapeutic categories, and operates the world’s third-largest global generics business, providing patients around the globe with increased access to affordable, high-quality medicines. Actavis is an industry leader in research and development, with one of the broadest development pipelines in the pharmaceutical industry and a leading position in the submission of generic product applications globally.
With commercial operations in approximately 100 countries, Actavis is committed to working with physicians, healthcare providers and patients to deliver innovative and meaningful treatments that help people around the world live longer, healthier lives.
Actavis intends to adopt a new global name – Allergan – pending shareholder approval in 2015.
For more information, visit Actavis’ website at www.actavis.com.
Statements contained in this communication that refer to Actavis’ estimated or anticipated future results, including estimated synergies, or other non-historical facts are forward-looking statements that reflect Actavis’ current perspective of existing trends and information as of the date of this communication. Actual results may differ materially from Actavis’ current expectations depending upon a number of factors affecting Actavis’ business. These factors include, among others, the timing and success of product launches; the difficulty of predicting the timing or outcome of product development efforts and regulatory agency approvals or actions, if any; market acceptance of and continued demand for Actavis’ products; difficulties or delays in manufacturing; and such other risks and uncertainties detailed in Actavis’ periodic public filings with the Securities and Exchange Commission, including but not limited to Actavis plc’s Quarterly Report on Form 10-Q for the quarter ended March 31, 2015 and from time to time in Actavis’ other investor communications. Except as expressly required by law, Actavis disclaims any intent or obligation to update or revise these forward-looking statements.
i Camilleri M. Current and future pharmacological treatments for diarrhea-predominant irritable bowel syndrome. Expert Opinion on Pharmacotherapy. 2013;14:1151.
ii Grundmann O, Yoon SL. Irritable bowel syndrome: epidemiology, diagnosis, and treatment: an update for health-care practitioners. Journal of Gastroenterology and Hepatology. 2010;25:691–699.
iii Eluxadoline Xifaxin Summary Final. November 2014.
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Keep an eye on Synergy Pharmaceuticals (SGYP) this month: Analysts like it, its shares have waned since a big spike this summer, and the official filing of its star product is expected any day.
When the New York-based pharmaceutical company, which specializes in gastrointestinal therapy, announced that it passed clinical trials on its flagship drug plecanatide this summer, shares rocketed 95%.
But today analysts appear mystified at why the stock has receded 45% from its July high, especially with plecanatide’s new drug application with the Food and Drug Administration expected this month. (It’s currently trading below $6, and the consensus price target is over $13, according to data provided by Bloomberg.)
Synergy should be raking in $600 million from plecanatide, a daily tablet that treats patients with irritable bowel syndrome (IBS), within five years of obtaining FDA approval (expected in 2017, according to equity research firm BTIG. Synergy currently has a market capitalization of just $645 million.
BTIG’s $11 price target is also buoyed by roughly $142 million on the balance sheet, as well as newly appointed management including CFO Gary Sender and COO Troy Hamilton, both former executives at pharma success story Shire (SHPG). Though Shire shares are down just under 4% over the past 12 month, they have rocketed 112% over the past two years.
Synergy also stands to benefit from a growing demand for gastrointestinal treatments, feeding the appetite of Big Pharma for potential acquisitions, according to BTIG.
“With about 45 million Americans suffering from chronic constipation and IBS, and major companies like Allergan(AGN) and Valeant (VRX) focusing their marketing efforts on GI treatments, it seems logical to imagine SGYP as a takeover candidate,” BTIG analyst Timothy Chiang wrote in a November report.
Whether or not this leads to a buyout or another stock surge, Synergy certainly can be counted on for a healthy dose of small-cap volatility as its chief product takes the final steps toward reaching its customers.
Synergy Pharmaceuticals Announces Successful End-of-Phase 2 Meeting with FDA for Plecanatide in Irritable Bowel Syndrome with Constipation
Pivotal Phase 3 IBS-C Program to be Initiated in the Fourth Quarter of 2014
NEW YORK– Synergy Pharmaceuticals Inc. (NASDAQ:SGYP) today announced that it has successfully completed an End-of-Phase 2 meeting with the U.S. Food and Drug Administration (FDA) on its lead drug plecanatide for the treatment of irritable bowel syndrome with constipation (IBS-C). Agreement was reached with the FDA for the plecanatide pivotal phase 3 IBS-C clinical development program that is scheduled to begin in the fourth quarter of this year.
“We are very pleased with the outcome of our meeting with the FDA and have a clear path forward to start the IBS-C registration program with plecanatide this year,” said Dr. Gary S. Jacob, Chairman and CEO of Synergy. “The pivotal phase 3 IBS-C trials will include both 3.0 mg and 6.0 mg plecanatide, which are consistent with the doses currently being evaluated in our phase 3 chronic idiopathic constipation (CIC) program. Plecanatide has demonstrated a clinical dose-response for efficacy with an excellent tolerability profile that is observed across trials. This is an important advantage as we look to bring two doses to market in both indications and provide physicians with options for addressing individual patient needs.”
Synergy’s pivotal phase 3 IBS-C clinical development program will consist of two registration trials, each including 1,050 patients who will receive either placebo, 3.0 mg or 6.0 mg plecanatide. IBS-C patients successfully completing either of the 12-week placebo-controlled registration trials will be offered enrollment into a long-term safety trial in order to complement and support the ongoing long-term safety database for the CIC indication.
About Plecanatide
Plecanatide is Synergy’s lead uroguanylin analog in late-stage clinical development to treat patients with CIC and IBS-C. Uroguanylin is a natural gastrointestinal (GI) hormone produced by humans in the small intestine and plays a key role in regulating the normal functioning of the digestive tract through its activity on the guanylate cyclase-C (GC-C) receptor. The GC-C receptor is known to be a primary source for stimulating a variety of beneficial physiological responses. Orally administered plecanatide mimics uroguanylin’s functions by binding to and activating the GC-C receptor to stimulate fluid and ion transit required for normal bowel function. Synergy has successfully completed a phase 2b trial of plecanatide in 951 patients with CIC and is currently enrolling patients in two pivotal phase 3 CIC trials. The company also recently announced positive top-line data results from a phase 2b dose-ranging study with plecanatide in patients with IBS-C.
About Synergy Pharmaceuticals
Synergy Pharmaceuticals (NASDAQ:SGYP) is a biopharmaceutical company focused on the development of novel therapies based on the natural human hormone, uroguanylin, to treat GI diseases and disorders. Synergy has created two unique analogs of uroguanylin – plecanatide and SP-333 – designed to mimic the natural hormone’s activity on the GC-C receptor and target a variety of GI conditions. SP-333 is currently in phase 2 development for opioid-induced constipation and is also being explored for ulcerative colitis. For more information, please visit www.synergypharma.com.
Pfizer Inc. is in advanced talks to buy Allergan Plc for as much as $380 per share, according to people familiar with the matter, valuing the Botox maker at as high as $150 billion — if the U.S. government doesn’t get in the way of the drug industry’s largest-ever deal.
There has been 51 US company tax-inversion based relocations since 1982 with the rate picking up in the last 3 years (from Bloomberg Data). Many of these inversions in recent years have involved large pharma companies.
The companies aim to announce an agreement as soon as Monday, the people said, asking not to be identified because the discussions are private. The price being discussed is $370 to $380 per share, two of the people said. However, the U.S. Treasury Department’s letter on tax inversion deals, released on Wednesday, could delay the final agreement and change the terms of any transaction, another person said.
Pfizer shares sank 1.5 percent to $32.80 and Allergan fell 1.4 percent to $306.37 at 9:57 a.m. in New York on speculation that the deal could be hampered by the Treasury’s letter, which said the department is reviewing ways to address overseas acquisitions and plans to issue guidance later this week.
FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
FDA Cellular & Gene Therapy Guidances: Implications for CRSPR/Cas9 Trials
Reporter: Stephen J. Williams, PhD
The recent announcement by Editas CEO Katrine Bosley to pursue a CRSPR/Cas9 gene therapy trial to correct defects in an yet to be disclosed gene to treat one form of a rare eye disease called Leber congenital amaurosis (multiple mutant genes have been linked to the disease) have put an interesting emphasis on the need for a regulatory framework to initiate these trials. Indeed at the 2015 EmTechMIT Conference Editas CEO Katrine Bosley had mentioned this particular issue: the need for discourse with FDA and regulatory bodies to establish guidelines for design of clinical trials using the CRSPR gene editing tool.
To this effect, I have listed below, the multiple FDA Guidance Documents surrounding gene therapy to show that, in the past year, the FDA has shown great commitment to devise a regulatory framework for this therapeutic area.
FDA Guidance On Source Animal, Product, Preclinical and Clinical Issues Concerning the Use of Xenotranspantation Products in Humans – Implications for 3D BioPrinting of Regenerative Tissue
Reporter: Stephen J. Williams, Ph.D.
The FDA has submitted Final Guidance on use xeno-transplanted animal tissue, products, and cells into human and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.
This document is to provide guidance on the production, testing and evaluation of products intended for use in xenotransplantation. The guidance includes scientific questions that should be addressed by sponsors during protocol development and during the preparation of submissions to the Food and Drug Administration (FDA), e.g., Investigational New Drug Application (IND) and Biologics License Application (BLA). This guidance document finalizes the draft guidance of the same title dated February 2001.
For the purpose of this document, xenotransplantation refers to any procedure that involves the transplantation, implantation, or infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs. For the purpose of this document, xenotransplantation products include live cells, tissues or organs used in xenotransplantation. (See Definitions in section I.C.)
This document presents issues that should be considered in addressing the safety of viable materials obtained from animal sources and intended for clinical use in humans. The potential threat to both human and animal welfare from zoonotic or other infectious agents warrants careful characterization of animal sources of cells, tissues, and organs. This document addresses issues such as the characterization of source animals, source animal husbandry practices, characterization of xenotransplantation products, considerations for the xenotransplantation product manufacturing facility, appropriate preclinical models for xenotransplantation protocols, and monitoring of recipients of xenotransplantation products. This document recommends specific practices intended to prevent the introduction and spread of infectious agents of animal origin into the human population. FDA expects that new methods proposed by sponsors to address specific issues will be scientifically rigorous and that sufficient data will be presented to justify their use.
Examples of procedures involving xenotransplantation products include:
transplantation of xenogeneic hearts, kidneys, or pancreatic tissue to treat organ failure,
implantation of neural cells to ameliorate neurological degenerative diseases,
administration of human cells previously cultured ex vivo with live nonhuman animal antigen-presenting or feeder cells, and
extracorporeal perfusion of a patient’s blood or blood component perfused through an intact animal organ or isolated cells contained in a device to treat liver failure.
Imagine stripping out the failing components of an old car — the engine, transmission, exhaust system and all of those parts — leaving just the old body and other structural elements. Replace those old mechanical parts with a brand new electric, hydrogen powered, biofuel, nuclear or whatever kind of engine you want and now you have a brand new car. It has an old frame, but that’s okay. The frame wasn’t causing the problem, and it can live on for years, undamaged.
When challenged to design internal organs, tissue engineers are taking a similar approach, particularly with the most complex organs, like the heart, liver and kidneys. These organs have three dimensional structures that are elaborate, not just at the gross anatomic level, but in microscopic anatomy too. Some day, their complex connective tissue scaffolding, the stroma, might be synthesized from the needed collagen proteins with advanced 3-D printing. But biomedical engineering is not there yet, so right now the best candidate for organ scaffolding comes from one of humanity’s favorite farm animals: the pig.
Chimera alarmists connecting with anti-biotechnology movements might cringe at the thought of building new human organs starting with pig tissue, but if you’re using only the organ scaffolding and building a working organ from there, pig organs may actually be more desirable than those donated by humans.
How big is the anti-chimerite movement?
Unlike anti-GMO and anti-vaccination activists, there really aren’t too many anti-chemerites around. Nevertheless, there is a presence on the web of people who express concern about mixing of humans and non-human animals. Presently, much of their concern is focussed on the growing of human organs inside non-human animals, pigs included. One anti-chemerite has written that it could be a problem for the following reason:
Once a human organ is grown inside a pig, that pig is no longer fully a pig. And without a doubt, that organ will no longer be a fully human organ after it is grown inside the pig. Those receiving those organs will be allowing human-animal hybrid organs to be implanted into them. Most people would be absolutely shocked to learn some of the things that are currently being done in the name of science.
The blog goes on to express alarm about the use of human genes in rice and from there morphs into an off the shelf garden variety anti-GMO tirade, though with an an anti-chemeric current running through it. The concern about making pigs a little bit human and humans a little bit pig becomes a concern about making rice a little bit human. But the concern about fusing tissues and genes of humans and other species does not fit with the trend in modern medicine.
Utilization of pig tissue enters a new age
A porcine human ear for xenotransplantation. source: The Scientist
For decades, pig, bovine and other non-human tissues have been used in medicine. People are walking around with pig and cow heart valves. Diabetics used to get a lot of insulin from pigs and cows, although today, thanks to genetic engineering, they’re getting human insulin produced by microorganisms modified genetically to make human insulin, which is safer and more effective.
When it comes to building new organs from old ones, however, pig organs could actually be superior for a couple of reasons. For one thing, there’s no availability problem with pigs. Their hearts and other organs also have all of the crucial components of the extracellular matrix that makes up an organ’s scaffolding. But unlike human organs, the pig organs don’t tend to carry or transfer human diseases. That is a major advantage that makes them ideal starting material. Plus there is another advantage: typically, the hearts of human cadavers are damaged, either because heart disease is what killed the human owner or because resuscitation efforts aimed at restarting the heart of a dying person using electrical jolts and powerful drugs.
Rebuilding an old organ into a new one
How then does the process work? Whether starting with a donated human or pig organ, there are several possible methods. But what they all have in common is that only the scaffolding of the original organ is retained. Just like the engine and transmission of the old car, the working tissue is removed, usually using detergents. One promising technique that has been applied to engineer new hearts is being tested by researchers at the University of Pittsburgh. Detergents pumped into the aorta attached to a donated heart (donated by a human cadaver, or pig or cow). The pressure keeps the aortic valve closed, so the detergents to into the coronary arteries and through the myocardial (heart muscle) and endocardial (lining over the muscle inside the heart chambers) tissue, which thus gets dissolved over the course of days. What’s left is just the stroma tissue, forming a scaffold. But that scaffold has signaling factors that enable embryonic stem cells, or specially programed adult pleuripotent cells to become all of the needed cells for a new heart.
Eventually, 3-D printing technology may reach the point when no donated scaffolding is needed, but that’s not the case quite yet, plus with a pig scaffolding all of the needed signaling factors are there and they work just as well as those in a human heart scaffold. All of this can lead to a scenario, possibly very soon, in which organs are made using off-the-self scaffolding from pig organs, ready to produce a custom-made heart using stem or other cells donated by new organ’s recipient.
David Warmflash is an astrobiologist, physician, and science writer. Follow @CosmicEvolution to read what he is saying on Twitter.
And a Great Article in The Scientist by Dr. Ed Yong Entitled
To cope with a growing shortage of hearts, livers, and lungs suitable for transplant, some scientists are genetically engineering pigs, while others are growing organs in the lab.
.. where Joseph Vacanti and David Cooper figured that using
“engineered pigs without the a-1,3-galactosyltransferase gene that produces the a-gal residues. In addition, the pigs carry human cell-membrane proteins such as CD55 and CD46 that prevent the host’s complement system from assembling and attacking the foreign cells”
… thereby limiting rejection of the xenotransplated tissue.
In addition to issues related to animal virus transmission the issue of optimal scaffolds for organs as well as the advantages which 3D Printing would have in mass production of organs is discussed:
To Vacanti, artificial scaffolds are the future of organ engineering, and the only way in which organs for transplantation could be mass-produced. “You should be able to make them on demand, with low-cost materials and manufacturing technologies,” he says. That is relatively simple for organs like tracheas or bladders, which are just hollow tubes or sacs. Even though it is far more difficult for the lung or liver, which have complicated structures, Vacanti thinks it will be possible to simulate their architecture with computer models, and fabricate them with modern printing technology. (See “3-D Printing,” The Scientist, July 2012.) “They obey very ordered rules, so you can reduce it down to a series of algorithms, which can help you design them,” he says. But Taylor says that even if the architecture is correct, the scaffold would still need to contain the right surface molecules to guide the growth of any added cells. “It seems a bit of an overkill when nature has already done the work for us,” she says.
Other articles of FDA Guidance and 3D Bio Printing on this Open Access Journal Include:
New FDA Draft Guidance On Homologous Use of Human Cells, Tissues, and Cellular and Tissue-Based Products – Implications for 3D BioPrinting of Regenerative Tissue
Reporter: Stephen J. Williams, Ph.D.
The FDA recently came out with a Draft Guidance on use of human cells, tissues and cellular and tissue-based products (HCT/P) {defined in 21 CFR 1271.3(d)} and their use in medical procedures. Although the draft guidance was to expand on previous guidelines to prevent the introduction, transmission, and spread of communicable diseases, this updated draft may have implications for use of such tissue in the emerging medical 3D printing field.
In 21 CFR 1271.10, the regulations identify the criteria for regulation solely under section 361 of the PHS Act and 21 CFR Part 1271. An HCT/P is regulated solely under section 361 of the PHS Act and 21 CFR Part 1271 if it meets all of the following criteria (21 CFR 1271.10(a)):
The HCT/P is minimally manipulated;
The HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent;
The manufacture of the HCT/P does not involve the combination of the cells or tissues with another article, except for water, crystalloids, or a sterilizing, preserving, or storage agent, provided that the addition of water, crystalloids, or the sterilizing, preserving, or storage agent does not raise new clinical safety concerns with respect to the HCT/P; and
Either:
The HCT/P does not have a systemic effect and is not dependent upon the metabolic activity of living cells for its primary function; or
The HCT/P has a systemic effect or is dependent upon the metabolic activity of living cells for its primary function, and:
Is for autologous use;
Is for allogeneic use in a first-degree or second-degree blood relative; or
Is for reproductive use.
If an HCT/P does not meet all of the criteria in 21 CFR 1271.10(a), and the establishment that manufactures the HCT/P does not qualify for any of the exceptions in 21 CFR 1271.15, the HCT/P will be regulated as a drug, device, and/or biological product under the Federal Food, Drug and Cosmetic Act (FD&C Act), and/or section 351 of the PHS Act, and applicable regulations, including 21 CFR Part 1271, and pre-market review will be required.
1 Examples of HCT/Ps include, but are not limited to, bone, ligament, skin, dura mater, heart valve, cornea, hematopoietic stem/progenitor cells derived from peripheral and cord blood, manipulated autologous chondrocytes, epithelial cells on a synthetic matrix, and semen or other reproductive tissue. The following articles are not considered HCT/Ps: (1) Vascularized human organs for transplantation; (2) Whole blood or blood components or blood derivative products subject to listing under 21 CFR Parts 607 and 207, respectively; (3) Secreted or extracted human products, such as milk, collagen, and cell factors, except that semen is considered an HCT/P; (4) Minimally manipulated bone marrow for homologous use and not combined with another article (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the bone marrow); (5) Ancillary products used in the manufacture of HCT/P; (6) Cells, tissues, and organs derived from animals other than humans; (7) In vitro diagnostic products as defined in 21 CFR 809.3(a); and (8) Blood vessels recovered with an organ, as defined in 42 CFR 121.2 that are intended for use in organ transplantation and labeled “For use in organ transplantation only.” (21 CFR 1271.3(d))
Contains Nonbinding Recommendations Draft – Not for Implementation
Section 1271.10(a)(2) (21 CFR 1271.10(a)(2)) provides that one of the criteria for an HCT/P to be regulated solely under section 361 of the PHS Act is that the “HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.” As defined in 21 CFR 1271.3(c), homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor. This criterion reflects the Agency’s conclusion that there would be increased safety and effectiveness concerns for HCT/Ps that are intended for a non-homologous use, because there is less basis on which to predict the product’s behavior, whereas HCT/Ps for homologous use can reasonably be expected to function appropriately (assuming all of the other criteria are also met).2 In applying the homologous use criterion, FDA will determine what the intended use of the HCT/P is, as reflected by the the labeling, advertising, and other indications of a manufacturer’s objective intent, and will then apply the homologous use definition.
FDA has received many inquiries from manufacturers about whether their HCT/Ps meet the homologous use criterion in 21 CFR 1271.10(a)(2). Additionally, transplant and healthcare providers often need to know this information about the HCT/Ps that they are considering for use in their patients. This guidance provides examples of different types of HCT/Ps and how the regulation in 21 CFR 1271.10(a)(2) applies to them, and provides general principles that can be applied to HCT/Ps that may be developed in the future. In some of the examples, the HCT/Ps may fail to meet more than one of the four criteria in 21 CFR 1271.10(a).
III. QUESTIONS AND ANSWERS
What is the definition of homologous use?
Homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor (21 CFR 1271.3(c)), including when such cells or tissues are for autologous use. We generally consider an HCT/P to be for homologous use when it is used to repair, reconstruct, replace, or supplement:
Recipient cells or tissues that are identical (e.g., skin for skin) to the donor cells or tissues, and perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor; or,
Recipient cells that may not be identical to the donor’s cells, or recipient tissues that may not be identical to the donor’s tissues, but that perform one or more of the same basic functions in the recipient as the cells or tissues performed in the donor.3
3“Establishment Registration and Listing for Manufacturers of Human Cellular and Tissue-Based Products” 63 FR 26744 at 26749 (May 14, 1998).
Contains Nonbinding Recommendations Draft – Not for Implementation
1-1. A heart valve is transplanted to replace a dysfunctional heart valve. This is homologous use because the donor heart valve performs the same basic function in the donor as in the recipient of ensuring unidirectional blood flow within the heart.
1-2. Pericardium is intended to be used as a wound covering for dura mater defects. This is homologous use because the pericardium is intended to repair or reconstruct the dura mater and serve as a covering in the recipient, which is one of the basic functions it performs in the donor.
Generally, if an HCT/P is intended for use as an unproven treatment for a myriad of
diseases or conditions, the HCT/P is likely not intended for homologous use only.4
What does FDA mean by repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues?
Repair generally means the physical or mechanical restoration of tissues, including by covering or protecting. For example, FDA generally would consider skin removed from a donor and then transplanted to a recipient in order to cover a burn wound to be a homologous use. Reconstruction generally means surgical reassembling or re-forming. For example, reconstruction generally would include the reestablishment of the physical integrity of a damaged aorta.5 Replacement generally means substitution of a missing tissue or cell, for example, the replacement of a damaged or diseased cornea with a healthy cornea or the replacement of donor hematopoietic stem/progenitor cells in a recipient with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. Supplementation generally means to add to, or complete. For example, FDA generally would consider homologous uses to be the implantation of dermal matrix into the facial wrinkles to supplement a recipient’s tissues and the use of bone chips to supplement bony defects. Repair, reconstruction, replacement, and supplementation are not mutually exclusive functions and an HCT/P could perform more than one of these functions for a given intended use.
What does FDA mean by “the same basic function or functions” in the definition of homologous use?
For the purpose of applying the regulatory framework, the same basic function or functions of HCT/Ps are considered to be those basic functions the HCT/P performs in the body of the donor, which, when transplanted, implanted, infused, or transferred, the HCT/P would be expected to perform in the recipient. It is not necessary for the HCT/P in the recipient to perform all of the basic functions it performed in the donor, in order to
4 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).
5 “Current Good Tissue Practice for Human Cell, Tissue, and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement” 69 FR 68612 at 68643 (November 24, 2004) states, “HCT/Ps with claims for “reconstruction or repair” can be regulated solely under section 361 of the PHS Act, provided the HCT/P meets all the criteria in § 1271.10, including minimal manipulation and homologous use.”
Contains Nonbinding Recommendations Draft – Not for Implementation
meet the definition of homologous use. However, to meet the definition of homologous use, any of the basic functions that the HCT/P is expected to perform in the recipient must be a basic function that the HCT/P performed in the donor.
A homologous use for a structural tissue would generally be to perform a structural function in the recipient, for example, to physically support or serve as a barrier or conduit, or connect, cover, or cushion.
A homologous use for a cellular or nonstructural tissue would generally be a metabolic or biochemical function in the recipient, such as, hematopoietic, immune, and endocrine functions.
3-1. The basic functions of hematopoietic stem/progenitor cells (HPCs) include to form and to replenish the hematopoietic system. Sources of HPCs include cord blood, peripheral blood, and bone marrow.6
HPCs derived from peripheral blood are intended for transplantation into an individual with a disorder affecting the hematopoietic system that is inherited, acquired, or the result of myeloablative treatment. This is homologous use because the peripheral blood product performs the same basic function of reconstituting the hematopoietic system in the recipient.
HPCs derived from bone marrow are infused into an artery with a balloon catheter for the purpose of limiting ventricular remodeling following acute myocardial infarction. This is not homologous use because limiting ventricular remodeling is not a basic function of bone marrow.
A manufacturer provides HPCs derived from cord blood with a package insert stating that cord blood may be infused intravenously to differentiate into neuronal cells for treatment of cerebral palsy. This is not homologous use because there is insufficient evidence to support that such differentiation is a basic function of these cells in the donor.
3-2. The basic functions of the cornea include protecting the eye by forming its outermost layer and serving as the refracting medium of the eye. A corneal graft is transplanted to restore sight in a patient with corneal blindness. This is homologous use because a corneal graft performs the same basic functions in the donor as in the recipient.
3-3. The basic functions of a vein or artery include serving as a conduit for blood flow throughout the body. A cryopreserved vein or artery is used for arteriovenous access during hemodialysis. This is homologous use because the vein or artery is supplementing the vessel as a conduit for blood flow.
3-4. The basic functions of amniotic membrane include covering, protecting, serving as a selective barrier for the movement of nutrients between the external and in utero
6 Bone marrow meets the definition of an HCT/P only if is it more than minimally manipulated; intended by the manufacturer for a non-homologous use, or combined with certain drugs or devices.
Contains Nonbinding Recommendations Draft – Not for Implementation
environment, and to retain fluid in utero. Amniotic membrane is used for bone tissue replacement to support bone regeneration following surgery to repair or replace bone defects. This is not a homologous use because bone regeneration is not a basic function of amniotic membrane.
3-5. The basic functions of pericardium include covering, protecting against infection, fixing the heart to the mediastinum, and providing lubrication to allow normal heart movement within chest. Autologous pericardium is used to replace a dysfunctional heart valve in the same patient. This is not homologous use because facilitating unidirectional blood flow is not a basic function of pericardium.
Does my HCT/P have to be used in the same anatomic location to perform the same basic function or functions?
An HCT/P may perform the same basic function or functions even when it is not used in the same anatomic location where it existed in the donor.7 A transplanted HCT/P could replace missing tissue, or repair, reconstruct, or supplement tissue that is missing or damaged, either when placed in the same or different anatomic location, as long as it performs the same basic function(s) in the recipient as in the donor.
4-1. The basic functions of skin include covering, protecting the body from external force, and serving as a water-resistant barrier to pathogens or other damaging agents in the external environment. The dermis is the elastic connective tissue layer of the skin that provides a supportive layer of the integument and protects the body from mechanical stress.
An acellular dermal product is used for supplemental support, protection, reinforcement, or covering for a tendon. This is homologous use because in both anatomic locations, the dermis provides support and protects the soft tissue structure from mechanical stress.
An acellular dermal product is used for tendon replacement or repair. This is not homologous use because serving as a connection between muscle and bone is not a basic function of dermis.
4-2. The basic functions of amniotic membrane include serving as a selective barrier for the movement of nutrients between the external and in utero environment and to retain fluid in utero. An amniotic membrane product is used for wound healing of dermal ulcers and defects. This is not homologous use because wound healing of dermal lesions is not a basic function of amniotic membrane.
4-3. The basic functions of pancreatic islets include regulating glucose homeostasis within the body. Pancreatic islets are transplanted into the liver through the portal vein,
7 “Human Cells, Tissues, and Cellular and Tissue-Based Products; Establishment Registration and Listing” 66 FR 5447 at 5458 (January 19, 2001).
6
Contains Nonbinding Recommendations Draft – Not for Implementation
for preservation of endocrine function after pancreatectomy. This is homologous use because the regulation of glucose homeostasis is a basic function of pancreatic islets.
What does FDA mean by “intended for homologous use” in 21 CFR 1271.10(a)(2)?
The regulatory criterion in 21 CFR 1271.10(a)(2) states that the HCT/P is intended for homologous use only, as reflected by the labeling, advertising, or other indications of the manufacturer’s objective intent.
Labeling includes the HCT/P label and any written, printed, or graphic materials that supplement, explain, or are textually related to the product, and which are disseminated by or on behalf of its manufacturer.8 Advertising includes information, other than labeling, that originates from the same source as the product and that is intended to supplement, explain, or be textually related to the product (e.g., print advertising, broadcast advertising, electronic advertising (including the Internet), statements of company representatives).9
An HCT/P is intended for homologous use when its labeling, advertising, or other indications of the manufacturer’s objective intent refer to only homologous uses for the HCT/P. When an HCT/P’s labeling, advertising, or other indications of the manufacturer’s objective intent refer to non-homologous uses, the HCT/P would not meet the homologous use criterion in 21 CFR 1271.10(a)(2).
What does FDA mean by “manufacturer’s objective intent” in 21 CFR 1271.10(a)(2)?
A manufacturer’s objective intent is determined by the expressions of the manufacturer or its representatives, or may be shown by the circumstances surrounding the distribution of the article. A manufacturer’s objective intent may, for example, be shown by labeling claims, advertising matter, or oral or written statements by the manufacturer or its representatives. It may be shown by the circumstances that the HCT/P is, with the knowledge of the manufacturer or its representatives, offered for a purpose for which it is neither labeled nor advertised.
Inhibition of Topoisomerase (DNA) I (TOP1): DNA Damage Repair and Anticancer Therapy
Yang Xu and Chengtao Her *
School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Mail Drop 64-7520, Pullman, WA 99164, USA; E-Mail: davidxy22@vetmed.wsu.edu
* Author to whom correspondence should be addressed; E-Mail: cher@wsu.edu; Tel.: +1-509-335-7537; Fax: +1-509-335-4159.
Academic Editors: Wolf-Dietrich Heyer, Thomas Helleday and Fumio Hanaoka Received: 22 May 2015 / Accepted: 14 July 2015 / Published: 22 July 2015
Abstract: Most chemotherapy regimens contain at least one DNA-damaging agent that preferentially affects the growth of cancer cells. This strategy takes advantage of the differences in cell proliferation between normal and cancer cells. Chemotherapeutic drugs are usually designed to target rapid-dividing cells because sustained proliferation is a common feature of cancer [1,2]. Rapid DNA replication is essential for highly proliferative cells, thus blocking of DNA replication will create numerous mutations and/or chromosome rearrangements—ultimately triggering cell death [3]. Along these lines, DNA topoisomerase inhibitors are of great interest because they help to maintain strand breaks generated by topoisomerases during replication. In this article, we discuss the characteristics of topoisomerase (DNA) I (TOP1) and its inhibitors, as well as the underlying DNA repair pathways and the use of TOP1 inhibitors in cancer therapy.
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Type IB Topoisomerases and Inhibitors 1.1. TOP1
DNA topoisomerases resolve topological constraints that may arise from DNA strand separation and are therefore important for transcription and replication [4]. There are six topoisomerases in humans, classified as Type IA, IB and IIA. Type IA topoisomerases TOP3a and TOP3b cleave one DNA strand to relax only negative supercoiling. In addition, TOP3a forms the BTR complex with BLM and RMI1/2, which plays a role in the dissolution of double-Holliday junctions [5]. Type IIA topoisomerases TOP2a and TOP2b generate double-strand breaks on one DNA molecule to allow the passing of other DNA strands [6]. Topoisomerases are attractive drug targets in cancer therapy. For example, the commonly used anticancer agents doxorubicin and etoposide (VP-16) are TOP2 inhibitors [7]. Type IB topoisomerases include the nuclear TOP1 and mitochondrial TOP1mt [4]. TOP1 initiates the DNA relaxation by nicking one DNA strand. It then forms a TOP1-DNA cleavage complex (TOP1cc) by covalently linked to the 3′-phosphate end via its tyrosine residue Y723 (3′-P-Y). Following the resolution of topological entanglements and the removal of TOP1, the 5′-hydroxyl end is realigned with the 3′-end for religation. Each nicking-closing cycle enables the relaxation of one DNA supercoiling (Figure 1).
Figure 1. A schematic representation of strand passages catalyzed by three types of topoisomerases (adapted from ref. [8]).
TOP1 is essential for embryonic development in mammals [9]. Although TOP1 plays an important role in the deconvolution of supercoils arising amid DNA replication, the precise steps involved with
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the recruitment of TOP1 to topological constraints remains to be revealed. It appears that in yeast TOP1 travels at a distance of 600 bp ahead of the replication fork [10] and remains associated with the GINS-MCM complex [11]. However, the yeast TOP1 is distinct from its human counterpart in that it has little effect on fork progression or the firing of replication origin [12]. In humans, TOP1 binds to the regions of the pre-replicative complex in cells during the M, early G1, and G1/S phases of the cell cycle to control the firing of replication origins [12]. This difference may explain why yeast cells are viable in the absence of TOP1. In addition, TOP1 also has functions in transcription that are independent of its role in resolving DNA topological entanglements. First, TOP1 is known to repress transcription by binding to TFIID [13]. Second, inhibition of TOP1 can cause the induction of c-Jun in leukemia cells, suggesting its additional role in the control of transcription [14]. Furthermore, TOP1 interacts with the splicing factor ASF/SF2 by which it promotes the maturation of RNA—through suppressing the formation of R-loops (RNA-DNA hybrids)—and prevents collision between transcription bubble and replication fork [15,16]. It appears that the levels of TOP1 have to be dynamically regulated. In B cells, TOP1 is reduced by activation-induced cytidine deaminase (AID) to facilitate class-switch recombination (CSR) and somatic hypermutation (SHM) [17,18]. Although TOP1mt is important for mitochondrial integrity and metabolism, mice lacking mitochondrial TOP1mt are viable and fertile but they are associated with increased negative supercoiling of mtDNA [19,20].
1.2. TOP1 Inhibitors
Stabilization of TOP1cc by topoisomerase poison is detrimental to cells due to the disruption of DNA uncoiling, increased strand breaks, and unstable RNA transcripts as well as incomplete DNA replication [21]. The TOP1 inhibitor camptothecin (CPT), first isolated from the Chinese tree Camptotheca acuminate, was clinically used for cancer treatment long before it was identified as a TOP1 inhibitor [22]. Due to side effects, CPT is no longer used clinically and it has been replaced by more effective and safer TOP1 inhibitors [23]. Currently, CPT derivatives topotecan (trade name: Hycamtin) and irinotecan (CPT-11, trade name: Camptosar) are routinely used to treat colorectal, ovarian and lung cancers, while a few other TOP1 inhibitors are being tested in clinical trials.
CPT is a 5-ring alkaloid that is active in its closed E-ring (lactone) form but it is inactive with an open E-ring (carboxylate) at physiological and alkaline pH [24]. Therefore, CPT is not effective for inhibiting TOP1mt due to a higher pH mitochondrial environment. The inactive form of CPT tends to bind to serum albumin, which might be a reason for its side effects. CPT is highly specific for TOP1 and the binding is of relatively low affinity and can be reversed after drug removal. These features make the action of CPT controllable [24], and in fact CPT is widely used in studies of replication-associated DNA damage response. There are a few CPT derivatives and non-CPT TOP1 inhibitors [4,8,24]. For example, CPT derivatives Diflomotecan and S39625 were designed to stabilize the E-ring. Irinotecan has the bis-piperidine side chain to increase its water solubility, but it also contributes to some side effects. Non-CPTs—such as indolocarbazoles, phenanthrolines (e.g., ARC-111) and indenoisoquinolines—refer to drugs that have no typical CPT E-ring structures but they can still specifically target TOP1 and bind irreversibly to TOP1cc. Some of the CPT derivatives (i.e., Gimatecan and Belotecan) and non-CPTs (i.e., NSC 725776 and NSC 724998) are presently tested in clinical trials [23].
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How does CPT trap TOP1cc? Analysis of the crystal structure and modeling suggest that CPT-TOP1-DNA forms a ternary complex to prevent the two DNA ends from religation [25–27]. Although it is still controversial on how CPT is intercalated into DNA, it seems that CPT traps TOP1cc with a thymine (T) at the -1 position and a guanine (G) at the +1 position on the scissile strand, and it is therefore sequence-specific [28]. Three amino acid residues of the TOP1 enzyme, R364, D533 and N722, combined with DNA bases, contribute to the stabilization of the ternary complex by forming hydrogen bonds and hydrophobic interactions. It is of note that several point mutations, including N722S, in Camptotheca acuminata TOP1 confer resistance to CPT [29]. Interestingly, the same amino acids also contribute to the inhibition of TOP1 by non-CPT drugs [24].
Repair of TOP1 Poison-Induced DNA Lesions
As aforementioned, CPT-induced trapping of TOP1cc creates a single strand break with a free 5′-hydroxyl group, whereas the 3′-phosphate is connected to Y723 of TOP1 (3′-P-Y). At least two pathways contribute to the repair of DNA lesions created by TOP1 poison [30]. The tyrosyl-DNA-phosphodiesterase (TDP1) pathway starts with the ubiquitination and proteasome-mediated degradation of TOP1 in the CPT-TOP1-DNA complex to generate a 3′-P end linked to a short peptide [31]. TDP1 then cleaves the P-Y bond to release the 3′-P end; however, the 3′-P end cannot be directly ligated to the 5′-OH end because of the requirements of DNA ligases. The human polynucleotide kinase (PNKP) can process the DNA ends by functioning as both a 3′-phosphatase and a kinase to generate the required 3′-OH and 5′-P termini for direct ligation. The rest of the repair events can be best described by the single-strand break (SSB) repair pathway, which will be discussed below. Indeed, TDP1 and PNKP are tightly associated with the SSB repair machinery [32,33].
The endonuclease pathway requires multiple endonucleases to excise the DNA—usually at a few nucleotides away from the 3′-P-TOP1 end – on the scissile strand to release the DNA-TOP1 complex [30]. Initial studies were carried out to identify genes that functioned in CPT repair in the absence of TDP1 in yeast [34,35]. These studies led to the identification of RAD1-RAD10, SLX1-SLX4, MUS81-MMS4, MRE11-SAE2 as well as genes involved in recombination. The RAD1-RAD10 (human XPF/ERCC4-ERCC1) complex is a DNA structure-specific endonuclease that can act on 5′ overhang structures [36]. Interestingly, the cleavage site of XPF-ERCC1 is in the non-protruding DNA strand, about 3–4 nucleotides away from the 3′ end [36]. Therefore, trapped TOP1ccs can be removed by this endonuclease activity. Likewise, MUS81-MMS4 (human MUS81-EME1) can also cleave nicked duplex at the 5′ of the nick [37]. The SLX1-SLX4 endonuclease, although not tested on nicked duplexes, is able to process 3′ flap and other DNA structures [38,39]. In human cells, SLX4 also associates with XPF-ERCC1 and MUS81-EME1 endonucleases to process specific DNA intermediates [39,40]. Moreover, MRE11-RAD50 cleaves the 3′-P-Y bond and resects DNA to produce a 3′-OH end [41]. A direct role of SAE2 (human CtIP) in processing 3′-P-TOP1 is unknown, and its endonuclease activity appears to be limited to the 5′ flap or DNA “hairpin” structures [42,43]. Nonetheless, the endonuclease activity of CtIP is essential for processing CPT adducts [42]. In addition, like CtIP, the 5′ flap endonuclease RAD27 (human FEN1) seems to be unable to directly process 3′-P-TOP1 ends [44]. However, the gap endonuclease activity of FEN1 is important for processing stalled replication forks and CPT-induced adducts [45]. The role of FEN1 in SSB repair will be discussed further in the next section.
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During DNA replication, SSBs created by CPT are most likely converted to double-strand breaks (DSBs) by replication fork runoff. This conversion appears to be dependent on the proteolysis of TOP1 [46]. The repair of one-ended DSBs, as will be discussed in the next section, is largely dependent on homologous recombination (HR). However, low doses of CPT may also induce PARP1 and/or RAD51 dependent replication fork regression—generating no or few DSBs [47,48]. The regressed fork leads to the formation of a “chicken foot” DNA structure by newly synthesized strands [3,49,50]. The formation of regressed fork can be largely suppressed by ATR, EXO1, and DNA2 [51–53]. However, fork reversal can also be beneficial as it provides time for the repair of TOP1-induced DNA lesions by TDP1, thereby preventing DSB formation and the activation of error-prone non-homologous end-joining (NHEJ) [30].
Pathways Involved in the Repair of CPT-Induced DNA Lesions
Normal cells use DNA damage response (DDR) pathways to maintain genomic stability [54]. As aforementioned, SSB and DSB repair mechanisms are the two major DDR pathways that repair TOP1-induced DNA lesions. Paradoxically, cancer cells exploit DDR pathways to accumulate necessary genomic alterations for promoting proliferation. Furthermore, altered DDR and apoptotic responses in cancer cells are the major obstacles to successful chemotherapy. Thus, the delineation of TOP1-related SSB and DSB repair mechanisms is of great importance for identifying drug targets that can selectively affect cancer cell survival.
3.1. Single-Strand Break (SSB) Repair
Trapping of TOP1cc results in a 3′-P-TOP1 end and a 5′-OH terminus. Because the two ends cannot be directly religated, the persisting SSB is likely to be detected by PARP1 in which activated PARP1 catalyzes the synthesis of poly(ADP-ribose) (PAR) chains for recruiting repair proteins [55]. This reaction can be rapidly reversed by PARG, which hydrolyzes the PAR chains. The PAR chains at the SSB sites are important for the recruitment of XRCC1 that functions as a loading dock for other SSB repair proteins including TDP1 and PNKP. TDP1 generates 3′-P and PNKP converts 3′-P to 3′-OH, and PNKP also converts 5′-OH to 5′-P, making ends compatible for religation with no base loss. The rejoining of the 3′-OH and 5′-P ends is mainly mediated by LIG3, in which XRCC1 mediates the recruitment of LIG3.
If the trapped TOP1cc intermediates are processed by endonucleases, the initial SSBs will be converted to 3′-OH and 5′-OH ends with a gap over a few nucleotides (in the case of XPF-ERCC1, the loss is in the range of 3–4 nt), leading to the activation of PARP1 and XRCC1 recruitment. Consequentially, Pol3 recruited by XRCC1 can catalyze the gap filling, and PCNA-Polö/E also plays a role in this process [55]. If the 5′-OH is not processed by PNKP, the 5′-flap resulted from gap filling is likely to be removed by FEN1, which explains why FEN1 deficiency also leads to an increased CPT sensitivity. The final ligation is catalyzed by LIG1 because of the presence of PCNA.
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3.2. Double-Strand Break (DSB) Repair
Successful DSB repair requires concerted actions of proteins involved in DNA damage signaling and repair [54]. To repair TOP1 poison-induced DNA lesions, ATR signaling is required due to the runoff of replication fork and the presence of long single-strand DNA (ssDNA) [56]. The full activation of ATR follows a “two-man” rule—the ssDNA-ATRIP-dependent recruitment of ATR kinase and the RAD17 clamp loader/9-1-1/TOPBP1 mediator loading at the ssDNA-dsDNA junction. ATR phosphorylates CHEK1 to harness cell cycle arrest. If one-ended DSB is formed, ATM will be activated through the action of the MRE11-RAD50-NBS1 (MRN) complex. ATM mainly phosphorylates CHEK2 to mediate cell cycle arrest. Both ATM and ATR are able to phosphorylate hundreds of proteins in response to DSB formation [57]. One remarkable substrate is the histone H2AX, which can be phosphorylated by both kinases to yield g-H2AX. It is conceived that the propagation of g-H2AX signaling along the chromatin facilitates MDC1 recruitment and BRCA1 signaling via the MDC1-RNF8-RNF168-RAP80 ubiquitin cascade—events that are essential for HR [58].
The repair of TOP1 poison-induced DNA lesions is in essence the repair of one-ended DSBs, which facilitates the restoration of replication forks to restart DNA replication. It is important to note that one-ended DSB repair occurs in the S phase and relies on HR rather than NHEJ [59]. The first step in HR is end resection to generate a 3′-overhang for homology searching. A TOP1 cleavage in the leading strand may require end resection by the MRN-CtIP-BRCA1 and BLM-EXO1-DNA2 complexes [60], whereas a cleavage in the lagging strand automatically forms a 3′-overhang. Rad51 then associates with the 3′-ssDNA to form a nucleofilament for strand invasion, which leads to the formation of a D-loop structure [61]. This process continues with DNA synthesis, branch migration and the resolution of Holliday junction structures to reconstitute a functional replication fork [62]. TOP1 poisons can also lead to the formation of two-ended DSB if two replication forks collide into each other at the site of SSB. The repair of this type of DSBs is not aimed for fork restoration and can be accomplished by the classical DSB repair mechanisms [61].
3.3. Genes Involved in CPT-Induced Damage Repair
A long list of genes, in which mutations confer sensitivity to CPT in yeast, chicken or mammalian cells, has been compiled [24,30,63]. With no surprise, many genes involved in SSB and DSB repair are on the list, such as PARP1, XRCC1, PNKP, TDP1 for SSB repair; MRN, ATM-CHK2, ATR-CHK1 for DSB signaling; BRCA1/2, XRCC2, XRCC3 for HR. Most recently, the hMSH5-FANCJ complex has also been implicated to play a role in CPT-induced DNA damage response and repair [64]. Mutations in the binding partners of these repair factors are also likely to sensitize cells to CPT treatment. For example, depletion of the MRN-binding partner hnRNPUL increases the sensitivity to CPT [65]; and deficiencies in ZRANB3 and SPIDR, binding partners of PCNA and RAD51, cause CPT hypersensitivity in cancer cells [66–68]. In addition, the two DNA helicases BLM and WRN have also been implicated in the repair of CPT-induced DNA lesions [69,70]. Early studies revealed that chicken BLM knockout cells and human BLM-deficient fibroblasts showed increased sensitivity to CPT [71,72]. On the contrary, mouse BLM knockout embryonic stem cells showed mild resistance to
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CPT [73]. This discrepancy is likely attributable to the complexity of CPT-induced DNA lesion repair as well as different treatment conditions and experimental systems.
Interstrand crosslinks (ICLs) resemble CPT-induced lesions in that they block both replication and transcription [74]. They may induce replication fork reversal and fork collapse, which require DNA incision for lesion processing and HR for repair. ICL repair is accomplished by the coordinated actions of 17 Fanconi anemia (FA) genes whose mutations contribute to FA in patients [75]. Depletion of FANCP/SLX4 or FANCQ/XPF causes cellular sensitivity to CPT because they form an endonuclease complex involved in the repair of trapped TOP1cc [38]. Likewise, depletion of FANCS/BRCA1, FANCD1/BRCA2, FANCN/PALB2 or FANCO/RAD51C sensitizes cells to CPT because of their involvement in HR [76]. Accordingly, depletion of the FA core complex except FANCM—involved in fork reversal—is not expected to increase CPT sensitivity because they are unable to recognize the trapped TOP1cc [76]. However, the roles of FANCI, D2, J and FAN1 in the process are elusive due to conflicting reports presumably reflecting different experimental systems [76–78]. For example, in a multicolor competition assay, loss of FANCI or FAN1 rendered cells sensitive to CPT treatment [77]. However, this observation could not be recapitulated in studies performed with FANCI-deficient lymphoblasts and FAN1-depleted HEK293 cells [76,79], indicating that the involvement of these two genes in CTP sensitivity might be cell type specific.
It is interesting to note that the MMS22L-TONSL complex plays a prominent role in mediating CPT sensitivity [80–83]. Depletion of this complex impairs RAD51 foci formation and triggers G2/M arrest, indicating that the MMS22L-TONSL complex participates in HR repair. Furthermore, this complex associates with MCM, FACT, ASF1 and histones. FACT and ASF1 are histone chaperones that function in H2A/H2B and H3/H4 chromatin assembly and disassembly, respectively [84]. They recycle parental histones from old DNA strands unwound by MCM and incorporate them into newly synthesized DNA strands. FACT and ASF1 also function in checkpoint signaling; therefore the involvement of MMS22L-TONSL in CPT response implies the existence of a close association between HR, DNA damage signaling and replication restart.
TOP1 Inhibition in Cancer Treatment
The understanding of the function of TOP1 and the cellular effects of TOP1 inhibition has been a stepping-stone for the development of effective CPT derivatives in cancer therapy. Since TOP1 functions in normal and cancer cells, the use of low doses of TOP1 inhibitors are actively sought to treat cancers that heavily rely on the function of TOP1 for survival (e.g., highly malignant, rapid-dividing tumor cells). In fact, the FDA-approved CPT derivatives topotecan and irinotecan are currently used to treat ovarian and colorectal cancers, respectively [24].
Furthermore, the promising results from a Phase I trial have warranted further evaluation of the CPT derivative Diflomotecan in Phase II trials [85]. Other derivatives like Gimatecan, Lurtotecan and Exatecan are also being tested in clinical trials (Table 1). The non-CPT indolocarbazole BMS-250749 showed great anti-tumor activity against preclinical xenograft models [86], but no further evaluation beyond Phase I trials is presently available (Table 2). Another indolocarbazole compound Edotecarin has shown promising anti-tumor activity in xenograft models and it is now advanced to Phase II studies of patients with advanced solid tumors [87]. By contrast, Phenanthroline ARC-111 (topovale)
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was potently against human tumor xenografts and displayed anti-cancer activity in colon and Wilms’ tumors [88]; however, no result from Phase I clinical trials is available owing to profound bone marrow toxicity [89]. To date, indenoisoquinolines are the most promising non-CPT inhibitors in clinical trials. LMP400 (NSC 743400, indotecan) and LMP776 (NSC 725776, indimitecan) show significant anti-tumor activities in animal models and both are being evaluated in Phase I clinical trials for relapsed solid tumors and lymphomas [8,90].
Table 1. CPT derivatives in clinical trials [91].
Name Structure Clinical Trial Malignancy Reference
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Given the observation that CPT-mediated TOP1 inhibition provokes DNA repair activities, a synergistic effect is then anticipated on cancer cells by inhibition of TOP1 and downregulation of DNA repair activities. The rationale for this approach is to accelerate the accumulation of DNA breaks and trigger cellular apoptosis, probably through mitotic catastrophe [92]. Which DNA repair pathways can we exploit? Currently, the major interests are in SSB and DSB repair mechanisms. Indeed, PARP inhibitors can enhance the cytotoxicity of TOP1 inhibitors in cancer cell lines as well as in mouse models [93–96]. Phase I studies of combination therapy using PARP inhibitors veliparib or olaparib (FDA-approved) together with topotecan were carried out in patients with advanced solid tumors but showed some dose-dependent side effects [97,98]. TDP1 can be another potential target because it functions directly downstream of PARP1 in the repair of TOP1 poison-induced DNA lesions [99]. TDP1 inhibitors sensitize cells to CPT treatment in vitro [100,101], however in vivo evaluation is presently unavailable due to unsuitable properties of the compounds [102].
Table 2. Non-CPT derivatives in preclinical and clinical trials [91].
Name Structure Clinical Trial Malignancy Reference
Indolocarbazoles
(Edotecarin,
BMS-250749)
Phase II
(Edotecarin, Pfizer)
Stomach, breast
neoplasms
Preclinical
(BMS-250749)
Anti-tumor activity
in preclinical xenograft models
[86,87,103]
Phenanthridines
(ARC-111/topovale)
Anti-tumor activity
Preclinical in preclinical [88,89,103]
xenograft models
Indenoisoquinolines
(LMP400, LMP776)
Phase I Lymphomas [8,90,103]
DSB repair can be targeted by either inhibition of DSB signaling or inhibition of HR. ATM and ATR inhibitors can largely increase the sensitivity to CPT in cancer cells [104,105]. This can be explained by the fact that abrogation of the cell cycle arrest will allow cells with unreplicated or unrepaired chromosomes to enter mitosis thereby triggering mitotic catastrophe and cell death. Similarly, CHEK1 and CHEK2 inhibitors are tested in Phase I studies in combination with irinotecan [106,107]. Inhibitors that can directly block HR proteins are very limited [108]. This is partially attributed to the fact that HR genes are often mutated in cancer cells, thus diminishing the enthusiasm for developing HR inhibitors. One diterpenoid compound, however, was found to be able to inhibit the function of BRCA1 and render cytotoxicity in human prostate cancer cells [109]. Several RAD51 inhibitors have also been
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identified but have not been tested in cell lines [110]. Inhibition of BRCA1 and RAD51 can be also achieved indirectly by harnessing corresponding kinases [106]. Clearly, defective hMRE11 sensitizes colon cancer cells to CPT treatment [111]. Although MRE11-deficeint tumor xenografts failed to display significant growth inhibition by irinotecan alone, combining thymidine with irinotecan caused a dramatic growth delay [112].
TOP1 inhibitors might be also useful for treating cancers with BRCA1/2 mutations. The successful use of PARP inhibitors in treating BRCA1/2-deficient tumors has ignited a broad interest in searching for synthetic lethality among DNA damage response and repair genes [113,114]. In the PARP-BRCA1/2 example, the accumulation of SSBs by PARP inhibition would lead to the formation of DSBs during replication. In HR-deficient cells, DSBs can only be repaired by illegitimate (toxic) NHEJ—joining one-ended DSBs from different locations—leading to cell death [115,116]. However, resistance to PARP inhibitors can arise in BRCA1-deficient tumors during treatment from either genetic reversion of BRCA1 mutations or the loss of NHEJ [117–122]. Therefore, it would be beneficial to explore the possibility of developing a similar synthetic lethal strategy to use TOP1 inhibitors in the treatment of BRCA1/2-deficient tumors.
Figure 2. An overview of the effects of TOP1 inhibition is provided. Inhibitors and key DNA repair factors are highlighted.
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Conclusions
Trapping of TOP1 by inhibitors generates SSBs and DSBs that are repaired by their corresponding repair pathways (Figure 2). Therefore, developing effective TOP1 inhibitors not only provides powerful tools to study DNA replication and repair but also establishes a foundation to devise new synthetic lethal strategies for efficient cancer treatments. The accumulation of DNA strand breaks (SSBs and DSBs) by TOP1 inhibition in HR-deficient tumor cells is expected to enhance cytotoxicity. However, increased DNA repair activities in cancer cells can make TOP1 inhibitors less effective, so silencing of repair pathways in conjunction with the use of TOP1 inhibitors offers an attractive new means for cancer control. Since each tumor is unique, it would be advantageous to identify the individualities of DNA repair pathways or biomarkers reflecting the changes of DNA repair activities in tumor cells [92,123]. This will make it possible to achieve better and predictable prognosis through tailored therapeutic regimens. Given that TOP1 is essential for transcription and DNA replication, future design of novel TOP1 inhibitors and combinational therapy strategies should aim to increase therapeutic efficacy of the inhibitors, thus reducing side effects.
Acknowledgments
The work in the Her laboratory is supported by the NIH grant GM084353.
Author Contributions
Yang Xu and Chengtao Her wrote and revised the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest with the contents of this article.
Purpose: F14512 is a new topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells and increases topoisomerase II poisoning. F14512 is currently in Phase I/II clinical trial in patients with acute myeloid leukemia. The aim of this study was to investigate F14512 potential in a new clinical indication. Because of the many similarities between human and dog lymphomas, we sought to determine the tolerance, efficacy, PK/PD relationship of F14512 in this indication, and potential biomarkers that could be translated into human trials. Experimental design: Twenty-three dogs with stage III-IV naturally occurring lymphomas were enrolled in the Phase 1 dose-escalation trial which consisted of three cycles of F14512 intravenous injections. Endpoints included safety and therapeutic efficacy. Serial blood samples and tumor biopsies were obtained for PK/PD and biomarker studies. Results: Five dose levels were evaluated in order to determine the recommended dose. F14512 was well tolerated, with the expected dose-dependent hematological toxicity. F14512 induced an early decrease of tumoral lymph node cells, and a high response rate of 91% (21/23) with 10 complete responses, 11 partial responses, 1 stable disease and 1 progressive disease. Phosphorylation of histone H2AX was studied as a potential pharmacodynamic biomarker of F14512. Conclusions: This trial demonstrated that F14512 can be safely administered to dogs with lymphoma resulting in strong therapeutic efficacy. Additional evaluation of F14512 is needed to compare its efficacy with standards of care in dogs, and to translate biomarker and efficacy findings into clinical trials in humans.
Background: The J0509 (phase III study for chemotherapy-naive ED-SCLC) demonstrated amrubicin plus cisplatin (AP) was inferior to irinotecan plus cisplatin (IP). However, median overall survival (OS) of both AP and IP (15 and 17 mo) was more favorable than those of previous trials (9-12 mo), probably because switching to different topo-I or topo-II in the second-line therapy, especially the use of topo-II in IP arm, was frequent. This analysis aimed to investigate whether observed survival benefit of IP arm can be explained by the treatment switching, and how post-protocol chemotherapy affected the result of J0509. Methods: Two analysis sets from J0509 were used: all randomized 283 pts and 250 pts who received post-protocol chemotherapy. One pt without initiation date of second-line therapy was excluded. A rank-preserving structural failure time (RPSFT) model was used to estimate “causal survival benefit” that would have been observed if all pts had been followed with the same type of regimen as randomized throughout the follow-up period. Additionally, to assess the survival impact of second-line use of topo-II, OS after initiating second-line therapy (OS2) was analyzed by multivariate Cox models. Results: %treatment switching in IP arm and AP arm was 65.2% (92/141) and 43.7% (62/142). By RPSFT model, estimated OS excluding the effect of the treatment switching was 2.7-fold longer in IP (topo-I) arm than AP (topo-II) arm. This causal survival benefit was stronger than the original report of J0509 (nearly 1.4-fold extension by Cox model), indicating that re-challenging topo-I in IP arm appeared beneficial. The multivariate Cox analysis for OS2 (n = 250) revealed second-line use of topo-II was detrimental (hazard ratio, 1.5; 95%CI, 1.1-2.1). Among sensitive relapsed pts in IP arm, OS2 was favorable in the following order: irinotecan-based regimen > the other topo-I > topo-II. Conclusions: IP remains the standard therapy. Re-challenging topo-I, especially irinotecan-based topo-I, seemed beneficial for IP-sensitive pts. This result should be confirmed in further investigations with large sample size. Clinical trial information: 000000720.
Below is actively recruiting clinical trials evaluating topoisomerase inhibitors. Shown are only a few trials for a complete list from CancerTrials.gov please see this link:
Cyclin-dependent kinases (CDKs), in complex with their cyclin partners, modulate the transition through phases of the cell division cycle. Cyclin D–CDK complexes are important in cancer progression, especially for certain types of breast cancer. Fassl et al. discuss advances in understanding the biology of cyclin D–CDK complexes that have led to new concepts about how drugs that target these complexes induce cancer cell cytostasis and suggest possible combinations to widen the types of cancer that can be treated. They also discuss progress in overcoming resistance to cyclin D–CDK inhibitors and their possible application to diseases beyond cancer. —GKA
Structured Abstract
BACKGROUND
Cyclins and cyclin-dependent kinases (CDKs) drive cell division. Of particular importance to the cancer field are D-cyclins, which activate CDK4 and CDK6. In normal cells, the activity of cyclin D–CDK4/6 is controlled by the extracellular pro-proliferative or inhibitory signals. By contrast, in many cancers, cyclin D–CDK4/6 kinases are hyperactivated and become independent of mitogenic stimulation, thereby driving uncontrolled tumor cell proliferation. Mouse genetic experiments established that cyclin D–CDK4/6 kinases are essential for growth of many tumor types, and they represent potential therapeutic targets. Genetic and cell culture studies documented the dependence of breast cancer cells on CDK4/6. Chemical CDK4/6 inhibitors were synthesized and tested in preclinical studies. Introduction of these compounds to the clinic represented a breakthrough in breast cancer treatment and will likely have a major impact on the treatment of many other tumor types.
ADVANCES
Small-molecule CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) showed impressive results in clinical trials for patients with hormone receptor–positive breast cancers. Addition of CDK4/6 inhibitors to standard endocrine therapy substantially extended median progression-free survival and prolonged median overall survival. Consequently, all three CDK4/6 inhibitors have been approved for treatment of women with advanced or metastatic hormone receptor–positive breast cancers. In the past few years, the renewed interest in CDK4/6 biology has yielded several surprising discoveries. The emerging concept is that CDK4/6 kinases regulate a much wider set of cellular functions than anticipated. Consequently, CDK4/6 inhibitors, beyond inhibiting tumor cell proliferation, affect tumor cells and the tumor environment through mechanisms that are only beginning to be elucidated. For example, inhibition of CDK4/6 affects antitumor immunity acting both on tumor cells and on the host immune system. CDK4/6 inhibitors were shown to enhance the efficacy of immune checkpoint blockade in preclinical mouse cancer models. These new concepts are now being tested in clinical trials.
OUTLOOK
Palbociclib, ribociclib, and abemaciclib are being tested in more than 300 clinical trials for more than 50 tumor types. These trials evaluate CDK4/6 inhibitors in combination with a wide range of therapeutic compounds that target other cancer-relevant pathways. Several other combination treatments were shown to be efficacious in preclinical studies and will enter clinical trials soon. Another CDK4/6 inhibitor, trilaciclib, is being tested for its ability to shield normal cells of the host from cytotoxic effects of chemotherapy. New CDK4/6 inhibitors have been developed and are being assessed in preclinical and clinical trials. The major impediment in the therapeutic use of CDK4/6 inhibitors is that patients who initially respond to treatment often develop resistance and eventually succumb to the disease. Moreover, a substantial fraction of tumors show preexisting, intrinsic resistance to CDK4/6 inhibitors. One of the main challenges will be to elucidate the full range of resistance mechanisms. Even with the current, limited knowledge, one can envisage the principles of new, improved approaches to overcome known resistance mechanisms. Another largely unexplored area for future study is the possible involvement of CDK4/6 in other pathologic states beyond cancer. This will be the subject of intense studies, and it may extend the utility of CDK4/6 inhibitors to the treatment of other diseases.
Targeting cyclin D–CDK4/6 for cancer treatment.
D-cyclins (CycD) activate CDK4 and CDK6 in G1 phase of the cell cycle and promote cell cycle progression by phosphorylating the retinoblastoma protein RB1. RB1 inhibits E2F transcription factors; phosphorylation of RB1 activates E2F-driven transcription. In many cancers, CycD-CDK4/6 is constitutively activated and drives uncontrolled cell proliferation. The development of small-molecule CDK4/6 inhibitors provided a therapeutic tool to repress constitutive CycD-CDK4/6 activity and to inhibit cancer cell proliferation. As with several targeted therapies, tumors eventually develop resistance and resume cell proliferation despite CDK4/6 inhibition. New combination treatments, involving CDK4/6 inhibitors plus inhibition of other pathways, are being tested in the clinic to delay or overcome the resistance.
Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and their activating partners, D-type cyclins, link the extracellular environment with the core cell cycle machinery. Constitutive activation of cyclin D–CDK4/6 represents the driving force of tumorigenesis in several cancer types. Small-molecule inhibitors of CDK4/6 have been used with great success in the treatment of hormone receptor–positive breast cancers and are in clinical trials for many other tumor types. Unexpectedly, recent work indicates that inhibition of CDK4/6 affects a wide range of cellular functions such as tumor cell metabolism and antitumor immunity. We discuss how recent advances in understanding CDK4/6 biology are opening new avenues for the future use of cyclin D–CDK4/6 inhibitors in cancer treatment.
Cyclin D1, the activator of CDK4 and CDK6, was discovered in the early 1990s (1, 2). The role of cyclin D1 in oncogenesis was already evident at the time of its cloning, as it was also identified as the protein product of the PRAD1 oncogene, which is rearranged and overexpressed in parathyroid adenomas (3), and of the BCL1 oncogene, which is rearranged in B-lymphocytic malignancies (4). Subsequently, the remaining two D-type cyclins, D2 and D3, were discovered on the basis of their homology to cyclin D1 (1).
Cyclins serve as regulatory subunits of cyclin-dependent kinases (CDKs) (5). Shortly after the discovery of D-cyclins, CDK4 and CDK6 were identified as their kinase partners (6). Mouse gene knockout studies revealed that CDK4 and CDK6 play redundant roles in development, and combined ablation of CDK4 and CDK6 was found to result in embryonic lethality (7). The essentially identical phenotype was seen in cyclin D–knockout mice, thereby confirming the role of D-cyclins as CDK4/6 activators in vivo (8). Surprisingly, these analyses revealed that many normal nontransformed mammalian cell types can proliferate without any cyclin D–CDK4/6 activity (7, 8).
CDK4 and CDK6 are expressed at constant levels throughout the cell cycle. By contrast, D-cyclins are labile proteins that are transcriptionally induced upon stimulation of cells with growth factors. For this reason, D-cyclins are regarded as links between the cellular environment and the cell cycle machinery (6).
Cell cycle inhibitors play an important role in regulating the activity of cyclin D–CDK4/6 (Fig. 1). The INK inhibitors (p16INK4A, p15INK4B, p18INK4C, p19INK4D) bind to CDK4 or CDK6 and prevent their interaction with D-type cyclins, thereby inhibiting cyclin D–CDK4/6 kinase activity. By contrast, KIP/CIP inhibitors (p27KIP1, p57KIP2, p21CIP1), which inhibit the activity of CDK2-containing complexes, serve as assembly factors for cyclin D–CDK4/6 (6, 9). This was demonstrated by the observation that mouse fibroblasts devoid of p27KIP1 and p21CIP1 fail to assemble cyclin D–CDK4/6 complexes (10).
Fig. 1. Molecular events governing progression through the G1 phase of the cell cycle.
The mammalian cell cycle can be divided into G1, S (DNA synthesis), G2, and M (mitosis) phases. During G1 phase, cyclin D (CycD)–CDK4/6 kinases together with cyclin E (CycE)–CDK2 phosphorylate the retinoblastoma protein RB1. This activates the E2F transcriptional program and allows entry of cells into S phase. Members of the INK family of inhibitors (p16INK4A, p15INK4B, p18INK4C, and p19INK4D) inhibit cyclin D–CDK4/6; KIP/CIP proteins (p21CIP1, p27KIP1, and p57KIP2) inhibit cyclin E–CDK2. Cyclin D–CDK4/6 complexes use p27KIP1 and p21CIP1 as “assembly factors” and sequester them away from cyclin E–CDK2, thereby activating CDK2. Proteins that are frequently lost or down-regulated in cancers are marked with green arrows, overexpressed proteins with red arrows.
p27KIP1 can bind cyclin D–CDK4/6 in an inhibitory or noninhibitory mode, depending on p27KIP1 phosphorylation status. Cyclin D–p27KIP1-CDK4/6 complexes are catalytically inactive unless p27KIP1 is phosphorylated on Tyr88 and Tyr89 (11). Two molecular mechanisms may explain this switch. First, Tyr88/Tyr89 phosphorylation may dislodge the helix of p27KIP1 from the CDK active site and allow adenosine triphosphate (ATP) binding (12). Second, the presence of tyrosine-unphosphorylated p27KIP1 within the cyclin D–CDK4 complex prevents the activating phosphorylation of CDK4’s T-loop by the CDK-activating kinase (CAK) (12). Brk has been identified as a physiological kinase of p27KIP1 (13); Abl and Lyn can phosphorylate p27KIP1 in vitro, but their in vivo importance remains unclear (11, 14).
The activity of cyclin D–CDK4/6 is also regulated by proteolysis. Cyclin D1 is an unstable protein with a half-life of less than 30 min. At the end of G1 phase, cyclin D1 is phosphorylated at Thr286 by GSK3β (15). This facilitates association of cyclin D1 with the nuclear exportin CRM1 and promotes export of cyclin D1 from the nucleus to the cytoplasm (16). Subsequently, phosphorylated cyclin D1 becomes polyubiquitinated by E3 ubiquitin ligases, thereby targeting it for proteasomal degradation. Several substrate receptors of E3 ubiquitin ligases have been implicated in recognizing phosphorylated cyclin D1, including F-box proteins FBXO4 (along with αB crystallin), FBXO31, FBXW8, β-TrCP1/2, and SKP2 (17). The anaphase-promoting complex/cyclosome (APC/C) was also proposed to target cyclin D1 while F-box proteins FBXL2 and FBXL8 target cyclins D2 and D3 (17, 18). Surprisingly, the level and stability of cyclin D1 was unaffected by depletion of several of these proteins, indicating that some other E3 plays a rate-limiting role in cyclin D1 degradation (19). Indeed, recent studies reported that D-cyclins are ubiquitinated and targeted for proteasomal degradation by the E3 ubiquitin ligase CRL4, which uses AMBRA1 protein as its substrate receptor (20–22).
Cyclin D–CDK4/6 in cancer
Genomic aberrations of the cyclin D1 gene (CCND1) represent frequent events in different tumor types. The t(11;14)(q13;q32) translocation juxtaposing CCND1 with the immunoglobulin heavy-chain (IGH) locus represents the characteristic feature of mantle-cell lymphoma and is frequently observed in multiple myeloma or plasma cell leukemia (23, 24). Amplification of CCND1 is seen in many other malignancies—for example, in 13 to 20% of breast cancers (23, 24), more than 40% of head and neck squamous cell carcinomas, and more than 30% of esophageal squamous cell carcinomas (23). A higher proportion of cancers (e.g., up to 50% of mammary carcinomas) overexpress cyclin D1 protein (24). Also, cyclins D2 and D3, CDK4, and CDK6 are overexpressed in various tumor types (5, 9). Cyclin D–CDK4/6 can also be hyperactivated through other mechanisms such as deletion or inactivation of INK inhibitors, most frequently p16INK4A (5, 9, 23). Altogether, a very large number of human tumors contain lesions that hyperactivate cyclin D–CDK4/6 (5).
An oncogenic role for cyclin D–CDK4/6 has been supported by mouse cancer models. For example, targeted overexpression of cyclin D1 in mammary glands of transgenic mice led to the development of mammary carcinomas (25). Also, overexpression of cyclin D2, D3, or CDK4, or loss of p16INK4a resulted in tumor formation (9).
Conversely, genetic ablation of D-cyclins, CDK4, or CDK6 decreased tumor sensitivity (9). For instance, Ccnd1– or Cdk4-null mice, or knock-in mice expressing kinase-inactive cyclin D1–CDK4/6, were resistant to develop human epidermal growth factor receptor 2 (HER2)–driven mammary carcinomas (26–29). An acute, global shutdown of cyclin D1 in mice bearing HER2-driven tumors arrested tumor growth and triggered tumor-specific senescence while having no obvious impact on normal tissues (30). Likewise, an acute ablation of CDK4 arrested tumor cell proliferation and triggered tumor cell senescence in a KRAS-driven non–small-cell lung cancer (NSCLC) mouse model (31). These observations indicated that CDK4 and CDK6 might represent excellent therapeutic targets in cancer treatment.
CDK4/6 functions in cell proliferation and oncogenesis
The best-documented function of cyclin D–CDK4/6 in driving cell proliferation is phosphorylation of the retinoblastoma protein, RB1, and RB-like proteins, RBL1 and RBL2 (5, 6) (Fig. 1). Unphosphorylated RB1 binds and inactivates or represses E2F transcription factors. According to the prevailing model, phosphorylation of RB1 by cyclin D–CDK4/6 partially inactivates RB1, leading to release of E2Fs and up-regulation of E2F-transcriptional targets, including cyclin E. Cyclin E forms a complex with its kinase partner, CDK2, and completes full RB1 phosphorylation, leading to activation of the E2F transcriptional program and facilitating S-phase entry (5, 6). In normal, nontransformed cells, the activity of cyclin D–CDK4/6 is tightly regulated by the extracellular mitogenic milieu. This links inactivation of RB1 with mitogenic signals. In cancer cells carrying activating lesions in cyclin D–CDK4/6, the kinase is constitutively active, thereby decoupling cell division from proliferative and inhibitory signals (5).
This model has been questioned by the demonstration that RB1 exists in a monophosphorylated state throughout G1 phase and becomes inactivated in late G1 by cyclin E–CDK2, which “hyperphosphorylates” RB1 on multiple residues (32). However, recent single-cell analyses revealed that cyclin D–CDK4/6 activity is required for the hyperphosphorylation of RB1 throughout G1, whereas cyclin E/A–CDK maintains RB1 hyperphosphorylation in S phase (33). Moreover, phosphorylation of RB1 by cyclin D–CDK4/6 was shown to be required for normal cell cycle progression (34).
In addition to this kinase-dependent mechanism, up-regulation of D-cyclin expression and formation of cyclin D–CDK4/6 complexes lead to redistribution of KIP/CIP inhibitors from cyclin E–CDK2 complexes (which are inhibited by these proteins) to cyclin D–CDK4/6 (which use them as assembly factors), thereby activating the kinase activity of cyclin E–CDK2 (6). Cyclin E–CDK2 in turn phosphorylates RB1 and other cellular proteins and promotes cell cycle progression.
Cyclin D1–CDK4/6 directly phosphorylates, stabilizes, and activates the transcription factor FOXM1. This promotes cell cycle progression and protects cancer cells from entering senescence (35). Cyclin D–CDK4 also phosphorylates and inactivates SMAD3, which mediates transforming growth factor–β (TGF-β) antiproliferative response. CDK4/6-dependent phosphorylation of SMAD3 inhibits its transcriptional activity and disables the ability of TGF-β to induce cell cycle arrest (36). FZR1/CDH1, an adaptor protein of the APC complex, is another phosphorylation substrate of CDK4. Depletion of CDH1 in human cancer cells partially rescued the proliferative block upon CDK4/6 inhibition, and it cooperated with RB1 depletion in restoring full proliferation (37).
Cyclin D–CDK4/6 also phosphorylates and inactivates TSC2, a negative regulator of mTORC1, thereby resulting in mTORC1 activation. Conversely, inhibition of CDK4/6 led to decreased mTORC1 activity and reduced protein synthesis in cells representing different human tumor types. It was proposed that through TSC2 phosphorylation, activation of cyclin D–CDK4/6 couples cell growth with cell division (38). Consistent with this, the antiproliferative effect of CDK4/6 inhibition was reduced in cells lacking TSC2 (38).
MEP50, a co-regulatory factor of protein arginine-methyltransferase 5 (PRMT5), is phosphorylated by cyclin D1–CDK4. Through this mechanism, cyclin D1–CDK4/6 increases the catalytic activity of PRMT5/MEP50 (39). It was proposed that deregulation of cyclin D1–CDK4 kinase in tumor cells, by increasing PRMT5/MEP50 activity, reduces the expression of CUL4, a component of the E3 ubiquitin-ligase complex, and stabilizes CUL4 targets such as CDT1 (39). In addition, by stimulating PRMT5/MEP50-dependent arginine methylation of p53, cyclin D–CDK4/6 suppresses the expression of key antiproliferative and pro-apoptotic p53 target genes (40). Another study proposed that PRMT5 regulates splicing of the transcript encoding MDM4, a negative regulator of p53. CDK4/6 inhibition reduced PRMT5 activity and altered the pre-mRNA splicing of MDM4, leading to decreased levels of MDM4 protein and resulting in p53 activation. This, in turn, up-regulated the expression of a p53 target, p21CIP1, that blocks cell cycle progression (41).
During oncogenic transformation of hematopoietic cells, chromatin-bound CDK6 phosphorylates the transcription factors NFY and SP1 and induces the expression of p53 antagonists such as PRMT5, PPM1D, and MDM4 (42). Also, in acute myeloid leukemia cells expressing constitutively activated FLT3, CDK6 binds the promoter region of the FLT3 gene as well as the promoter of PIM1 pro-oncogenic kinase and stimulates their expression. Treatment of FLT3-mutant leukemic cells with a CDK4/6 inhibitor decreased FLT3 and PIM1 expression and triggered cell cycle arrest and apoptosis (43). The relevance of these various mechanisms in the context of human tumors is unclear and requires further study.
Mechanism of action of CDK4/6 inhibitors
Three small-molecule CDK4/6 inhibitors have been extensively characterized in preclinical studies: palbociclib and ribociclib, which are highly specific CDK4/6 inhibitors, and abemaciclib, which inhibits CDK4/6 and other kinases (Table 1). It has been assumed that these compounds act in vivo by directly inhibiting cyclin D–CDK4/6 (9). This simple model has been recently questioned by observations that palbociclib inhibits only cyclin D–CDK4/6 dimers, but not trimeric cyclin D–CDK4/6-p27KIP1 (44). However, it is unlikely that substantial amounts of cyclin D–CDK4 dimers ever exist in cells, because nearly all cyclin D–CDK4 in vivo is thought to be complexed with KIP/CIP proteins (11, 14, 44). Palbociclib also binds monomeric CDK4 (44). Surprisingly, treatment of cancer cells with palbociclib for 48 hours failed to inhibit CDK4 kinase, despite cell cycle arrest, but it inhibited CDK2 (44). Hence, palbociclib might prevent the formation of active CDK4-containing complexes (through binding to CDK4) and indirectly inhibit CDK2 by liberating KIP/CIP inhibitors. This model needs to be reconciled with several observations. First, treatment of cells with CDK4/6 inhibitors results in a rapid decrease of RB1 phosphorylation on cyclin D–CDK4/6-dependent sites, indicating an acute inhibition of CDK4/6 (45–47). Moreover, CDK4/6 immunoprecipitated from cells can be inhibited by palbociclib (48) and p21CIP-associated cyclin CDK4/6 kinase is also inhibited by treatment of cells with palbociclib (49). Lastly, CDK2 is dispensable for proliferation of several cancer cell lines (50, 51), hence the indirect inhibition of CDK2 alone is unlikely to be responsible for cell cycle arrest.
Name of compound
IC50
Other known targets
Stage of clinical development
Palbociclib (PD-0332991)
D1-CDK4, 11 nM;
D2-CDK6, 15 nM;
D3-CDK4, 9 nM
FDA-approved for HR+/HER2– advanced
breast cancer in combination with
endocrine therapy; phase 2/3 trials
for several other tumor types
Ribociclib (LEE011)
D1-CDK4, 10 nM;
D3-CDK6, 39 nM
FDA-approved for HR+/HER2– advanced
breast cancer in combination with
endocrine therapy; phase 2/3 trials
for several other tumor types
Abemaciclib (LY2835219)
D1-CDK4, 0.6 to 2 nM;
D3-CDK6, 8 nM
Cyclin T1–CDK9, PIM1, HIPK2, CDKL5,
CAMK2A, CAMK2D, CAMK2G,
GSK3α/β, and (at higher doses)
cyclin E/A–CDK2 and cyclin B–CDK1
FDA-approved for early (adjuvant) and
advanced HR+/HER2– breast cancer in
combination with endocrine therapy;
FDA-approved as monotherapy in advanced
HR+/HER2– breast cancer; phase 2/3 trials
for several other tumor types
Trilaciclib (G1T28)
D1-CDK4, 1 nM;
D3-CDK6, 4 nM
FDA-approved for small-cell lung cancer
to reduce chemotherapy-induced bone
marrow suppression; phase 2/3 trials
for other solid tumors
Lerociclib (G1T38)
D1-CDK4, 1 nM;
D3-CDK6, 2 nM
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and EGFR-mutant
non–small-cell lung cancer
SHR6390
CDK4, 12 nM;
CDK6, 10 nM
Phase 1/2/3 trials for HR+/HER2– advanced
breast cancer and other solid tumors
PF-06873600
CDK4, 0.13 nM (Ki),
CDK6, 0.16 nM (Ki)
CDK2, 0.09 nM (Ki)
Phase 2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
FCN-437
D1-CDK4, 3.3 nM;
D3-CDK6, 13.7 nM
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
Birociclib (XZP-3287)
Not reported
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
HS-10342
Not reported
Phase 1/2 trials for HR+/HER2– advanced
breast cancer and other solid tumors
This table lists major inhibitors of CDK4 and CDK6, half-maximal inhibitory concentration (IC50) for different cyclin D–CDK4/6 complexes (if known), other known targets, and the stage of clinical development. Ki, inhibitory constant.
Palbociclib, ribociclib, and abemaciclib were shown to block binding of CDK4 and CDK6 to CDC37, the kinase-targeting subunit of HSP90, thereby preventing access of CDK4/6 to the HSP90-chaperone system (52). Because the HSP90-CDC37 complex stabilizes several kinases (53), these observations suggest that CDK4/6 inhibitors, by disrupting the interaction between CDC37 and CDK4 or CDK6, might promote degradation of CDK4 and CDK6. However, depletion of CDK4/6 is typically not observed upon treatment with CDK4/6 inhibitors (54). More studies are needed to resolve these conflicting reports and to establish how CDK4/6 inhibitors affect the cell cycle machinery in cancer cells.
Validation of CDK4/6 inhibitors as anticancer agents
Consistent with the notion that RB1 represents the major rate-limiting substrate of cyclin D–CDK4/6 in cell cycle progression (55–57), palbociclib, ribociclib, and abemaciclib were shown to block proliferation of several RB1-positive cancer cell lines, but not cell lines that have lost RB1 expression (46, 58, 59). Breast cancer cell lines representing the luminal, estrogen receptor–positive (ER+) subtype were shown to be most susceptible to cell proliferation arrest upon palbociclib treatment (45). Palbociclib, ribociclib, abemaciclib, and another CDK4/6 inhibitor, lerociclib, were demonstrated to display potent antitumor activity in xenografts of several tumor types, including breast cancers (46, 60–62). Palbociclib and abemaciclib cross the blood-brain barrier and inhibit growth of intracranial glioblastoma (GBM) xenografts, with abemaciclib being more efficient in reaching the brain (63, 64). Recently, additional CDK4/6 inhibitors were shown to exert therapeutic effects in mouse xenograft models of various cancer types, including SHR6390 (65), FCN-437 (66), and compound 11 (67); the latter two were reported to cross the blood-brain barrier. In most in vivo studies, the therapeutic effect was dependent on expression of intact RB1 protein in tumor cells (46, 63). However, antitumor effects of palbociclib were also reported in bladder cancer xenografts independently of RB1 status; this was attributed to decreased phosphorylation of FOXM1 (68).
Tumor cell senescence upon CDK4/6 inhibition
In addition to blocking cell proliferation, inhibition of CDK4/6 can also trigger tumor cell senescence (63), which depends on RB1 and FOXM1 (35, 54). The role of RB1 in enforcing cellular senescence is well established (69). In addition, cyclin D–CDK4/6 phosphorylates and activates FOXM1, which has anti-senescence activity (35, 70). Senescence represents a preferred therapeutic outcome to cell cycle arrest, as it may lead to a durable inhibition of tumor growth.
It is not clear what determines the extent of senescence upon treatment of cancer cells with CDK4/6 inhibitors. A recent study showed that inhibition of CDK4/6 leads to an RB1-dependent increase in reactive oxygen species (ROS) levels, resulting in activation of autophagy, which mitigates the senescence of breast cancer cells in vitro and in vivo (71). Co-treatment with palbociclib plus autophagy inhibitors strongly augmented the ability of CDK4/6 inhibitors to induce tumor cell senescence and led to sustained inhibition of cancer cell proliferation in vitro and of xenograft growth in vivo (71). Decreased mTOR signaling after long-term CDK4/6 inhibition was shown to be essential for the induction of senescence in melanoma cells, and activation of mTORC1 overrode palbociclib-induced senescence (72). Others postulated that expression of the chromatin-remodeling enzyme ATRX and degradation of MDM2 determines the choice between quiescence and senescence upon CDK4/6 inhibition (73). Inhibition of CDK4 causes dissociation of the deubiquitinase HAUSP/USP7 from MDM2, thereby driving autoubiquitination and proteolytic degradation of MDM2, which in turn promotes senescence. This mechanism requires ATRX, which suggests that expression of ATRX can be used to predict the senescence response (73). Two additional proteins that play a role in this process are PDLIM7 and type II cadherin CDH18. Expression of CDH18 correlated with a sustained response to palbociclib in a phase 2 trial for patients with liposarcoma (74).
Markers predicting response to CDK4/6 inhibition
Only tumors with intact RB1 respond to CDK4/6 inhibitor treatment by undergoing cell cycle arrest or senescence (9, 58). In addition, “D-cyclin activating features” (CCND1 translocation, CCND2 or CCND3 amplification, loss of the CCND1-3 3′-untranslated region, and deletion of FBXO31 encoding an F-box protein implicated in cyclin D1 degradation) were shown to confer a strong response to abemaciclib in cancer cell lines (58). Moreover, co-deletion of CDKN2A and CDKN2C (encoding p16INK4A/p19ARF and p18INK4C, respectively) confers palbociclib sensitivity in glioblastoma (75). Thr172 phosphorylation of CDK4 and Tyr88 phosphorylation of p27KIP1 (both associated with active cyclin D–CDK4) correlate with sensitivity of breast cancer cell lines or tumor explants to palbociclib (76, 77). Surprisingly, in PALOMA-1, PALOMA-2, and PALOMA-3 trials (78–80), and in another independent large-scale study (81), CCND1 gene amplification or elevated levels of cyclin D1 mRNA or protein were not predictive of palbociclib efficacy. Conversely, overexpression of CDK4, CDK6, or cyclin E1 is associated with resistance of tumors to CDK4/6 inhibitors (see below).
Synergy of CDK4/6 inhibitors with other compounds
Several preclinical studies have documented the additive or synergistic effects of combining CDK4/6 inhibitors with inhibitors of the receptor tyrosine kinases as well as phosphoinositide 3-kinase (PI3K), RAF, or MEK (Table 2). This synergism might be because these pathways impinge on the cell cycle machinery through cyclin D–CDK4/6 (82–86). In some cases, the effect was seen in the presence of specific genetic lesions, such as EGFR, BRAFV600E, KRAS, and PIK3CA mutations (59, 87–89) (Table 2). When comparing different dosing regimens, continuous treatment with a MEK inhibitor with intermittent palbociclib resulted in more complete tumor responses than other combination schedules (90). Treatment with CDK4/6 inhibitors sensitized cancer cells to ionizing radiation (63) or cisplatin (68). The synergism with platinum-based chemotherapy was attributed to the observation that upon this treatment, CDK6 phosphorylates and stabilizes the FOXO3 transcription factor, thereby promoting tumor cell survival. Consequently, inhibition of CDK6 increases platinum sensitivity by enhancing tumor cell death (91).
In several instances, co-treatment with CDK4/6 inhibitors prevented the development of resistance to other compounds or inhibited the proliferation of resistant tumor cells. Co-treatment of melanoma patient-derived xenografts (PDXs) with ribociclib plus the RAF inhibitor encorafenib delayed or prevented development of encorafenib resistance (92). PDXs that acquired encorafenib resistance remained sensitive to the combination of encorafenib plus ribociclib (59). Treatment of BRAFV600E-mutant melanoma xenografts with palbociclib plus the BRAFV600E inhibitor PLX4720 prevented development of resistance (89). BRAFV600E-mutant melanoma cell lines that acquired resistance to the BRAFV600E inhibitor vemurafenib remained sensitive to palbociclib or abemaciclib, and xenografts underwent senescence and tumor regression upon CDK4/6 inhibition (72, 93). Treatment of ALK-mutant, ALK kinase inhibitor–resistant neuroblastoma xenografts with palbociclib restored the sensitivity to these compounds (94). A combination of PI3K and CDK4/6 inhibitors overcame the intrinsic and acquired resistance of breast cancers to PI3K inhibitors and resulted in regression of PIK3CA-mutant xenografts (88).
Up-regulation of cyclin D1 expression was shown to mediate acquired resistance of HER2+ tumors to anti-HER2 therapies in a mouse breast cancer model (95). Treatment of mice bearing trastuzumab-resistant tumors or PDXs of resistant HER2+ mammary carcinomas with abemaciclib restored the sensitivity of tumors to HER2 inhibitors and inhibited tumor cell proliferation. Moreover, in the case of treatment-naïve tumors, co-administration of abemaciclib significantly delayed the development of resistance to anti-HER2 therapies (95).
Several anticancer treatments, such as chemotherapy, target dividing cells. Because CDK4/6 inhibitors block tumor cell proliferation, they might impede the effects of chemotherapy. Indeed, several reports have documented that co-administration of CDK4/6 inhibitors antagonized the antitumor effects of compounds that act during S phase (doxorubicin, gemcitabine, methotrexate, mercaptopurine) or mitosis (taxanes) (96, 97). However, some authors reported synergistic effects (98, 99), although the molecular underpinnings are unclear.
A recent report documented that administration of CDK4/6 inhibitors prior to taxanes inhibited tumor cell proliferation and impeded the effect of taxanes (100). By contrast, administration of taxanes first (or other chemotherapeutic compounds that act on mitotic cells or cells undergoing DNA synthesis), followed by CDK4/6 inhibitors, had a strong synergistic effect. The authors showed that by repressing the E2F-dependent transcriptional program, CDK4/6 inhibitors impaired the expression of genes required for DNA-damage repair via homologous recombination. Because treatment of cancer cells with chemotherapy triggers DNA damage, the impairment of DNA-damage repair induced cytotoxicity, thereby explaining the synergistic effect (100).
Cells with impaired homologous recombination rely on poly-(ADP-ribose) polymerase (PARP) for double-stranded DNA-damage repair, which renders them sensitive to PARP inhibition. Indeed, a strong synergistic effect has been demonstrated between CDK4/6 inhibitors and PARP inhibitors in PDX-derived cell lines (100). Such synergy was also reported for ovarian cancer cells (101). Another study found that inhibition of CDK4/6 resulted in down-regulation of PARP levels (102).
Protection against chemotherapy-induced toxicity
Administration of palbociclib to mice induced reversible quiescence in hematopoietic stem/progenitor cells (HSPCs). This effect protected mice from myelosuppression after total-body irradiation. Moreover, treatment of tumor-bearing mice with CDK4/6 inhibitors together with irradiation mitigated radiation-induced toxicity without compromising the therapeutic effect (103). Co-administration of a CDK4/6 inhibitor, trilaciclib, with cytotoxic chemotherapy (5-FU, etoposide) protected animals from chemotherapy-induced exhaustion of HSPCs, myelosuppression, and apoptosis of bone marrow (60, 104). These observations led to phase 2 clinical trial, which evaluated the effects of trilaciclib administered prior to etoposide and carboplatin for treatment of small-cell lung cancer. Trilaciclib improved myelopreservation while having no adverse effect on antitumor efficacy (105). A similar phase 2 clinical trial investigating trilaciclib in combination with gemcitabine and carboplatin chemotherapy in patients with metastatic triple-negative breast cancer (TNBC) did not observe a significant difference in myelosuppression. However, this study demonstrated an overall survival benefit of the combination therapy (106, 107).
Metabolic function of CDK4/6 in cancer cells
The role of CDK4/6 in tumor metabolism is only starting to be appreciated (Fig. 2A). Treatment of pancreatic cancer cells with CDK4/6 inhibitors was shown to induce tumor cell metabolic reprogramming (108). CDK4/6 inhibition increased the numbers of mitochondria and lysosomes, activated mTOR, and increased the rate of oxidative phosphorylation, likely through an RB1-dependent mechanism (108). Combined inhibition of CDK4/6 and mTOR strongly suppressed tumor cell proliferation (108). Moreover, CDK4/6 can phosphorylate and inactivate TFEB, the master regulator of lysosomogenesis, and through this mechanism reduce lysosomal numbers. Conversely, CDK4/6 inhibition activated TFEB and increased the number of lysosomes (109). Another mechanism linking CDK4/6 and lysosomes was provided by the observation that treatment of TNBC cells with CDK4/6 inhibitors decreased mTORC1 activity and impaired the recruitment of mTORC1 to lysosomes (110). Consistent with the idea that mTORC1 inhibits lysosomal biogenesis, CDK4/6 inhibition increased the number of lysosomes in tumor cells. Because an increased lysosomal biomass underlies some cases of CDK4/6 inhibitor resistance (see below) (111), stimulation of lysosomogenesis by CDK4/6 inhibitors might limit their clinical efficacy by inducing resistance.
Fig. 2. CDK4 and CDK6: More than cell cycle kinases.
Although the role of CDK4 and CDK6 in cell cycle progression has been well documented, both kinases regulate several other functions that are only now starting to be unraveled. (A) Inhibition of CDK4/6 (CDK4/6i) affects lysosome and mitochondrial numbers as well as oxidative phosphorylation. Cyclin D3–CDK6 phosphorylates glycolytic enzymes 6-phosphofructokinase (PFKP) and pyruvate kinase M2 (PKM2), thereby controlling ROS levels via the pentose phosphate (PPP) and serine synthesis pathways. (B) Inhibition of CDK4/6 affects antitumor immunity, acting both within cancer cells and on the immune system of the host. In tumor cells, inhibition of CDK4/6 impedes expression of an E2F target, DNA methyltransferase (DNMT). DNMT inhibition reduces methylation of endogenous retroviral genes (ERV) and increases intracellular levels of double-stranded RNA (dsRNA) (114). In effector T cells, inhibition of CDK4/6 stimulates NFAT transcriptional activity and enhances secretion of IFN-γ and interleukin 2 (IL-2) (115).
Lastly, CDK4/6 inhibition impaired lysosomal function and the autophagic flux in cancer cells. It was argued that this lysosomal dysfunction was responsible for the senescent phenotype in CDK4/6 inhibitor–treated cells (110). Because lysosomes are essential for autophagy, the authors co-treated TNBC xenografts with abemaciclib plus an AMPK activator, A769662 (which induces autophagy), and found that this led to cancer cell death and subsequent regression of tumors (110).
Cyclin D3–CDK6 phosphorylates and inhibits two rate-limiting glycolytic enzymes, 6-phosphofructokinase and pyruvate kinase M2. This redirects glycolytic intermediates into the pentose phosphate pathway (PPP) and serine synthesis pathway. Through this mechanism, cyclin D3–CDK6 promotes the production of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) and helps to neutralize ROS (112). Treatment of tumors expressing high levels of cyclin D3–CDK6 (such as leukemias) with CDK4/6 inhibitors reduced the PPP- and serine-synthesis pathway flow, thereby depleting the antioxidants NADPH and GSH. This increased ROS levels and triggered tumor cell apoptosis (112).
Another link between cyclin D–CDK4/6 in metabolism and cancer was provided by the observation that livers of obese/diabetic mice up-regulate cyclin D1 expression (113). Treatment of these mice with an antidiabetic compound, metformin, reduced liver cyclin D1 levels and largely protected mice against development of hepatocellular carcinoma. Also, genetic ablation of cyclin D1 protected obese/diabetic mice from liver cancer, and administration of palbociclib inhibited liver cancer progression. These treatments had no effect on tumors in nonobese animals (113). These observations raise the possibility of using antidiabetic compounds with CDK4/6 inhibitors for treatment of liver cancers in obese patients.
CDK4/6 inhibitors and antitumor immune responses
Several recent reports have started to unravel how inhibition of CDK4/6 influences antitumor immune responses, acting both on tumor cells as well as on the tumor immune environment (Fig. 2B). Treatment of breast cancer–bearing mice or breast cancer cells with abemaciclib activated expression of endogenous retroviral elements in tumor cells, thereby increasing the levels of double-stranded RNA. This, in turn, stimulated production of type III interferons and increased presentation of tumor antigens. Hence, CDK4/6 inhibitors, by inducing viral gene expression, trigger antiviral immune responses that help to eliminate the tumor (114).
Inhibition of CDK4/6 also affects the immune system by impeding the proliferation of CD4+FOXP3+ regulatory T cells (Tregs), which normally inhibit the antitumor response. Because cytotoxic CD8+ T cells are less affected by CDK4/6 inhibition, abemaciclib treatment decreases the Treg/CD8+ ratio of intratumoral T cells and facilitates tumor cell killing by cytotoxic CD8+ T cells (114).
Inhibition of CDK4/6 also resulted in activation of T cells through derepression of NFAT signaling. NFAT4 (and possibly other NFATs) are phosphorylated by cyclin D3–CDK6 (115). Inhibition of CDK4/6 decreased phosphorylation of NFATs, resulting in their nuclear translocation and enhanced transcriptional activity. This caused up-regulation of NFAT targets, resulting in T cell activation, which enhanced the antitumor immune response. In addition, CDK4/6 inhibitors increased the infiltration of effector T cells into tumors, likely because of elevated levels of chemokines CXCL9 and CXCL10 after CDK4/6 inhibitor treatment (115). Abemaciclib treatment also induced inflammatory and activated T cell phenotypes in tumors and up-regulated the expression of immune checkpoint proteins CD137, PD-L1, and TIM-3 on CD4+ and CD8+ cells (116).
CDK4/6 inhibition also caused up-regulation of PD-L1 protein expression in tumor cells (117, 118). This effect was shown to be independent of RB1 status in the tumor. Mechanistically, CDK4/6 phosphorylates and stabilizes SPOP, which promotes PD-L1 polyubiquitination and degradation (118). Cyclin D–CDK4 also represses expression of PD-L1 through RB1. Specifically, cyclin D–CDK4/6-mediated phosphorylation of RB1 on S249/T252 promotes binding of RB1 to NF-κB protein p65, and this represses the expression of a subset NF-κB–regulated genes, including PD-L1 (119).
These observations prompted tests of the efficacy of combining CDK4/6 inhibitors with antibodies that elicit immune checkpoint blockade. Indeed, treatment of mice bearing autochthonous breast cancers, or cancer allografts, with CDK4/6 inhibitors together with anti-PD-1/PD-L1 antibodies enhanced the efficacy of immune checkpoint blockade and led to complete tumor regression in a high proportion of animals (114, 115, 118). Conversely, activation of the cyclin D–CDK4 pathway by genomic lesions in human melanomas correlated with resistance to anti–PD-1 therapy (117).
Some authors did not observe synergy when abemaciclib was administered concurrently with immune checkpoint inhibitors in allograft tumor models (116, 120). However, a strong synergistic antitumor effect was detected when abemaciclib was administered first (and continued) and anti–PD-L1 antibody was administered later. The combined treatment induced immunological memory, as mice that underwent tumor regression were resistant to rechallenge with the same tumor (116). Abemaciclib plus anti–PD-L1 treatment increased infiltration of CD4+ and CD8+ T cells into tumors, and increased the expression of major histocompatibility complex class I (MHC-I) and MHC-II on tumor cells and on macrophages and MHC-I on dendritic cells (116). In the case of anti–CTLA-4 plus anti–PD-1 treatment in melanoma allograft model, the synergistic effect was observed when immune checkpoint inhibitor treatment was started first, followed by abemaciclib (120).
The synergistic antitumor effect of PI3K and CDK4/6 inhibitors in TNBC is mediated, in part, by enhancement of tumor immunogenicity (121). Combined treatment of TNBC cells with ribociclib plus the PI3K inhibitor apelisib synergistically up-regulated the expression of immune-related pathways in tumor cells, including proteins involved in antigen presentation. Co-treatment of tumor-bearing mice also decreased proliferation of CD4+FOXP3+ Treg cells, increased activation of intratumoral CD4+ and CD8+ T cells, increased the frequency of tumor-infiltrating NKT cells, and decreased the numbers of intratumoral immunosuppressive myeloid-derived suppressor cells. Moreover, combined treatment strongly augmented the response to immune checkpoint therapy with PD-1 and CTLA-4 antibodies (121).
Single-cell RNA sequencing of human melanomas identified an immune resistance program expressed by tumor cells that correlates with T cell exclusion from the tumor mass and immune evasion by tumor cells. The program can predict the response of tumors to immune checkpoint inhibitors. Treatment of human melanoma cells with abemaciclib repressed this program in an RB1-dependent fashion (120).
Together, these findings indicate that CDK4/6 inhibitors may convert immunologically “cold” tumors into “hot” ones. The most pressing issue is to validate these findings in a clinical setting. The utility of combining CDK4/6 inhibitors with PD-1 or PD-L1 antibodies is currently being evaluated in several clinical trials. Note that the effects of CDK4/6 inhibition on the immune system of the host are independent of tumor cell RB1 status, raising the possibility of using CDK4/6 inhibitors to also boost the immune response against RB1-negative tumors.
CDK4/6 inhibitors in clinical trials
Table 3 summarizes major clinical trials with CDK4/6 inhibitors. Given early preclinical data indicating that breast cancers—in particular, the hormone receptor–positive ones—are very sensitive to CDK4/6 inhibition (as discussed above), many clinical trials have focused on this cancer type. Most studies have evaluated CDK4/6 inhibitors administered together with anti-estrogens (the aromatase inhibitors letrozole or anastrozole, or the estrogen receptor antagonist fulvestrant) for treatment of advanced/metastatic HR+/HER2– breast cancers in postmenopausal women. Addition of CDK4/6 inhibitors significantly extended median progression-free survival (78, 122–130) and prolonged median overall survival (131–134). Moreover, abemaciclib has shown clinical activity when administered as a single agent (135). Consequently, palbociclib, ribociclib, and abemaciclib have been approved by the US Food and Drug Administration (FDA) for treatment of patients with advanced/metastatic HR+/HER2– breast cancer (Box 1). A recent phase 3 clinical trial, MonarchE, evaluated abemaciclib plus standard endocrine therapy in treatment of patients with early-stage, high-risk, lymph node–positive HR+/HER2– breast cancer. Addition of abemaciclib reduced the risk of breast cancer recurrence (136). This is in contrast to the similar PALLAS study reported this year, which found no benefit of adding palbociclib to endocrine therapy for women with early-stage breast cancer (137). Analysis of patient populations in these two trials may help to explain the different outcomes. It is also possible that the favorable outcome of the MonarchE study reflects a broader spectrum of kinases inhibited by abemaciclib. The utility of CDK4/6 inhibitors in early-stage breast cancer remains unclear and is being addressed in ongoing clinical trials (PALLAS, PENELOPE-B, EarLEE-1, MonarchE) (138).
CDK4/6 inhibitor
Trial name
Trial details
Treatment
Patients
Outcome
Ref.
Other outcomes
Palbociclib
PALOMA-1
Randomized
phase 2
Aromatase inhibitor
letrozole alone
(standard of care)
versus letrozole
plus palbociclib
Postmenopausal women
with advanced ER+/HER2–
breast cancer who had
not received any systemic
treatment for their
advanced disease
Addition of palbociclib markedly
increased median PFS from
10.2 months in the
letrozole group to
20.2 months in the
palbociclib plus
letrozole group
On the basis of this result, palbociclib
received a “Breakthrough Therapy”
designation status from FDA and was
granted accelerated approval, in
combination with letrozole, for the
treatment of ER+/HER2– metastatic
breast cancer
Palbociclib
PALOMA-2
Double-blind
phase 3
Palbociclib plus
letrozole as first-
line therapy
Postmenopausal women
with ER+/HER2–
breast cancer
Addition of palbociclib strongly
increased median PFS:
14.5 months in the placebo-
letrozole group versus
24.8 months in the
palbociclib-letrozole group
Palbociclib was equally efficacious in
patients with luminal A and B breast
cancers, and there was no single
biomarker associated with the lack of
clinical benefit, except for RB1 loss;
CDK4 amplification was associated
with endocrine resistance, but this
was mitigated by addition of
palbociclib; tumors with high levels
of FGFR2 and ERBB3 mRNA
displayed greater PFS gain
after addition of palbociclib (79)
Palbociclib
PALOMA-3
Randomized
phase 3
Estrogen receptor
antagonist
fulvestrant plus
placebo versus
fulvestrant plus
palbociclib
Women with HR+/HER2–
metastatic breast cancer
that had progressed on
previous endocrine therapy
The study demonstrated a
substantial prolongation
of median PFS in the palbociclib-
treated group: 4.6 months in the
placebo plus fulvestrant group
versus 9.5 months in the
palbociclib plus fulvestrant
group; addition of palbociclib
also extended median overall
survival from 28.0 months
(placebo-fulvestrant) to
34.9 months (palbociclib-
fulvestrant); estimated rate
of survival at 3 years was
41% versus 50%, respectively
Palbociclib in
patients with
early breast
cancer at high
risk of recurrence
Ongoing
Ribociclib
MONA
LEESA-2
Randomized
phase 3
Ribociclib plus
letrozole versus
placebo plus
letrozole
First-line treatment for
postmenopausal women
with HR+/HER2– recurrent
or metastatic breast
cancer who had not
received previous
systemic therapy for
advanced disease
At 18 months, PFS
was 42.2% in the
placebo-letrozole
group and 63.0%
in the ribociclib-
letrozole group
Patients with advanced
(metastatic or recurrent)
HR+/HER2– breast cancer
who have either received no
treatment for the advanced
disease or previously
received a single line of
endocrine therapy for the
advanced disease
Addition of ribociclib significantly
extended median PFS, from
12.8 months (placebo-fulvestrant)
to 20.5 months (ribociclib-
fulvestrant); overall survival at
42 months was also extended
from 45.9% (placebo-fulvestrant)
to 57.8% (ribociclib-fulvestrant)
Ribociclib versus
placebo together
with an anti-
estrogen tamoxifen
or an aromatase
inhibitor (letrozole
or anastrozole)
Premenopausal and
perimenopausal women
with HR+/HER2– advanced
breast cancer who had not
received previous treatment
with CDK4/6 inhibitors
Ribociclib significantly increased
median PFS from 13.0 months in
the placebo-endocrine therapy
group to 23.8 months in the
ribociclib-endocrine therapy
group; overall survival was also
strongly prolonged in the ribociclib
group (estimated overall survival
at 42 months was 46.0% for the
placebo group and 70.2% in the
ribociclib group)
Ribociclib in the
treatment of early-
stage, high-risk
HR+/HER2–
breast cancers
Ongoing
Abemaciclib
MONARCH 1
Phase 2 trial
Abemaciclib as a
single agent
Women with HR+/HER2–
metastatic breast cancer
who had progressed on or
after prior endocrine therapy
and had 1 or 2 chemotherapy
regimens in the metastatic
setting
Abemaciclib exhibited promising activity
in these heavily pretreated patients
with poor prognosis; median
PFS was 6.0 months and overall
survival 17.7 months
The most common adverse events
were diarrhea, fatigue, and
nausea (136)
Abemaciclib
MONARCH 2
Double-blind
phase 3
Abemaciclib in
combination
with fulvestrant
Women with HR+/HER2– breast
cancer who had progressed
while receiving endocrine
therapy, or while receiving
first-line endocrine therapy for
metastatic disease
Addition of abemaciclib significantly
increased PFS from 9.3 months in
the placebo-fulvestrant to 16.4 in
the abemaciclib-fulvestrant group;
median overall survival was also
extended from 37.3 months
to 46.7 months
Abemaciclib plus
an aromatase
inhibitor
(anastrozole
or letrozole)
Postmenopausal women
with advanced HR+/HER2–
breast cancer who had
no prior systemic therapy
in the advanced setting
Addition of abemaciclib prolonged
PFS from 14.8 months (in
the placebo-aromatase
inhibitor group) to 28.2 months
(abemaciclib-aromatase
inhibitor group)
Patients with HR+/HER2–
lymph node–positive,
high-risk early
breast cancer
Preliminary analysis indicates that
addition of abemaciclib resulted
in a significant improvement of
invasive disease-free survival
and of distant relapse-
free survival
Chemotherapy alone
(gemcitabine and
carboplatin),
versus concurrent
administration of
trilaciclib plus
chemotherapy,
versus
administration of
trilaciclib prior to
chemotherapy
(to mitigate the
cytotoxic effect of
chemotherapy on
bone marrow)
Patients with recurrent or
metastatic triple-negative
breast cancer who had no
more than two previous
lines of chemotherapy
Addition of trilaciclib did not offer
detectable myeloprotection, but
resulted in increased overall
survival (from 12.8 months in the
chemotherapy-only group to
20.1 months in the concurrent
trilaciclib and chemotherapy
group and 17.8 months in trilaciclib
before chemotherapy group)
Approved by FDA in 2016, in combination with fulvestrant for the treatment of hormone receptor–positive, HER2-negative (HR+/HER2–) advanced or metastatic breast cancer in women with disease progression following endocrine therapy. Approved in 2017 for the treatment of HR+/HER2– advanced or metastatic breast cancer in combination with an aromatase inhibitor as initial endocrine-based therapy in postmenopausal women.
Palbociclib is administered at a dose of 125 mg (given orally) daily for 3 weeks followed by 1 week off, or 200 mg daily for 2 weeks followed by 1 week off. The rate-limiting toxicities are neutropenia, thrombocytopenia, and anemia.
Ribociclib
Approved by FDA in 2017, in combination with an aromatase inhibitor as initial endocrine-based therapy for the treatment of postmenopausal women with HR+/HER2– advanced or metastatic breast cancer. In 2018, the FDA expanded the indication for ribociclib in combination with an aromatase inhibitor for pre/perimenopausal women with HR+/HER2– advanced or metastatic breast cancer, as initial endocrine-based therapy. FDA also approved ribociclib in combination with fulvestrant for postmenopausal women with HR+/HER2– advanced or metastatic breast cancer, as initial endocrine-based therapy or following disease progression on endocrine therapy.
Ribociclib is administered at a dose of 600 mg (given orally) daily for 3 weeks followed by 1 week off. The main toxicities are neutropenia and thrombocytopenia.
Abemaciclib
Approved by FDA in 2017, in combination with fulvestrant for women with HR+/HER2– advanced or metastatic breast cancer with disease progression following endocrine therapy. In addition, abemaciclib was approved as monotherapy for women and men with HR+/HER2– advanced or metastatic breast cancer with disease progression following endocrine therapy and prior chemotherapy in the metastatic setting. Approved by FDA in 2018 in combination with an aromatase inhibitor as initial endocrine-based therapy for postmenopausal women with HR+/HER2– advanced or metastatic breast cancer. Approved by FDA in 2021 for adjuvant treatment of early-stage HR+/HER2– breast cancer in combination with endocrine therapy.
Abemaciclib is administered at a dose of 200 mg (given orally) every 12 hours. The dose-limiting toxicity is fatigue. Neutropenia is also observed but is not rate-limiting. Other severe side effects include diarrhea and nausea.
Currently, palbociclib is being used in 164 active or recruiting clinical trials, ribociclib in 69 trials, and abemaciclib in 98 trials for more than 50 tumor types (139). These trials evaluate combinations of CDK4/6 inhibitors with a wide range of compounds (Table 4). Trials with trilaciclib test the benefit of this compound in preserving bone marrow and the immune system.
Additional target
Inhibitor
Immune checkpoint inhibitor
Tumor type
Trial identifier
Palbociclib
Aromatase
Letrozole, anastrozole,
exemestane
HR+ breast cancer, HR+ ovarian
cancer, metastatic breast cancer,
metastatic endometrial cancer
tumors with ERK1/2
mutations, glioblastoma,
metastatic cancer
NCT04534283,
NCT04391595,
NCT02857270
Trilaciclib
Proliferating cells
Chemotherapy
SCLC: This trial evaluates the
potential clinical benefit of
trilaciclib in preventing
chemotherapy-induced
myelosuppression in patients
receiving chemotherapy
NCT04504513
Proliferating cells +
PD-L1
Carboplatin + etoposide
Atezolizumab
SCLC: This trial investigates the
potential clinical benefit of trilaciclib
in preserving the bone marrow and
the immune system, and enhancing
antitumor efficacy when
administered with chemotherapy
NCT03041311
Proliferating cells
Topotecan
SCLC: This trial investigates the
potential clinical benefit of
trilaciclib in preserving the
bone marrow and the immune
system, and enhancing the
antitumor efficacy of chemotherapy
when administered prior
to chemotherapy
NCT02514447
Proliferating cells
Carboplatin + gemcitabine
Metastatic TNBC: This study
investigates the potential
clinical benefit of trilaciclib in
preserving the bone marrow
and the immune system, and
enhancing the antitumor efficacy
of chemotherapy when administered
prior to chemotherapy
NCT02978716
Lerociclib
ER
ER antagonist: fulvestrant
HR+/HER2– metastatic
breast cancer
NCT02983071
EGFR
Osimertinib
EGFR mutant NSCLC
NCT03455829
SHR6390
ER
ER antagonist: fulvestrant
HR+/HER2– recurrent/
metastatic breast cancer
NCT03481998
Aromatase
Letrozole, anastrozole
HR+/HER2– recurrent/
metastatic breast cancer
NCT03966898,
NCT03772353
EGFR/HER2
Pyrotinib
HER2+ gastric cancer, HER2+
metastatic breast cancer
NCT04095390,
NCT03993964
AR
AR antagonists: SHR3680
metastatic TNBC
NCT03805399
PF-06873600
Endocrine therapy
Single agent and then
in combination with
endocrine therapy
HR+/HER2– metastatic breast
cancer, ovarian and fallopian tube
cancer, TNBC and other tumors
Although CDK4/6 inhibitors represent very effective agents in cancer treatment, nearly all patients eventually develop resistance and succumb to the disease. Moreover, a substantial fraction of tumors show intrinsic resistance to treatment with CDK4/6 inhibitors (Fig. 3).
Fig. 3. Mechanisms of cancer cell resistance to CDK4/6 inhibition.
Known mechanisms include loss of RB1, activation of pathways impinging on CycD-CDK4/6, amplification of the CDK4/6 genes and overexpression of CDK6 protein, activation of CycE-CDK2, and lysosomal sequestration of CDK4/6 inhibitors. Blank pieces of the puzzle denote additional mechanisms that remain to be discovered.
The best-documented mechanism of preexisting and acquired resistance is the loss of RB1 (71, 81, 140). Acquired RB1 loss has been detected in PDXs (141), in circulating tumor DNA (ctDNA) (142, 143), and in tumors from patients treated with CDK4/6 inhibitors (144, 145). However, RB1 mutations are likely subclonal and are seen in only 5 to 10% of patients (143, 145).
Increased expression of CDK6 was shown to underlie acquired resistance to CDK4/6 inhibitors. Amplification of the CDK6 gene and the resulting overexpression of CDK6 protein were found in abemaciclib-resistant ER+ breast cancer cells (146) and in ctDNA of patients with ER+ breast cancers that progressed during treatment with palbociclib plus endocrine therapy (147). Also, CDK4 gene amplification conferred insensitivity to CDK4/6 inhibition in GBM and sarcomas (148–150), whereas overexpression of CDK4 protein was associated with resistance to endocrine therapy in HR+ breast cancers (79).
Resistant breast cancer cells can also up-regulate the expression of CDK6 through suppression of the TGF-β/SMAD4 pathway by the microRNA miR-432-5p. In this mechanism, exosomal expression of miR-432-5p mediates the transfer of the resistance phenotype between neighboring cell populations (151). Another mechanism of CDK6 up-regulation in ER+ breast cancers is the loss of FAT1, which represses CDK6 expression via the Hippo pathway. Loss of FAT1 triggers up-regulation of CDK6 expression by the Hippo pathway effectors TAZ and YAP. Moreover, genomic alterations in other components of the Hippo pathway, although rare, are also associated with reduced sensitivity to CDK4/6 inhibitors (81).
Genetic lesions that activate pathways converging on D-type cyclins can cause resistance to CDK4/6 inhibitors. These include (i) FGFR1/2 gene amplification or mutational activation, detected in ctDNA from patients with ER+ breast cancers that progressed upon treatment with palbociclib plus endocrine therapy (147); (ii) hyperactivation of the MAPK pathway in resistant prostate adenocarcinoma cells, possibly due to increased production of EGF by cancer cells (152); and (iii) increased secretion of FGF in palbociclib-resistant KRAS-mutant NSCLC cells, which stimulates FGFR1 signaling in an autocrine or paracrine fashion, resulting in activation of ERK1/2 and mTOR as well as up-regulation of D-cyclin, CDK6, and cyclin E expression (153). Analyses of longitudinal tumor biopsies from a melanoma patient revealed an activating mutation in the PIK3CA gene that conferred resistance to ribociclib plus MEK inhibitor treatment (154). It is possible that these lesions elevate the cellular levels of active cyclin D–CDK4/6 complexes, thereby increasing the threshold for CDK4/6 inhibition.
Formation of a noncanonical cyclin D1–CDK2 complex was shown to represent another mechanism of acquired CDK4/6 inhibitor resistance. Such a complex was observed in palbociclib-treated ER+ breast cancer cells and was implicated in overcoming palbociclib-induced cell cycle arrest (141). Also, depletion of AMBRA1 promoted the interaction of D-cyclins with CDK2, resulting in resistance to CDK4/6 inhibitors (20, 22); it remains to be seen whether this represents an intrinsic or acquired resistance mechanism in human tumors.
Genetic analyses revealed that activation of cyclin E can bypass the requirement for cyclin D–CDK4/6 in development and tumorigenesis (155, 156). Hence, it comes as no surprise that increased activity of cyclin E–CDK2 is responsible for a large proportion of intrinsic and acquired resistance to CDK4/6 inhibitors. Several different mechanisms can activate cyclin E–CDK2 kinase in resistant tumor cells: (i) Down-regulation of KIP/CIP inhibitors results in increased activity of cyclin E–CDK (54, 157). (ii) Loss of PTEN expression, which activates AKT signaling, leads to nuclear exclusion of p27KIP1. This in turn prevents access of p27KIP1 to CDK2, resulting in increased CDK2 kinase activity (144). (iii) Activation of the PI3K/AKT pathway causes decreased levels of p21CIP1. Co-treatment of melanoma PDXs with MDM2 inhibitors (which up-regulate p21CIP1 via p53) sensitized intrinsically resistant tumor cells to CDK4/6 inhibitors (158). (iv) Up-regulation of cyclin D1 levels triggers sequestration of KIP/CIP inhibitors from cyclin E–CDK2 to cyclin D–CDK4/6, thereby activating the former (158). (v) Amplification of the CCNE1 gene and increased levels of cyclin E1 protein result in elevated activity of E-CDK2 kinase (141). (vi) mTOR signaling has been shown to up-regulate cyclin E1 (and D1) in KRAS-mutated pancreatic cancer cells; CDK2 activity was essential for CDK4/6 inhibitor resistance in this setting (159). (vii) Up-regulation of PDK1 results in activation of the AKT pathway, which increases the expression of cyclins E and A and activates CDK2 (160). (viii) In CDK4/6 inhibitor–resistant melanoma cells, high levels of RNA-binding protein FXR1 increase translation of the amino acid transporter SLC36A1. Up-regulation of SLC36A1 expression activates mTORC1, which in turn increases CDK2 expression (161). All these lesions are expected to allow cell proliferation, despite CDK4/6 inhibition, as a consequence of the activation of the downstream cell cycle kinase CDK2.
The role for cyclin E–CDK2 in CDK4/6 inhibitor resistance has been confirmed in clinical trials. In patients with advanced ER+ breast cancer treated with palbociclib and letrozole or fulvestrant, the presence of proteolytically cleaved cytoplasmic cyclin E in tumor tissue conferred strongly shortened progression-free survival (71). Moreover, analyses of PALOMA-3 trial for patients with ER+ breast cancers revealed lower efficacy of palbociclib plus fulvestrant in patients displaying high cyclin E mRNA levels in metastatic biopsies (80). Amplification of the CCNE1 gene was detected in ctDNA of patients with ER+ breast cancers that progressed on palbociclib plus endocrine therapy (147). Also, amplification of the CCNE2 gene (encoding cyclin E2) was seen in a fraction of CDK4/6 inhibitor–resistant HR+ mammary carcinomas (145, 162).
Collectively, these analyses indicate that resistant cells may become dependent on CDK2 for cell cycle progression. Indeed, depletion of CDK2 or inhibition of CDK2 kinase activity in combination with CDK4/6 inhibitors blocked proliferation of CDK4/6 inhibitor–resistant cancer cells (111, 141, 158–161). Recently, two CDK2-specific inhibitors, PF-07104091 (163) and BLU0298 (164), have been reported. PF-07104091 is now being tested in a phase 2 clinical trial in combination with palbociclib plus antiestrogens. Another recent study identified a novel compound, PF-3600, that inhibits CDK4/6 and CDK2 (165). PF3600 had potent antitumor effects against xenograft models of intrinsic and acquired resistance to CDK4/6 inhibition (165). A phase 2 clinical trial is currently evaluating this compound as a single agent and in combination with endocrine therapy in patients with HR+/HER2– breast cancer and other cancer types.
Whole-exome sequencing of 59 HR+/HER2– metastatic breast tumors from patients treated with CDK4/6 inhibitors and anti-estrogens revealed eight alterations that likely conferred resistance: RB1 loss; amplification of CCNE2 or AURKA; activating mutations or amplification of AKT1, FGFR2, or ERBB2; activating mutations in RAS genes; and loss of ER expression. The frequent activation of AURKA (in 27% of resistant tumors) raises the possibility of combining CDK4/6 inhibitors with inhibitors of Aurora A kinase to overcome resistance (145).
In contrast to ER+ mammary carcinomas, TNBCs are overall resistant to CDK4/6 inhibition (45). A subset of TNBCs display high numbers of lysosomes, which causes sequestration of CDK4/6 inhibitors into the expanded lysosomal compartment, thereby preventing their action on nuclear CDK4/6. Preclinical studies revealed that lysosomotropic agents that reverse the lysosomal sequestration (such as chloroquine, azithromycin, or siramesine) render TNBC cells fully sensitive to CDK4/6 inhibition (71, 111). These observations now need to be tested in clinical trials for TNBC patients.
Outlook
Although D-cyclins and CDK4/6 were discovered 30 years ago, several aspects of cyclin D–CDK4/6 biology, such as their role in antitumor immunity, are only now starting to be appreciated. The full range of cyclin D–CDK4/6 functions in tumor cells remains unknown. It is likely that these kinases play a much broader role in cancer cells than is currently appreciated. Hence, the impact of CDK4/6 inhibition on various aspects of tumorigenesis requires further study. Also, treatment of patients with CDK4/6 inhibitors likely affects several aspects of host physiology, which may be relevant to cancer progression.
In the next years, we will undoubtedly witness the development and testing of new CDK4/6 inhibitors. Because activation of CDK2 represents a frequent CDK4/6 inhibitor resistance mechanism, compounds that inhibit CDK4/6 and CDK2 may prevent or delay the development of resistance. Conversely, selective compounds that inhibit CDK4 but not CDK6 may allow more aggressive dosing, as they are expected not to result in bone marrow toxicity caused by CDK6 inhibition. New, less basic CDK4/6 inhibitor compounds (111) may escape lysosomal sequestration and may be efficacious against resistant cancer types such as TNBC. Degrader compounds, which induce proteolysis of cyclin D rather than inhibit cyclin D–CDK4/6 kinase, may have superior properties, as they would extinguish both CDK4/6-dependent and -independent functions of D-cyclins in tumorigenesis. Moreover, dissolution of cyclin D–CDK4/6 complexes is expected to liberate KIP/CIP inhibitors, which would then inhibit CDK2. D-cyclins likely play CDK-independent functions in tumorigenesis—for example, by regulating gene expression (166). However, their role in tumor biology and the utility of targeting these functions for cancer treatment remain largely unexplored.
An important challenge will be to test and identify combinatorial treatments involving CDK4/6 inhibitors for the treatment of different tumor types. CDK4/6 inhibitors trigger cell cycle arrest of tumor cells and, in some cases, senescence. It will be essential to identify combination treatments that convert CDK4/6 inhibitors from cytostatic compounds to cytotoxic ones, which would unleash the killing of tumor cells. Genome-wide high-throughput screens along with analyses of mouse cancer models and PDXs will help to address this point. Another largely unexplored area of cyclin D–CDK4/6 biology is the possible involvement of these proteins in other pathologies, such as metabolic disorders. Research in this area may extend the use of CDK4/6 inhibitors to treatment of other diseases. All these unresolved questions ensure that CDK4/6 biology will remain an active area of basic, translational, and clinical research for several years to come.
CDK inhibitors and Breast Cancer
The U.S. Food and Drug Administration today granted accelerated approval to Ibrance (palbociclib) to treat advanced (metastatic) breast cancer inr postmenopausal women with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer who have not yet received an endocrine-based therapy. It is to be used in combination with letrozole, another FDA-approved product used to treat certain kinds of breast cancer in postmenopausal women.
See Dr. Melvin Crasto’s blog posts on the announcement of approval of Ibrance (palbociclib) at
Palbociclib and LY2835219 are both cyclin-dependent kinase (CDK) 4/6 inhibitors. CDK4 and CDK6 are kinases that, together with cyclin D1, facilitate the transition of dividing cells from the G1 to the S (synthesis) phase of the cell cycle. Preclinical studies have shown that breast cancer cells rely on CDK4 and CDK6 for division and growth, and that selective CDK4/6 inhibitors can arrest the cells at this G1/S phase checkpoint.
The results of the phase II trial of palbociclib and phase I trial of LY2835219 both indicated that hormone receptor (HR)-positive disease appears to be the best marker to predict patient response.
LY2835219 Phase I Trial Demonstrates Early Activity
The CDK4/6 inhibitor LY2835219 has demonstrated early activity in heavily pretreated women with metastatic breast cancer. Nineteen percent of these women (9 out of 47) had a partial response and 51% (24 out of 47) had stable disease following monotherapy with the oral CDK4/6 inhibitor. Patients had received a median of seven prior therapies, and 75% had metastatic disease in the lung, liver, or brain. The median age of patients was 55 years.
All of the partial responses were in patients with HR-positive disease. The overall response rate for this patient subset was 25% (9 of 36 patients). Twenty of the patients with stable disease had HR-positive disease, with 13 patients having stable disease lasting 24 weeks or more.
Despite treatment, disease progression occurred in 23% of the patients.
These results were presented at a press briefing by Amita Patnaik, MD, associate director of clinical research at South Texas Accelerated Research Therapeutics in San Antonio, Texas, at the 2014 American Association for Cancer Research (AACR) Annual Meeting, held April 5–9, in San Diego.
The phase I trial of LY2835219 enrolled 132 patients with five different tumor types, including metastatic breast cancer. Patients received 150-mg to 200-mg doses of the oral drug every 12 hours.
The overall disease control rate was 70% for all patients and 81% among the 36 HR-positive patients.
The median progression-free survival (PFS) was 5.8 months for all patients and 9.1 months for HR-positive patients. Patnaik noted that the median PFS is still a moving target, as 18 patients, all with HR-positive disease, remain on therapy.
“The data are rather encouraging for a very heavily pretreated patient population,” said Patnaik during the press briefing.
Even though the trial was not designed to compare efficacy based on breast cancer subpopulations, the results in HR-positive tumors are particularly encouraging, according to Patnaik.
Common adverse events thought to be treatment-related were diarrhea, nausea, fatigue, vomiting, and neutropenia. These adverse events occurred in 5% or less of patients at grade 3 or 4 toxicity, except neutropenia, which occurred as a grade 3 or 4 toxicity in 11% of patients. Patnaik noted during the press briefing that the neutropenia was uncomplicated and did not result in discontinuation of therapy by any of the patients.
Palbociclib Phase II Data “Impressive”
The addition of the oral CDK4/6 inhibitor palbociclib resulted in an almost doubling of PFS in first-line treatment of postmenopausal metastatic breast cancer patients with HR-positive disease compared with a control population. The patients in this trial were not previously treated for their metastatic breast cancer, unlike the patient population in the phase I LY2835219 trial.
Patients receiving the combination of palbociclib at 125 mg once daily plus letrozole at 2.5 mg once daily had a median PFS of 20.2 months compared with a median of 10.2 months for patients treated with letrozole alone (hazard ratio = 0.488; P = .0004).
Richard S. Finn, MD, assistant professor of medicine at the University of California, Los Angeles, presented the data from the phase II PALOMA-1 trial at a press briefing at the AACR Annual Meeting.
A total of 165 patients were randomized 1:1 to either the experimental arm or control arm.
Forty-three percent of patients in the combination arm had an objective response compared with 33% of patients in the control arm.
Overall survival (OS), a secondary endpoint in this trial, was encouraging but the results are still preliminary, said Finn during the press briefing. The median OS was 37.5 months in the palbociclib arm compared with 33.3 months in the letrozole alone arm (P = .21). Finn noted that long-term follow-up is necessary to establish the median OS. “This first look of the survival data is encouraging. This is a front-line study, and it is encouraging that there is early [separation] of the curves,” he said.
No new toxicities were reported since the interim trial results. Common adverse events included leukopenia, neutropenia, and fatigue. The neutropenia could be quickly resolved and was uncomplicated and not accompanied by fever, said Finn.
Palbociclib is currently being tested in two phase III clinical trials: The PALOMA-3 trial is testing the combination of palbociclib with letrozole and fulvestrant in late-stage metastatic breast cancer patients who have failed endocrine therapy. The PENELOPE-B trial is testing palbociclib in combination with standard endocrine therapy in HR-positive breast cancer patients with residual disease after neoadjuvant chemotherapy and surgery.
References
Patnaik A, Rosen LS, Tolaney SM, et al. Clinical activity of LY2835219, a novel cell cycle inhibitor selective for CDK4 and CDK6, in patients with metastatic breast cancer. American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT232.
Finn RS, Crown JP, Lang I, et al. Final results of a randomized phase II study of PD 0332991, a cyclin-dependent kinase (CDK)-4/6 inhibitor, in combination with letrozole vs letrozole alone for first-line treatment of ER+/HER2-advanced breast cancer (PALOMA-1; TRIO-18). American Association for Cancer Research Annual Meeting 2014; April 5–9, 2014; San Diego. Abstr CT101.
This article has been cited by other articles in PMC.
Cyclin-dependent kinases (CDKs) drive cell cycle progression and control transcriptional processes. The dysregulation of multiple CDK family members occurs commonly in human cancer; in particular, the cyclin D-CDK4/6-retinoblastoma protein (RB)-INK4 axis is universally disrupted, facilitating cancer cell proliferation and prompting long-standing interest in targeting CDK4/6 as an anticancer strategy. Most agents that have been tested inhibit multiple cell cycle and transcriptional CDKs and have carried toxicity. However, several selective and potent inhibitors of CDK4/6 have recently entered clinical trial. PD0332991, the first to be developed, resulted from the introduction of a 2-aminopyridyl substituent at the C2-position of a pyrido(2,3-d)pyrimidin-7-one backbone, affording exquisite selectivity toward CDK4/6.1 PD0332991 arrests cells in G1 phase by blocking RB phosphorylation at CDK4/6-specfic sites and does not inhibit the growth of RB-deficient cells.2 Phase I studies conducted in patients with advanced RB-expressing cancers demonstrated mild side effects and dose-limiting toxicities of neutropenia and thrombocytopenia, with prolonged stable disease in 25% of patients.3,4 In cyclin D1-translocated mantle cell lymphoma, PD0332991 extinguished CDK4/6 activity in patients’ tumors, resulting in markedly reduced proliferation, and translating to more than 1 year of stability or response in 5 of 17 cases.5
Two recent papers from the Knudsen laboratory make several important observations that will help guide the continued clinical development of CDK4/6 inhibitors. In the study by Dean et al., surgically resected patient breast tumors were grown on a tissue culture matrix in the presence or absence of PD0332991. Crucially, these cultures retained associated stromal components known to play important roles in cancer pathogenesis and therapeutic sensitivities, as well as key histological and molecular features of the primary tumor, including expression of ER, HER2 and Ki-67. Similar to results in breast cancer cell lines,6 the authors demonstrate that only RB-positive tumors have growth inhibition in response to PD0332991, irrespective of ER or HER2 status, while tumors lacking RB were completely resistant. This result underscores RB as the predominant target of CDK4/6 in breast cancer cells and the primary marker of drug response in primary patient-derived tumors. As expected, RB-negative tumors routinely demonstrated robust expression of p16INK4A; however, p16INK4A expression was not always a surrogate marker for RB loss, supporting the importance of direct screening of tumors for RB expression to select patients appropriate for CDK4/6 inhibitor clinical trials.
In the second study, McClendon et al. investigated the efficacy of PD0332991 in combination with doxorubicin in triple-negative breast cancer cell lines. Again, RB functionality was paramount in determining response to either PD0332991 monotherapy or combination treatment. In RB-deficient cancer cells, CDK4/6 inhibition had no effect in either instance. However, in RB-expressing cancer cells, CDK4/6 inhibition and doxorubicin provided a cooperative cytostatic effect, although doxorubicin-induced cytotoxicity was substantially reduced, assessed by markers for mitotic catastrophe and apoptosis. Additionally, despite cytostatic cooperativity, CDK4/6 inhibition maintained the viability of RB-proficient cells in the presence of doxorubicin, which repopulated the culture after removal of drug. These results reflect previous data demonstrating that ectopic expression of p16INK4A can protect cells from the lethal effects of DNA damaging and anti-mitotic chemotherapies.7 Similar results have been reported in MMTV-c-neu mice bearing RB-proficient HER2-driven tumors, where PD0332991 compromised carboplatin-induced regressions,8 suggesting that DNA-damaging treatments should not be combined concomitantly with CDK4/6 inhibition in RB-proficient tumors.
To combine CDK4/6 inhibition with cytotoxics, sequential treatment may be considered, in which CDK4/6 inhibition is followed by DNA damaging chemotherapy; cells relieved of G1 arrest may synchronously enter S phase, where they may be most susceptible to agents disrupting DNA synthesis. Release of myeloma cells from a prolonged PD0332991-mediated G1 block leads to S phase synchronization; interestingly, all scheduled gene expression is not completely restored (including factors critical to myeloma survival such as IRF4), further favoring apoptotic responses to cytotoxic agents.9 Furthermore, in RB-deficient tumors, CDK4/6 inhibitors may be used to maximize the therapeutic window between transformed and non-transformed cells treated with chemotherapy. In contrast to RB-deficient cancer cells, RB-proficient non-transformed cells arrested in G1 in response to PD0332991 are afforded protection from DNA damaging agents, thereby reducing associated toxicities, including bone marrow suppression.8
In summary, the current work provides evidence for RB expression as a determinant of response to CDK4/6 inhibition in primary tumors and highlights the complexity of combining agents targeting the cell cycle machinery with DNA damaging treatments.
To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.
Keywords: ER, PD0332991, breast cancer, cell cycle, ex vivo
Breast cancer is a highly heterogeneous disease.1–4 Such heterogeneity is known to influence patient response to both standard of care and experimental therapeutics. In regards to biomarker-driven treatment of breast cancers, it was initially recognized that the presence of the estrogen receptor α (ER) in a fraction of breast cancer cells was associated with the response to tamoxifen and similar anti-estrogenic therapies.5,6 Since this discovery, subsequent marker analyses and gene expression profiling studies have further divided breast cancer into a series of distinct subtypes that harbor differing and often divergent therapeutic sensitivities.1–3 While clearly important in considering the use of several current standard of care therapies, these markers, or molecular sub-types, do not necessarily predict the response to new therapeutic approaches that are currently undergoing clinical development. Thus, there is the continued need for functional analyses of drug response and the definition of new markers that can be used to direct treatment strategies.
Currently, all preclinical cancer models are associated with specific limitations. It is well known that cell culture models lack the tumor microenvironment known to have a significant impact on tumor biology and therapeutic response.7–9 Xenograft models are dependent on the host response for the engraftment of tumor cells in non-native tissues, which do not necessarily recapitulate the nuances of complex tumor milieu.10 In addition, genetically engineered mouse models, while enabling the tumor to develop in the context of the host, can develop tumors that do not mirror aspects of human disease.10 Furthermore, it remains unclear whether any preclinical model truly represents the panoply of breast cancer subtypes that are observed in the clinic. Herein, we utilized a primary human tumor explant culture approach to interrogate drug response, as well as specific determinants of therapeutic response, in an unselected series of breast cancer cases.
Triple-negative breast cancer (TNBC) is an aggressive disease that lacks established markers to direct therapeutic intervention. Thus, these tumors are routinely treated with cytotoxic chemotherapies (e.g., anthracyclines), which can cause severe side effects that impact quality of life. Recent studies indicate that the retinoblastoma tumor suppressor (RB) pathway is an important determinant in TNBC disease progression and therapeutic outcome. Furthermore, new therapeutic agents have been developed that specifically target the RB pathway, potentially positioning RB as a novel molecular marker for directing treatment. The current study evaluates the efficacy of pharmacological CDK4/6 inhibition in combination with the widely used genotoxic agent doxorubicin in the treatment of TNBC. Results demonstrate that in RB-proficient TNBC models, pharmacological CDK4/6 inhibition yields a cooperative cytostatic effect with doxorubicin but ultimately protects RB-proficient cells from doxorubicin-mediated cytotoxicity. In contrast, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient TNBC cells to doxorubicin-mediated cytotoxicity, indicating that the effects of doxorubicin are indeed dependent on RB-mediated cell cycle control. Finally, the ability of CDK4/6 inhibition to protect TNBC cells from doxorubicin-mediated cytotoxicity resulted in recurrent populations of cells specifically in RB-proficient cell models, indicating that CDK4/6 inhibition can preserve cell viability in the presence of genotoxic agents. Combined, these studies suggest that while targeting the RB pathway represents a novel means of treatment in aggressive diseases such as TNBC, there should be a certain degree of caution when considering combination regimens of CDK4/6 inhibitors with genotoxic compounds that rely heavily on cell proliferation for their cytotoxic effects.
Click on Video Link for Dr. Tolaney slidepresentation of recent data with CDK4/6 inhibitor trial results https://youtu.be/NzJ_fvSxwGk
Sara Tolaney, MD, MPH, a breast oncologist with the Susan F. Smith Center for Women’s Cancers at Dana-Farber Cancer Institute, gives an overview of phase I clinical trials and some of the new drugs being tested to treat breast cancer. This talk was originally given at the Metastatic Breast Cancer Forum at Dana-Farber on Oct. 5, 2013.
A great article on current clinical trials and explanation of cdk inhibitors by Sneha Phadke, DO; Alexandra Thomas, MD at the site OncoLive
cdk4/6 inhibitor Ibrance Has Favorable Toxicity and Adverse Event Profile
As discussed in earlier posts and the Introduction to this chapter on Cytotoxic Chemotherapeutics, most anti-cancer drugs developed either to target DNA, DNA replication, or the cell cycle usually have similar toxicity profile which can limit their therapeutic use. These toxicities and adverse events usually involve cell types which normally exhibit turnover in the body, such as myeloid and lymphoid and granulocytic series of blood cells, epithelial cells lining the mucosa of the GI tract, as well as follicular cells found at hair follicles. This understandably manifests itself as common toxicities seen with these types of agents such as the various cytopenias in the blood, nausea vomiting diarrhea (although there are effects on the chemoreceptor trigger zone), and alopecia.
It was felt that the cdk4/6 inhibitors would show serious side effects similar to other cytotoxic agents and this definitely may be the case as outlined below:
For full details, please see Pharmacology/Toxicology review by Dr. Wei Chen The nonclinical studies adequately support the safety of oral administration of palbociclib for the proposed indication and the recommendation from the team is for approval. Non-clinical studies of palbociclib included safety pharmacology studies, genotoxicity
studies, reproductive toxicity studies, pharmacokinetic studies, toxicokinetic studies and repeat-dose general toxicity studies which were conducted in rats and dogs. The pivotal toxicology studies were conducted in compliance with Good Laboratory Practice regulation.
Pharmacology:
As described above, palbociclib is an inhibitor of CDK4 and CDK6. Palbociclib modulates downstream targets of CDK4 and CDK6 in vitro and induces G1 phase cell cycle arrest and therefore acts to inhibit DNA synthesis and cell proliferation. Combination of palbociclib with anti-estrogen agents demonstrated synergistic inhibition
of cell proliferation in ER+ breast cancer cells. Palbociclib showed anti-tumor efficacy in animal tumor model studies. Safety pharmacology studies with palbociclib demonstrated adverse effects on both the respiratory and cardiovascular function of dogs at a dose of 125mg/day (four times and 50-times the human clinical exposure
respectively) based on mean unbound Cmax.
General toxicology:
Palbociclib was studied in single dose toxicity studies and repeated dose studies in rats and dogs. Adverse effects in the bone marrow, lymphoid tissues, and male reproductive organs were observed at clinically relevant exposures. Partial to complete reversibility of toxicities to the hematolymphopoietic and male reproductive systems was demonstrated following a recovery period (4-12 weeks), with the exception of the male reproductive organ findings in dogs. Gastrointestinal, liver, kidney, endocrine/metabolic (altered glucose metabolism), respiratory, ocular, and adrenal effects were also seen.
Genetic toxicology:
Palbociclib was evaluated for potential genetic toxicity in in vitro and in vivo studies. The Ames bacterial mutagenicity assay in the presence or absence of metabolic activation demonstrated non-mutagenicity. In addition, palbociclib did not induce chromosomal aberrations in cultured human peripheral blood lymphocytes in the presence or absence of metabolic activation. Palbociclib was identified as aneugenic based on kinetochore analysis of micronuclei formation in an In vitro assay in CHO-WBL cells. In addition, palbociclib was shown to induce micronucleus formation in male rats at doses 100
mg/kg/day (10x human exposure at the therapeutic dose) in an in vivo rat micronucleus assay.
Reproductive toxicology: No effects on estrous cycle and no reproductive toxicities were noticed in standard assays.
Pharmacovigilance (note please see PDF for more information)
Deaths Associated With Trials: Although a few deaths occurred during some trials no deaths were attributed to the drug.
Non-Serious Adverse Events:
(note a reviewers comment below concerning incidence of pulmonary embolism is a combination trial with letrazole)
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