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The Nobel Prize in Chemistry 2020: Emmanuelle Charpentier & Jennifer A. Doudna

Reporters: Stephen J. Williams, Ph.D. and Aviva Lev-Ari, PhD, RN

 

UPDATED on 11/12/2020

Harvard’s Jack Szostak congratulates former advisee Jennifer Doudna

It was a toast from one Nobel laureate to another, sweetened by the pride of a mentor to a prized student.

When Jennifer Doudna Ph.D. ’89 was honored on Wednesday with the Nobel Prize in chemistry for her work on the CRISPR gene-editing technique, she became the second person to gain such an honor from the lab of Jack Szostak, a genetics professor at Harvard Medical School and Massachusetts General Hospital, and professor of chemistry and chemical biology at Harvard’s Faculty of Arts and Sciences.

Szostak, who won the Nobel Prize in physiology or medicine in 2009 for work on how telomere caps keep the body’s chromosomes from breaking down, advised Doudna’s doctoral work on RNA and on Wednesday raised a glass in honor of Doudna, now at the University of California, Berkeley. In a tweet, Szostak expressed his delight at seeing someone he once guided through her early scientific steps soar to science’s highest reaches:

Doudna received the prize together with Emmanuelle Charpentier, for their work discovering and developing CRISPR as a precise gene-editing tool. In just the eight years since the pair announced their discovery the use of the technique has rapidly spread to a host of fields, allowing researchers to alter the code of life and develop resistant crops, new medical therapies, and even anticipate curing inherited diseases.

 

UPDADTED on 11/2/2020

 

Announcement of the Nobel Prize in Chemistry 2020

Live webcast from the press conference where the Royal Swedish Academy of Sciences will announce the Nobel Prize in Chemistry 2020.

 

 

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry 2020 to

Emmanuelle Charpentier
Max Planck Unit for the Science of Pathogens, Berlin, Germany

Jennifer A. Doudna
University of California, Berkeley, USA

“for the development of a method for genome editing”

Genetic scissors: a tool for rewriting the code of life

Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true.

Researchers need to modify genes in cells if they are to find out about life’s inner workings. This used to be time-consuming, difficult and sometimes impossible work. Using the CRISPR/Cas9 genetic scissors, it is now possible to change the code of life over the course of a few weeks.

“There is enormous power in this genetic tool, which affects us all. It has not only revolutionised basic science, but also resulted in innovative crops and will lead to ground-breaking new medical treatments,” says Claes Gustafsson, chair of the Nobel Committee for Chemistry.

As so often in science, the discovery of these genetic scissors was unexpected. During Emmanuelle Charpentier’s studies of Streptococcus pyogenes, one of the bacteria that cause the most harm to humanity, she discovered a previously unknown molecule, tracrRNA. Her work showed that tracrRNA is part of bacteria’s ancient immune system, CRISPR/Cas, that disarms viruses by cleaving their DNA.

Charpentier published her discovery in 2011. The same year, she initiated a collaboration with Jennifer Doudna, an experienced biochemist with vast knowledge of RNA. Together, they succeeded in recreating the bacteria’s genetic scissors in a test tube and simplifying the scissors’ molecular components so they were easier to use.

In an epoch-making experiment, they then reprogrammed the genetic scissors. In their natural form, the scissors recognise DNA from viruses, but Charpentier and Doudna proved that they could be controlled so that they can cut any DNA molecule at a predetermined site. Where the DNA is cut it is then easy to rewrite the code of life.

Since Charpentier and Doudna discovered the CRISPR/Cas9 genetic scissors in 2012 their use has exploded. This tool has contributed to many important discoveries in basic research, and plant researchers have been able to develop crops that withstand mould, pests and drought. In medicine, clinical trials of new cancer therapies are underway, and the dream of being able to cure inherited diseases is about to come true. These genetic scissors have taken the life sciences into a new epoch and, in many ways, are bringing the greatest benefit to humankind.

Illustrations

The illustrations are free to use for non-commercial purposes. Attribute ”© Johan Jarnestad/The Royal Swedish Academy of Sciences”

Illustration: Using the genetic scissors (pdf)
Illustration: Streptococcus’ natural immune system against viruses:CRISPR/Cas9 pdf)
Illustration: CRISPR/Cas9 genetic scissors (pdf)

Read more about this year’s prize

Popular information: Genetic scissors: a tool for rewriting the code of life (pdf)
Scientific Background: A tool for genome editing (pdf)

Emmanuelle Charpentier, born 1968 in Juvisy-sur-Orge, France. Ph.D. 1995 from Institut Pasteur, Paris, France. Director of the Max Planck Unit for the Science of Pathogens, Berlin, Germany.

Jennifer A. Doudna, born 1964 in Washington, D.C, USA. Ph.D. 1989 from Harvard Medical School, Boston, USA. Professor at the University of California, Berkeley, USA and Investigator, Howard Hughes Medical Institute.

SOURCE

https://www.nobelprize.org/prizes/chemistry/2020/press-release/

 

Nobel Prize in Chemistry awarded to scientists who discovered CRISPR gene editing tool for ‘rewriting the code of life’

(CNN)The Nobel Prize in Chemistry has been awarded to Emmanuelle Charpentier and Jennifer A. Doudna for the development of a method for genome editing.

They discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using these, researchers can change the DNA of animals, plants and micro-organisms with extremely high precision.
Before announcing the winners on Wednesday, Göran K. Hansson, secretary-general for the Royal Swedish Academy of Sciences, said that this year’s prize was about “rewriting the code of life.”
The American biochemist Jennifer A. Doudna (left) and French microbiologist Emmanuelle Charpentier, pictured together in 2016.
 
The CRISPR/Cas9 gene editing tools have revolutionized the molecular life sciences, brought new opportunities for plant breeding, are contributing to innovative cancer therapies and may make the dream of curing inherited diseases come true, according to a press release from the Nobel committee.
 
 
There have also been some ethical concerns around the CRISPR technology, however.
Charpentier, a French microbiologist, and Doudna, an American biochemist, are the first women to jointly win the Nobel Prize in Chemistry, and the sixth and seventh women to win the chemistry prize.
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Jennifer Doudna wins 2020 Nobel Prize in chemistry

 

First Day in a Nobel Life: Jennifer Doudna

12,365 views
Oct 7, 2020
 
Scenes from day that UC Berkeley Professor Jennifer Doudna won the Nobel Prize For the full story, visit: https://news.berkeley.edu/2020/10/07/… University of California, Berkeley, biochemist Jennifer Doudna today won the 2020 Nobel Prize in Chemistry, sharing it with colleague Emmanuelle Charpentier for the co-development of CRISPR-Cas9, a genome editing breakthrough that has revolutionized biomedicine. CRISPR-Cas9 allows scientists to rewrite DNA — the code of life — in any organism, including human cells, with unprecedented efficiency and precision. The groundbreaking power and versatility of CRISPR-Cas9 has opened up new and wide-ranging possibilities across biology, agriculture and medicine, including the treatment of thousands of intractable diseases. Doudna and Charpentier, director of the Max Planck Institute for Infection Biology, will share the 10 million Swedish krona (more than $1 million) prize. “This great honor recognizes the history of CRISPR and the collaborative story of harnessing it into a profoundly powerful engineering technology that gives new hope and possibility to our society,” said Doudna. “What started as a curiosity‐driven, fundamental discovery project has now become the breakthrough strategy used by countless researchers working to help improve the human condition. I encourage continued support of fundamental science as well as public discourse about the ethical uses and responsible regulation of CRISPR technology.” Video by Clare Major & Roxanne Makasdjian
SOURCE

 

Jennifer Doudna wins 2020 Nobel Prize in chemistry

 

Jennifer Doudna in the PBS Movie CRISPR

Our critically-acclaimed documentary HUMAN NATURE is now streaming on NETFLIX. #HumanNatureFilm. Find out more about the film on our website.

 

Other Articles on the Nobel Prize in this Open Access Journal Include:

2020 Nobel Prize for Physiology and Medicine for Hepatitis C Discovery goes to British scientist Michael Houghton and US researchers Harvey Alter and Charles Rice

CONTAGIOUS – About Viruses, Pandemics and Nobel Prizes at the Nobel Prize Museum, Stockholm, Sweden 

AACR Congratulates Dr. William G. Kaelin Jr., Sir Peter J. Ratcliffe, and Dr. Gregg L. Semenza on 2019 Nobel Prize in Physiology or Medicine

2018 Nobel Prize in Physiology or Medicine for contributions to Cancer Immunotherapy to James P. Allison, Ph.D., of the University of Texas, M.D. Anderson Cancer Center, Houston, Texas. Dr. Allison shares the prize with Tasuku Honjo, M.D., Ph.D., of Kyoto University Institute, Japan

2017 Nobel prize in chemistry given to Jacques Dubochet, Joachim Frank, and Richard Henderson  for developing cryo-electron microscopy

2016 Nobel Prize in Chemistry awarded for development of molecular machines, the world’s smallest mechanical devices, the winners: Jean-Pierre Sauvage, J. Fraser Stoddart and Bernard L. Feringa

Correspondence on Leadership in Genomics and other Gene Curations: Dr. Williams with Dr. Lev-Ari

Programming life: An interview with Jennifer Doudna by Michael Chui, a partner of the McKinsey Global Institute

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Double Mutant PI3KA Found to Lead to Higher Oncogenic Signaling in Cancer Cells

Curator: Stephen J. Williams, PhD

PIK3CA (Phosphatidylinsitol 4,5-bisphosphate (PIP2) 3-kinase catalytic subunit α) is one of the most frequently mutated oncogenes in various tumor types ([1] and http://www.sanger.ac.uk/genetics/CGP/cosmic). Oncogenic mutations leading to the overactivation of PIK3CA, especially in context in of inactivating PTEN mutations, result in overtly high signaling activity and associated with the malignant phenotype.

In a Perspective article (Double trouble for cancer gene: Double mutations in an oncogene enhance tumor growth) in the journal Science[2], Dr. Alex Toker discusses the recent results of Vasan et al. in the same issue of Science[3] on the finding that double mutations in the same allele of PIK3CA are more frequent in cancer genomes than previously identified and these double mutations lead to increased PI3K pathway activation, increased tumor growth, and increased sensitivity to PI3K inhibitors in human breast cancer.

 

 

From Dr. Melvin Crasto blog NewDrugApprovals.org

Alpelisib: PIK3CA inhibitor:

Alpelisib: New PIK3CA inhibitor approved for HER2 negative metastatic breast cancer

 

FDA approves first PI3K inhibitor for breast cancer

syn https://newdrugapprovals.org/2018/06/25/alpelisib-byl-719/

Today, the U.S. Food and Drug Administration approved Piqray (alpelisib) tablets, to be used in combination with the FDA-approved endocrine therapy fulvestrant, to treat postmenopausal women, and men, with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated, advanced or metastatic breast cancer (as detected by an FDA-approved test) following progression on or after an endocrine-based regimen.

The FDA also approved the companion diagnostic test, therascreen PIK3CA RGQ PCR Kit, to detect the PIK3CA mutation in a tissue and/or a liquid biopsy. Patients who are negative by

May 24, 2019

Today, the U.S. Food and Drug Administration approved Piqray (alpelisib) tablets, to be used in combination with the FDA-approved endocrine therapy fulvestrant, to treat postmenopausal women, and men, with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated, advanced or metastatic breast cancer (as detected by an FDA-approved test) following progression on or after an endocrine-based regimen.

The FDA also approved the companion diagnostic test, therascreen PIK3CA RGQ PCR Kit, to detect the PIK3CA mutation in a tissue and/or a liquid biopsy. Patients who are negative by the therascreen test using the liquid biopsy should undergo tumor biopsy for PIK3CA mutation testing.

“Piqray is the first PI3K inhibitor to demonstrate a clinically meaningful benefit in treating patients with this type of breast cancer. The ability to target treatment to a patient’s specific genetic mutation or biomarker is becoming increasingly common in cancer treatment, and companion diagnostic tests assist oncologists in selecting patients who may benefit from these targeted treatments,” said Richard Pazdur, M.D., director of the FDA’s Oncology Center of Excellence and acting director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “For this approval, we employed some of our newer regulatory tools to streamline reviews without compromising the quality of our assessment. This drug is the first novel drug approved under the Real-Time Oncology Review pilot program. We also used the updated Assessment Aid, a multidisciplinary review template that helps focus our written review on critical thinking and consistency and reduces time spent on administrative tasks.”

Metastatic breast cancer is breast cancer that has spread beyond the breast to other organs in the body (most often the bones, lungs, liver or brain). When breast cancer is hormone-receptor positive, patients may be treated with anti-hormonal treatment (also called endocrine therapy), alone or in combination with other medicines, or chemotherapy.

The efficacy of Piqray was studied in the SOLAR-1 trial, a randomized trial of 572 postmenopausal women and men with HR-positive, HER2-negative, advanced or metastatic breast cancer whose cancer had progressed while on or after receiving an aromatase inhibitor. Results from the trial showed the addition of Piqray to fulvestrant significantly prolonged progression- free survival (median of 11 months vs. 5.7 months) in patients whose tumors had a PIK3CA mutation.

Common side effects of Piqray are high blood sugar levels, increase in creatinine, diarrhea, rash, decrease in lymphocyte count in the blood, elevated liver enzymes, nausea, fatigue, low red blood cell count, increase in lipase (enzymes released by the pancreas), decreased appetite, stomatitis, vomiting, weight loss, low calcium levels, aPTT prolonged (blood clotting taking longer to occur than it should), and hair loss.

Health care professionals are advised to monitor patients taking Piqray for severe hypersensitivity reactions (intolerance). Patients are warned of potentially severe skin reactions (rashes that may result in peeling and blistering of skin or mucous membranes like the lips and gums). Health care professionals are advised not to initiate treatment in patients with a history of severe skin reactions such as Stevens-Johnson Syndrome, erythema multiforme, or toxic epidermal necrolysis. Patients on Piqray have reported severe hyperglycemia (high blood sugar), and the safety of Piqray in patients with Type 1 or uncontrolled Type 2 diabetes has not been established. Before initiating treatment with Piqray, health care professionals are advised to check fasting glucose and HbA1c, and to optimize glycemic control. Patients should be monitored for pneumonitis/interstitial lung disease (inflammation of lung tissue) and diarrhea during treatment. Piqray must be dispensed with a patient Medication Guide that describes important information about the drug’s uses and risks.

Piqray is the first new drug application (NDA) for a new molecular entity approved under the Real-Time Oncology Review (RTOR) pilot program, which permits the FDA to begin analyzing key efficacy and safety datasets prior to the official submission of an application, allowing the review team to begin their review and communicate with the applicant earlier. Piqray also used the updated Assessment Aid (AAid), a multidisciplinary review template intended to focus the FDA’s written review on critical thinking and consistency and reduce time spent on administrative tasks. With these two pilot programs, today’s approval of Piqray comes approximately three months ahead of the Prescription Drug User Fee Act (PDUFA) VI deadline of August 18, 2019.

The FDA granted this application Priority Review designation. The FDA granted approval of Piqray to Novartis. The FDA granted approval of the therascreen PIK3CA RGQ PCR Kit to QIAGEN Manchester, Ltd.

https://www.fda.gov/news-events/press-announcements/fda-approves-first-pi3k-inhibitor-breast-cancer?utm_campaign=052419_PR_FDA%20approves%20first%20PI3K%20inhibitor%20for%20breast%20cancer&utm_medium=email&utm_source=Eloqua

 

Alpelisib

(2S)-1-N-[4-methyl-5-[2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl]-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide

PDT PAT WO 2010/029082

CHEMICAL NAMES: Alpelisib; CAS 1217486-61-7; BYL-719; BYL719; UNII-08W5N2C97Q; BYL 719
MOLECULAR FORMULA: C19H22F3N5O2S
MOLECULAR WEIGHT: 441.473 g/mol
  1. alpelisib
  2. 1217486-61-7
  3. BYL-719
  4. BYL719
  5. UNII-08W5N2C97Q
  6. BYL 719
  7. Alpelisib (BYL719)
  8. (S)-N1-(4-Methyl-5-(2-(1,1,1-trifluoro-2-methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-1,2-dicarboxamide
  9. NVP-BYL719

Alpelisib is an orally bioavailable phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antineoplastic activity. Alpelisib specifically inhibits PI3K in the PI3K/AKT kinase (or protein kinase B) signaling pathway, thereby inhibiting the activation of the PI3K signaling pathway. This may result in inhibition of tumor cell growth and survival in susceptible tumor cell populations. Activation of the PI3K signaling pathway is frequently associated with tumorigenesis. Dysregulated PI3K signaling may contribute to tumor resistance to a variety of antineoplastic agents.

Alpelisib has been used in trials studying the treatment and basic science of Neoplasms, Solid Tumors, BREAST CANCER, 3rd Line GIST, and Rectal Cancer, among others.

 

SYN 2

POLYMORPHS

https://patents.google.com/patent/WO2012175522A1/en

(S)-pyrrolidine-l,2-dicarboxylic acid 2-amide l-(4-methyl-5-[2-(2,2,2-trifluoro-l,l- dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl)-amidei hereafter referred to as compound I,

is an alpha-selective phosphatidylinositol 3 -kinase (PI3K) inhibitor. Compound I was originally described in WO 2010/029082, wherein the synthesis of its free base form was described. There is a need for additional solid forms of compound I, for use in drug substance and drug product development. It has been found that new solid forms of compound I can be prepared as one or more polymorph forms, including solvate forms. These polymorph forms exhibit new physical properties that may be exploited in order to obtain new pharmacological properties, and that may be utilized in drug substance and drug product development. Summary of the Invention

In one aspect, provided herein is a crystalline form of the compound of formula I, or a solvate of the crystalline form of the compound of formula I, or a salt of the crystalline form of the compound of formula I, or a solvate of a salt of the crystalline form of the compound of formula I. In one embodiment, the crystalline form of the compound of formula I has the polymorph form SA, SB, Sc, or SD.

In another aspect, provided herein is a pharmaceutical composition comprising a crystalline compound of formula I. In one embodiment of the pharmaceutical composition, the crystalline compound of formula I has the polymorph form SA, SB,Sc, or So.

In another aspect, provided herein is a method for the treatment of disorders mediated by PI3K, comprising administering to a patient in need of such treatment an effective amount of a crystalline compound of formula I, particularly SA, SB, SC,or SD .

In yet another aspect, provided herein is the use of a crystalline compound of formula I, particularly SA, SB, SC, or SD, for the preparation of a medicament for the treatment of disorders mediated by PI3K.

 

Source: https://newdrugapprovals.org/?s=alpelisib&submit=

 

Pharmacology and Toxicology from drugbank.ca

Indication

Alpelisib is indicated in combination with fulvestrant to treat postmenopausal women, and men, with advanced or metastatic breast cancer.Label This cancer must be hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, and PIK3CA­ mutated.Label The cancer must be detected by an FDA-approved test following progression on or after an endocrine-based regimen.Label

Associated Conditions

Contraindications & Blackbox Warnings

Learn about our commercial Contraindications & Blackbox Warnings data.

LEARN MORE

 

Pharmacodynamics

Alpelisib does not prolong the QTcF interval.Label Patients taking alpelisib experience a dose dependent benefit from treatment with a 51% advantage of a 200mg daily dose over a 100mg dose and a 22% advantage of 300mg once daily over 150mg twice daily.6 This suggests patients requiring a lower dose may benefit from twice daily dosing.6

Mechanism of action

Phosphatidylinositol-3-kinase-α (PI3Kα) is responsible for cell proliferation in response to growth factor-tyrosine kinase pathway activation.3 In some cancers PI3Kα’s p110α catalytic subunit is mutated making it hyperactive.3 Alpelisib inhibits (PI3K), with the highest specificity for PI3Kα.Label

TARGET ACTIONS ORGANISM
APhosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform inhibitor Humans

Absorption

Alpelisib reached a peak concentration in plasma of 1320±912ng/mL after 2 hours.4 Alpelisib has an AUClast of 11,100±3760h ng/mL and an AUCINF of 11,100±3770h ng/mL.4 A large, high fat meal increases the AUC by 73% and Cmax by 84% while a small, low fat meal increases the AUC by 77% and Cmax by 145%.Label

Volume of distribution

The apparent volume of distribution at steady state is 114L.Label

Protein binding

Alpelisib is 89% protein bound.Label

Metabolism

Alpelisib is metabolized by hydrolysis reactions to form the primary metabolite.Label It is also metabolized by CYP3A4.Label The full metabolism of Alpelisib has yet to be determined but a series of reactions have been proposed.4,5 The main metabolic reaction is the substitution of an amine group on alpelisib for a hydroxyl group to form a metabolite known as M44,5 or BZG791.Label Alpelisib can also be glucuronidated to form the M1 and M12 metabolites.4,5

Hover over products below to view reaction partners

Route of elimination

36% of an oral dose is eliminated as unchanged drug in the feces and 32% as the primary metabolite BZG791 in the feces.Label 2% of an oral dose is eliminated in the urine as unchanged drug and 7.1% as the primary metabolite BZG791.Label In total 81% of an oral dose is eliminated in the feces and 14% is eliminated in the urine.Label

Half-life

The mean half life of alprelisib is 8 to 9 hours.Label

Clearance

The mean apparent oral clearance was 39.0L/h.4 The predicted clearance is 9.2L/hr under fed conditions.Label

Adverse Effects

Learn about our commercial Adverse Effects data.

LEARN MORE

 

Toxicity

LD50 and Overdose

Patients experiencing an overdose may present with hyperglycemia, nausea, asthenia, and rash.Label There is no antidote for an overdose of alpelisib so patients should be treated symptomatically.Label Data regarding an LD50 is not readily available.MSDS In clinical trials, patients were given doses of up to 450mg once daily.Label

Pregnancy, Lactation, and Fertility

Following administration in rats and rabbits during organogenesis, adverse effects on the reproductive system, such as embryo-fetal mortality, reduced fetal weights, and increased incidences of fetal malformations, were observed.Label Based on these findings of animals studies and its mechanism of action, it is proposed that alpelisib may cause embryo-fetal toxicity when administered to pregnant patients.Label There is no data available regarding the presence of alpelisib in breast milk so breast feeding mothers are advised not to breastfeed while taking this medication and for 1 week after their last dose.Label Based on animal studies, alpelisib may impair fertility of humans.Label

Carcinogenicity and Mutagenicity

Studies of carcinogenicity have yet to be performed.Label Alpelisib has not been found to be mutagenic in the Ames test.Label It is not aneugenic, clastogenic, or genotoxic in further assays.Label

Affected organisms

Not Available

Pathways

Not Available

Pharmacogenomic Effects/ADRs 

 

Not Available

 

Source: https://www.drugbank.ca/drugs/DB12015

References

  1. Yuan TL, Cantley LC: PI3K pathway alterations in cancer: variations on a theme. Oncogene 2008, 27(41):5497-5510.
  2. Toker A: Double trouble for cancer gene. Science 2019, 366(6466):685-686.
  3. Vasan N, Razavi P, Johnson JL, Shao H, Shah H, Antoine A, Ladewig E, Gorelick A, Lin TY, Toska E et al: Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kalpha inhibitors. Science 2019, 366(6466):714-723.

 

 

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Systems Biology analysis of Transcription Networks, Artificial Intelligence, and High-End Computing Coming to Fruition in Personalized Oncology

Curator: Stephen J. Williams, Ph.D.

In the June 2020 issue of the journal Science, writer Roxanne Khamsi has an interesting article “Computing Cancer’s Weak Spots; An algorithm to unmask tumors’ molecular linchpins is tested in patients”[1], describing some early successes in the incorporation of cancer genome sequencing in conjunction with artificial intelligence algorithms toward a personalized clinical treatment decision for various tumor types.  In 2016, oncologists Amy Tiersten collaborated with systems biologist Andrea Califano and cell biologist Jose Silva at Mount Sinai Hospital to develop a systems biology approach to determine that the drug ruxolitinib, a STAT3 inhibitor, would be effective for one of her patient’s aggressively recurring, Herceptin-resistant breast tumor.  Dr. Califano, instead of defining networks of driver mutations, focused on identifying a few transcription factors that act as ‘linchpins’ or master controllers of transcriptional networks withing tumor cells, and in doing so hoping to, in essence, ‘bottleneck’ the transcriptional machinery of potential oncogenic products. As Dr. Castilano states

“targeting those master regulators and you will stop cancer in its tracks, no matter what mutation initially caused it.”

It is important to note that this approach also relies on the ability to sequence tumors  by RNA-seq to determine the underlying mutations which alter which master regulators are pertinent in any one tumor.  And given the wide tumor heterogeneity in tumor samples, this sequencing effort may have to involve multiple biopsies (as discussed in earlier posts on tumor heterogeneity in renal cancer).

As stated in the article, Califano co-founded a company called Darwin-Health in 2015 to guide doctors by identifying the key transcription factors in a patient’s tumor and suggesting personalized therapeutics to those identified molecular targets (OncoTarget™).  He had collaborated with the Jackson Laboratory and most recently Columbia University to conduct a $15 million 3000 patient clinical trial.  This was a bit of a stretch from his initial training as a physicist and, in 1986, IBM hired him for some artificial intelligence projects.  He then landed in 2003 at Columbia and has been working on identifying these transcriptional nodes that govern cancer survival and tumorigenicity.  Dr. Califano had figured that the number of genetic mutations which potentially could be drivers were too vast:

A 2018 study which analyzed more than 9000 tumor samples reported over 1.5 million mutations[2]

and impossible to develop therapeutics against.  He reasoned that you would just have to identify the common connections between these pathways or transcriptional nodes and termed them master regulators.

A Pan-Cancer Analysis of Enhancer Expression in Nearly 9000 Patient Samples

Chen H, Li C, Peng X, et al. Cell. 2018;173(2):386-399.e12.

Abstract

The role of enhancers, a key class of non-coding regulatory DNA elements, in cancer development has increasingly been appreciated. Here, we present the detection and characterization of a large number of expressed enhancers in a genome-wide analysis of 8928 tumor samples across 33 cancer types using TCGA RNA-seq data. Compared with matched normal tissues, global enhancer activation was observed in most cancers. Across cancer types, global enhancer activity was positively associated with aneuploidy, but not mutation load, suggesting a hypothesis centered on “chromatin-state” to explain their interplay. Integrating eQTL, mRNA co-expression, and Hi-C data analysis, we developed a computational method to infer causal enhancer-gene interactions, revealing enhancers of clinically actionable genes. Having identified an enhancer ∼140 kb downstream of PD-L1, a major immunotherapy target, we validated it experimentally. This study provides a systematic view of enhancer activity in diverse tumor contexts and suggests the clinical implications of enhancers.

 

A diagram of how concentrating on these transcriptional linchpins or nodes may be more therapeutically advantageous as only one pharmacologic agent is needed versus multiple agents to inhibit the various upstream pathways:

 

 

From: Khamsi R: Computing cancer’s weak spots. Science 2020, 368(6496):1174-1177.

 

VIPER Algorithm (Virtual Inference of Protein activity by Enriched Regulon Analysis)

The algorithm that Califano and DarwinHealth developed is a systems biology approach using a tumor’s RNASeq data to determine controlling nodes of transcription.  They have recently used the VIPER algorithm to look at RNA-Seq data from more than 10,000 tumor samples from TCGA and identified 407 transcription factor genes that acted as these linchpins across all tumor types.  Only 20 to 25 of  them were implicated in just one tumor type so these potential nodes are common in many forms of cancer.

Other institutions like the Cold Spring Harbor Laboratories have been using VIPER in their patient tumor analysis.  Linchpins for other tumor types have been found.  For instance, VIPER identified transcription factors IKZF1 and IKF3 as linchpins in multiple myeloma.  But currently approved therapeutics are hard to come by for targets with are transcription factors, as most pharma has concentrated on inhibiting an easier target like kinases and their associated activity.  In general, developing transcription factor inhibitors in more difficult an undertaking for multiple reasons.

Network-based inference of protein activity helps functionalize the genetic landscape of cancer. Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH, Califano A:. Nature genetics 2016, 48(8):838-847 [3]

Abstract

Identifying the multiple dysregulated oncoproteins that contribute to tumorigenesis in a given patient is crucial for developing personalized treatment plans. However, accurate inference of aberrant protein activity in biological samples is still challenging as genetic alterations are only partially predictive and direct measurements of protein activity are generally not feasible. To address this problem we introduce and experimentally validate a new algorithm, VIPER (Virtual Inference of Protein-activity by Enriched Regulon analysis), for the accurate assessment of protein activity from gene expression data. We use VIPER to evaluate the functional relevance of genetic alterations in regulatory proteins across all TCGA samples. In addition to accurately inferring aberrant protein activity induced by established mutations, we also identify a significant fraction of tumors with aberrant activity of druggable oncoproteins—despite a lack of mutations, and vice-versa. In vitro assays confirmed that VIPER-inferred protein activity outperforms mutational analysis in predicting sensitivity to targeted inhibitors.

 

 

 

 

Figure 1 

Schematic overview of the VIPER algorithm From: Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH, Califano A: Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nature genetics 2016, 48(8):838-847.

(a) Molecular layers profiled by different technologies. Transcriptomics measures steady-state mRNA levels; Proteomics quantifies protein levels, including some defined post-translational isoforms; VIPER infers protein activity based on the protein’s regulon, reflecting the abundance of the active protein isoform, including post-translational modifications, proper subcellular localization and interaction with co-factors. (b) Representation of VIPER workflow. A regulatory model is generated from ARACNe-inferred context-specific interactome and Mode of Regulation computed from the correlation between regulator and target genes. Single-sample gene expression signatures are computed from genome-wide expression data, and transformed into regulatory protein activity profiles by the aREA algorithm. (c) Three possible scenarios for the aREA analysis, including increased, decreased or no change in protein activity. The gene expression signature and its absolute value (|GES|) are indicated by color scale bars, induced and repressed target genes according to the regulatory model are indicated by blue and red vertical lines. (d) Pleiotropy Correction is performed by evaluating whether the enrichment of a given regulon (R4) is driven by genes co-regulated by a second regulator (R4∩R1). (e) Benchmark results for VIPER analysis based on multiple-samples gene expression signatures (msVIPER) and single-sample gene expression signatures (VIPER). Boxplots show the accuracy (relative rank for the silenced protein), and the specificity (fraction of proteins inferred as differentially active at p < 0.05) for the 6 benchmark experiments (see Table 2). Different colors indicate different implementations of the aREA algorithm, including 2-tail (2T) and 3-tail (3T), Interaction Confidence (IC) and Pleiotropy Correction (PC).

 Other articles from Andrea Califano on VIPER algorithm in cancer include:

Resistance to neoadjuvant chemotherapy in triple-negative breast cancer mediated by a reversible drug-tolerant state.

Echeverria GV, Ge Z, Seth S, Zhang X, Jeter-Jones S, Zhou X, Cai S, Tu Y, McCoy A, Peoples M, Sun Y, Qiu H, Chang Q, Bristow C, Carugo A, Shao J, Ma X, Harris A, Mundi P, Lau R, Ramamoorthy V, Wu Y, Alvarez MJ, Califano A, Moulder SL, Symmans WF, Marszalek JR, Heffernan TP, Chang JT, Piwnica-Worms H.Sci Transl Med. 2019 Apr 17;11(488):eaav0936. doi: 10.1126/scitranslmed.aav0936.PMID: 30996079

An Integrated Systems Biology Approach Identifies TRIM25 as a Key Determinant of Breast Cancer Metastasis.

Walsh LA, Alvarez MJ, Sabio EY, Reyngold M, Makarov V, Mukherjee S, Lee KW, Desrichard A, Turcan Ş, Dalin MG, Rajasekhar VK, Chen S, Vahdat LT, Califano A, Chan TA.Cell Rep. 2017 Aug 15;20(7):1623-1640. doi: 10.1016/j.celrep.2017.07.052.PMID: 28813674

Inhibition of the autocrine IL-6-JAK2-STAT3-calprotectin axis as targeted therapy for HR-/HER2+ breast cancers.

Rodriguez-Barrueco R, Yu J, Saucedo-Cuevas LP, Olivan M, Llobet-Navas D, Putcha P, Castro V, Murga-Penas EM, Collazo-Lorduy A, Castillo-Martin M, Alvarez M, Cordon-Cardo C, Kalinsky K, Maurer M, Califano A, Silva JM.Genes Dev. 2015 Aug 1;29(15):1631-48. doi: 10.1101/gad.262642.115. Epub 2015 Jul 30.PMID: 26227964

Master regulators used as breast cancer metastasis classifier.

Lim WK, Lyashenko E, Califano A.Pac Symp Biocomput. 2009:504-15.PMID: 19209726 Free

 

Additional References

 

  1. Khamsi R: Computing cancer’s weak spots. Science 2020, 368(6496):1174-1177.
  2. Chen H, Li C, Peng X, Zhou Z, Weinstein JN, Liang H: A Pan-Cancer Analysis of Enhancer Expression in Nearly 9000 Patient Samples. Cell 2018, 173(2):386-399 e312.
  3. Alvarez MJ, Shen Y, Giorgi FM, Lachmann A, Ding BB, Ye BH, Califano A: Functional characterization of somatic mutations in cancer using network-based inference of protein activity. Nature genetics 2016, 48(8):838-847.

 

Other articles of Note on this Open Access Online Journal Include:

Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

 

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Cholesterol-busting gut bacteria affect people’s cardiac health

Reporter: Irina Robu, PhD

Scientists at Broad Institute of MIT and Harvard University, has discovered a group a gut bacterium that can metabolize enough cholesterol to affect metabolism. Their study in Cell Host and Microbe, found that bacteria in the intestines have lower cholesterol levels in their blood. Cholesterol is a key biological molecule that functions as a structural component of all animal cell membranes and is a precursor of steroid hormones, vitamin D, and bile acids. Two main sources of cholesterol are thought to influence concentrations of this metabolite in serum: endogenous cholesterol synthesized in the liver and exogenous cholesterol derived from dietary components of animal origin.

The study shows a roadmap of how enzymes and microbial genes can manipulate metabolism and impact human health. The concept that bacteria can metabolize cholesterol is been known for a long time, but not enough has been known of which species of bacteria was doing this. However, isolating cholesterol metabolizing bacteria and growing them in the lab proved to be difficult.

The idea that bacteria can metabolize cholesterol isn’t a new one; in the early 1900s, scientists reported the existence of bacteria that could chemically transform cholesterol into a compound called coprostanol. Coprostanol-generating bacteria have been found in the guts of rats, baboons, pigs, and even humans, but the biology of these bacteria was poorly understood.

The scientists genetically engineered bacteria in the lab to produce genetically engineered bacteria in the lab to produce four enzymes of interest. Yet, they focused on one gene named Intestinal Stool Metabolism (IsmA) that could metabolize cholesterol. Furthermore, individuals with the IsmA gene had, on average, cholesterol levels in the blood that were 2.7 mg/dL lower than those without any copies of the IsmA genes in their microbiomes. This is a larger average effect on blood cholesterol than human genes such as HMGCR and PCSK9, which are known to alter a person’s risk of high cholesterol levels and are targeted by some FDA-approved cholesterol drugs.

SOURCE

https://www.broadinstitute.org/news/cholesterol-busting-gut-bacteria-may-affect-people%E2%80%99s-cardiac-health

 

 

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Live Conference Coverage AACR 2020 in Real Time: Monday June 22, 2020 Late Day Sessions

 

Reporter: Stephen J. Williams, PhD

 

Follow Live in Real Time using

#AACR20

@pharma_BI

@AACR

 

Register for FREE at https://www.aacr.org/

 

AACR VIRTUAL ANNUAL MEETING II

 

June 22-24: Free Registration for AACR Members, the Cancer Community, and the Public
This virtual meeting will feature more than 120 sessions and 4,000 e-posters, including sessions on cancer health disparities and the impact of COVID-19 on clinical trials

 

This Virtual Meeting is Part II of the AACR Annual Meeting.  Part I was held online in April and was centered only on clinical findings.  This Part II of the virtual meeting will contain all the Sessions and Abstracts pertaining to basic and translational cancer research as well as clinical trial findings.

 

REGISTER NOW

 

 

 

Virtual Educational Session

Prevention Research, Science Policy, Epidemiology, Survivorship

Carcinogens at Home: Science and Pathways to Prevention

Chemicals known to cause cancer are used and released to the environment in large volumes, exposing people where they live, work, play, and go to school. The science establishing an important role for such exposures in the development of cancers continues to strengthen, yet cancer prevention researchers are largely unfamiliar with the data drawn upon in identifying carcinogens and making decisions about their use. Characterizing and reducing harmful exposures and accelerating the devel

Julia Brody, Kathryn Z. Guyton, Polly J. Hoppin, Bill Walsh, Mary H. Ward

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Educational Session

Tumor Biology, Molecular and Cellular Biology/Genetics, Clinical Research Excluding Trials

EMT Still Matters: Let’s Explore! – Dedicated to the Memory of Isaiah J. Fidler

During carcinoma progression, initially benign epithelial cells acquire the ability to invade locally and disseminate to distant tissues by activating epithelial-mesenchymal transition (EMT). EMT is a cellular process during which epithelial cells lose their epithelial features and acquire mesenchymal phenotypes and behavior. Growing evidence supports the notion that EMT programs during tumor progression are usually activated to various extents and often partial and reversible, thus pr

Jean-Paul Thiery, Heide L Ford, Jing Yang, Geert Berx

DETAILS

Monday, June 22

1:30 PM – 3:00 PM EDT

Virtual Educational Session

Tumor Biology, Experimental and Molecular Therapeutics, Molecular and Cellular Biology/Genetics

One of These Things Is Not Like the Other: The Many Faces of Senescence in Cancer

Cellular senescence is a stable cell growth arrest that is broadly recognized to act as a barrier against tumorigenesis. Senescent cells acquire a senescence-associated secretory phenotype (SASP), a transcriptional response involving the secretion of inflammatory cytokines, immune modulators, and proteases that can shape the tumor microenvironment. The SASP can initially stimulate tumor immune surveillance and reinforce growth arrest. However, if senescent cells are not removed by the

Clemens A Schmitt, Andrea Alimonti, René Bernards

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Monday, June 22

1:30 PM – 3:00 PM EDT

Virtual Educational Session

Clinical Research Excluding Trials, Molecular and Cellular Biology/Genetics

Recent Advances in Applications of Cell-Free DNA

The focus of this educational session will be on recent developments in cell-free DNA (cfDNA) analysis that have the potential to impact the care of cancer patients. Tumors continually shed DNA into the circulation, where it can be detected as circulating tumor DNA (ctDNA). Analysis of ctDNA has become a routine part of care for a subset of patients with advanced malignancies. However, there are a number of exciting potential applications that have promising preliminary data but that h

Michael R Speicher, Maximilian Diehn, Aparna Parikh

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Methods Workshop

Clinical Research Excluding Trials, Clinical Trials, Experimental and Molecular Therapeutics, Molecular and Cellular Biology/Genetics

Translating Genetics and Genomics to the Clinic and Population

This session will describe how advances in understanding cancer genomes and in genetic testing technologies are being translated to the clinic. The speakers will illustrate the clinical impact of genomic discoveries for diagnostics and treatment of common tumor types in adults and in children. Cutting-edge technologies for characterization of patient and tumor genomes will be described. New insights into the importance of patient factors for cancer risk and outcome, including predispos

Heather L. Hampel, Gordana Raca, Jaclyn Biegel, Jeffrey M Trent

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Monday, June 22

1:30 PM – 3:22 PM EDT

Virtual Educational Session

Regulatory Science and Policy, Drug Development, Epidemiology

Under-representation in Clinical Trials and the Implications for Drug Development

The U.S. Food and Drug Administration relies on data from clinical trials to determine whether medical products are safe and effective. Ideally, patients enrolled in those trials are representative of the population in which the product will be used if approved, including people of different ages, races, ethnic groups, and genders. Unfortunately, with few patients enrolling in clinical trials, many groups are not well-represented in clinical trials. This session will explore challenges

Ajay K. Nooka, Nicole J. Gormley, Kenneth C Anderson, Ruben A. Mesa, Daniel J. George, Yelak Biru, RADM Richardae Araojo, Lola A. Fashoyin-Aje

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Cancer Chemistry

Targeted Protein Degradation: Target Validation Tools and Therapeutic Opportunity

This educational session will cover the exciting emerging field of targeted protein degradation. Key learning topics will include: 1. an introduction to the technology and its relevance to oncology; 2. PROTACS, degraders, and CELMoDs; 3. enzymology and protein-protein interactions in targeted protein degraders; 4. examples of differentiated biology due to degradation vs. inhibition; 5. how to address questions of specificity; and 6. how the field is approaching challenges in optimizing therapies

George Burslem, Mary Matyskiela, Lyn H. Jones, Stewart L Fisher, Andrew J Phillips

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Bioinformatics and Systems Biology, Experimental and Molecular Therapeutics, Drug Development, Molecular and Cellular Biology/Genetics

Obstacles and opportunities for protein degradation drug discovery

Lyn H. Jones
  • PROTACs ubiquitin mediated by E3 ligases;  first discovered by DeShaies and targeted to specific proteins
  • PROTACs used in drug discovery against a host of types of targets including kinases and membrane receptors
  • PROTACs can be modular but lack molecular structural activity relationships
  • can use chemical probes for target validation
  • four requirements: candidate exposure at site of action (for example lipophilicity for candidates needed to cross membranes and accumulate in lysosomes), target engagement (ternary occupancy as measured by FRET), functional pharmacology, relevant phenotype
  • PROTACs hijack the proteosomal degradation system

Proteolysis-targeting chimeras as therapeutics and tools for biological discovery

George Burslem
  • first PROTAC developed to coopt the VHL ubiquitin ligase system which degrades HIF1alpha but now modified for EREalpha
  • in screen for potential PROTACS there were compounds which bound high affinity but no degradation so phenotypic screening very important
  • when look at molecular dynamics can see where PROTAC can add additional protein protein interaction, verifed by site directed mutagenesis
  • able to target bcr-Abl
  • he says this is a rapidly expanding field because of all the new E3 ligase targets being discovered

Expanding the horizons of cereblon modulators

Mary Matyskiela

Translating cellular targeted protein degradation to in vivo models using an enzymology framework

Stewart L Fisher
  • new targeting compounds have an E3 ligase binding domain, a target binding domain and a linker domain
  • in vivo these compounds are very effective; BRD4 degraders good invitro and in vivo with little effect on body weight
  • degraders are essential activators of E3 ligases as these degraders bring targets in close proximity so activates a catalytic cycle of a multistep process (has now high turnover number)
  • in enzymatic pathway the degraders make a productive complex so instead of a kcat think of measuring a kprod or productivity of degraders linked up an E3 ligase
  • the degraders are also affecting the rebound protein synthesis; so Emax never to zero and see a small rebound of protein synthesis

 

Data-Driven Approaches for Choosing Combinatorial Therapies

Drug combinations remain the gold standard for treating cancer, as they significantly outperform single agents. However, due to the enormous size of drug combination space, it is virtually impossible to interrogate all possible combinations. This session will discuss approaches to identify novel combinations using both experimental and computational approaches. Speakers will discuss i) approaches to drug screening in cell lines, the impact of the microenvironment, and attempts to more

Bence Szalai, James E Korkola, Lisa Tucker-Kellogg, Jeffrey W Tyner

DETAILS

Monday, June 22

3:45 PM – 5:21 PM EDT

Virtual Educational Session

Tumor Biology

Cancer Stem Cells and Therapeutic Resistance

Cancer stem cells are a subpopulation of cells with a high capacity for self-renewal, differentiation and resistance to therapy. In this session, we will define cancer stem cells, discuss cellular plasticity, interactions between cancer stem cells and the tumor microenvironment, and mechanisms that contribute to therapeutic resistance.

Robert S Kerbel, Dolores Hambardzumyan, Jennifer S. Yu

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Drug Development, Experimental and Molecular Therapeutics

Molecular Imaging in Cancer Research

This session will cover the fundamentals as well as the major advances made in the field of molecular imaging. Topics covered will include the basics for optical, nuclear, and ultrasound imaging; the pros and cons of each modality; and the recent translational advancements. Learning objectives include the fundamentals of each imaging modality, recent advances in the technology, the processes involved to translate an imaging agent from bench to bedside, and how molecular imaging can gui

Julie Sutcliffe, Summer L Gibbs, Mark D Pagel, Katherine W Ferrara

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Tumor Biology, Immunology, Experimental and Molecular Therapeutics, Drug Development

Tumor Endothelium: The Gatekeepers of Tumor Immune Surveillance

Tumor-associated endothelium is a gatekeeper that coordinates the entry and egress of innate and adaptive immune cells within the tumor microenvironment. This is achieved, in part, via the coordinated expression of chemokines and cell adhesion molecules on the endothelial cell surface that attract and retain circulating leukocytes. Crosstalk between adaptive immune cells and the tumor endothelium is therefore essential for tumor immune surveillance and the success of immune-based thera

Dai Fukumura, Maria M Steele, Wen Jiang, Andrew C Dudley

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Immunology, Experimental and Molecular Therapeutics

Novel Strategies in Cancer Immunotherapy: The Next Generation of Targets for Anticancer Immunotherapy

T-cell immunotherapy in the form of immune checkpoint blockade or cellular T-cell therapies has been tremendously successful in some types of cancer. This success has opened the door to consider what other modalities or types of immune cells can be harnessed for exert antitumor functions. In this session, experts in their respective fields will discuss topics including novel approaches in immunotherapy, including NK cells, macrophage, and viral oncotherapies.

Evanthia Galanis, Kerry S Campbell, Milan G Chheda, Jennifer L Guerriero

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Tumor Biology, Drug Development, Immunology, Clinical Research Excluding Trials

Benign Cells as Drivers of Cancer Progression: Fat and Beyond

Carcinomas develop metastases and resistance to therapy as a result of interaction with tumor microenvironment, composed of various nonmalignant cell types. Understanding the complexity and origins of tumor stromal cells is a prerequisite for development of effective treatments. The link between obesity and cancer progression has revealed the engagement of adipose stromal cells (ASC) and adipocytes from adjacent fat tissue. However, the molecular mechanisms through which they stimulate

Guojun Wu, Matteo Ligorio, Mikhail Kolonin, Maria T Diaz-Meco

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Clinical Research Excluding Trials, Experimental and Molecular Therapeutics, Tumor Biology

Dharma Master Jiantai Symposium on Lung Cancer: Know Thy Organ – Lessons Learned from Lung and Pancreatic Cancer Research

The term “cancer” encompasses hundreds of distinct disease entities involving almost every possible site in the human body. Effectively interrogating cancer, either in animals models or human specimens, requires a deep understanding of the involved organ. This includes both the normal cellular constituents of the affected tissue as well as unique aspects of tissue-specific tumorigenesis. It is critical to “Know Thy Organ” when studying cancer. This session will focus on two of the most

Trudy G Oliver, Hossein Borghaei, Laura Delong Wood, Howard C Crawford

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Methods Workshop

Clinical Trials

Clinical Trial Design: Part 1: Novel Approaches and Methods in Clinical Trial Design

Good clinical trial design has always had to balance the competing interests of effectively and convincingly answering the question with the limitations imposed by scarce resources, complex logistics, and risks and potential benefits to participants. New targeted therapies, immuno-oncology, and novel combination treatments add new challenges on top of the old ones. This session will introduce these concerns and 1) suggest ways to consider what outcomes are relevant, 2) how we can best

Mary W. Redman, Nolan A. Wages, Susan G Hilsenbeck, Karyn A. Goodman

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Methods Workshop

Tumor Biology, Drug Development

High-Throughput Screens for Drivers of Progression and Resistance

The sequencing of human cancers now provides a landscape of the genetic alterations that occur in human cancer, and increasingly knowledge of somatic genetic alterations is becoming part of the evaluation of cancer patients. In some cases, this information leads directly to the selection of particular therapeutic approaches; however, we still lack the ability to decipher the significance of genetic alterations in many cancers. This session will focus on recent developments that permit the identification of molecular targets in specific cancers. This information, coupled with genomic characterization of cancer, will facilitate the development of new therapeutic agents and provide a path to implement precision cancer medicine to all patients.

William C Hahn, Mark A Dawson, Mariella Filbin, Michael Bassik

DETAILS

Monday, June 22

3:45 PM – 5:15 PM EDT

Defining a cancer dependency map

William C Hahn

Introduction

William C Hahn

Genome-scale CRISPR screens in 3D spheroids identify cancer vulnerabilities

Michael Bassik

Utilizing single-cell RNAseq and CRISPR screens to target cancer stem cells in pediatric brain tumors

Mariella Filbin
  • many gliomas are defined by discreet mutational spectra that also discriminates based on age and site as well (for example many cortical tumors have mainly V600E Braf mutations while thalamus will be FGFR1
  • they did single cell RNAseq on needle biopsy from 7 gliomas which gave about 3500 high quality single cells; obtained full length RNA
  • tumors clustered mainly where the patient it came from but had stromal cell contamination probably so did a deconvolution?  Copy number variation showed which were tumor cells and did principle component analysis
  • it seems they used a human glioma model as training set
  • identified a stem cell like glioma cell so concentrated on the genes altered in these for translational studies
  • developed multiple PDX models from patients
  • PDX transcriptome closest to patient transcriptome but organoid grown in serum free very close while organoids grown in serum very distinct transcriptome
  • developed a CRISPR barcoded library to determine genes for survival genes
  • pulled out BMI1  and EZH2 (polycomb complex proteins) as good targets

Virtual Methods Workshop

Prevention Research, Survivorship, Clinical Research Excluding Trials, Epidemiology

Implementation Science Methods for Cancer Prevention and Control in Diverse Populations: Integration of Implementation Science Methods in Care Settings

Through this Education Session we will use examples from ongoing research to provide an overview of implementation science approaches to cancer prevention and control research. We draw on examples to highlight study design approaches, research methods, and real-world solutions when applying implementation science to achieve health equity. Approaches to defining change in the care setting and measuring sustained changes are also emphasized. Using real examples of patient navigation prog

Graham A Colditz, Sanja Percac-Lima, Nathalie Huguet

DETAILS

Monday, June 22

3:45 PM – 5:30 PM EDT

Virtual Educational Session

Regulatory Science and Policy, Epidemiology

COVID-19 and Cancer: Guidance for Clinical Trial Conduct and Considerations for RWE

This session will consider the use of real-world evidence in the context of oncology clinical trials affected by the COVID-19 pandemic. Key aspects of the FDA’s recent “Guidance on Conduct of Clinical Trials of Medical Products of Medical Products during COVID-19 Public Health Emergency” will be discussed, including telemedicine, accounting for missing data, obtaining laboratory tests and images locally, using remote informed consent procedures, and additional considerations for contin

Wendy Rubinstein, Paul G. Kluetz, Amy P. Abernethy, Jonathan Hirsch, C.K. Wang

 

 

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Important but Unseen Human Embryo Developmental Stages Mimicked in Lab

 

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Scientists have created embryo-like structures that mimic a crucial yet not much known stage of human development. The structures, created from stem cells and called gastruloids, are the first to form a 3D assembly that lays out how the body will take shape. The gastruloids developed rudimentary components of a heart and nervous system, but lacked the components to form a brain and other cell types that would make them capable of becoming a viable fetus.

Human embryos take a momentous leap in their third week, when the largely homogeneous ball of cells starts to differentiate and develop specific characteristics of the body parts they will become, a process known as gastrulation. During this process, the embryo elongates and lays down a body plan with a head and tail, often called the head-to-tail axis. But scientists have never seen this process live in action. That is partly because many countries have regulations that stop embryos from being grown in the laboratory for research beyond 14 days.

Over the past years, several research groups have cultured embryonic stem-cell structures that model when cells start to differentiate. The latest model developed at the University of Cambridge, UK and their collaborators in the Netherlands, Showed for the first time what happens when the blueprint for the body’s development is laid out, around 18–21 days after conception. Genetic analysis showed that the cells formed were those that would eventually go on to form muscles in the trunk, vertebrae, heart and other organs.

If everything is done properly, the cells develop into 3D structures on their own — and then spontaneously mimic the gastrulation process. Although they display certain key features of a 21-day-old embryo, the gastruloids reach that stage after just 72 hours and survive for maximum 4 days before collapsing. Scientists will probably use the model to make structures that are even more realistic representations of early development.

The model could help scientists to understand the role of genetics and environmental factors in different disorders. The artificial structures make it possible to avoid ethical concerns about doing research on human embryos. But as the structures become more advanced and life-like, there may be ethical restrictions.

SOURCE

David Cyranoski

References for Original Study

https://www.nature.com/articles/d41586-020-01757-z?utm_source=Nature+Briefing

 

Other References:

https://pubmed.ncbi.nlm.nih.gov/32528178/

https://pubmed.ncbi.nlm.nih.gov/22804578/

https://pubmed.ncbi.nlm.nih.gov/24973948/

https://pubmed.ncbi.nlm.nih.gov/27419872/

https://pubmed.ncbi.nlm.nih.gov/28179190/

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Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 27, 2020 Minisymposium on Signaling in Cancer 11:45am-1:30 pm

Reporter: Stephen J. Williams, PhD.

SESSION VMS.MCB01.01 – Emerging Signaling Vulnerabilities in Cancer
April 27, 2020, 11:45 AM – 1:30 PM
Virtual Meeting: All Session Times Are U.S. EDT
DESCRIPTION

All session times are U.S. Eastern Daylight Time (EDT). Access to AACR Virtual Annual Meeting I sessions are free with registration. Register now at http://www.aacr.org/virtualam2020

Session Type

Virtual Minisymposium

Track(s)

Molecular and Cellular Biology/Genetics

16 Presentations
11:45 AM – 1:30 PM
– Chairperson

J. Silvio Gutkind. UCSD Moores Cancer Center, La Jolla, CA

11:45 AM – 1:30 PM
– Chairperson

  • in 80’s and 90’s signaling focused on defects and also oncogene addiction.  Now the field is switching to finding vulnerabilities in signaling cascades in cancer

Adrienne D. Cox. University of North Carolina at Chapel Hill, Chapel Hill, NC

11:45 AM – 11:55 AM
– Introduction

J. Silvio Gutkind. UCSD Moores Cancer Center, La Jolla, CA

11:55 AM – 12:05 PM
1085 – Interrogating the RAS interactome identifies EFR3A as a novel enhancer of RAS oncogenesis

Hema Adhikari, Walaa Kattan, John F. Hancock, Christopher M. Counter. Duke University, Durham, NC, University of Texas MD Anderson Cancer Center, Houston, TX

Abstract: Activating mutations in one of the three RAS genes (HRAS, NRAS, and KRAS) are detected in as much as a third of all human cancers. As oncogenic RAS mediates it tumorigenic signaling through protein-protein interactions primarily at the plasma membrane, we sought to document the protein networks engaged by each RAS isoform to identify new vulnerabilities for future therapeutic development. To this end, we determined interactomes of oncogenic HRAS, NRAS, and KRAS by BirA-mediated proximity labeling. This analysis identified roughly ** proteins shared among multiple interactomes, as well as a smaller subset unique to a single RAS oncoprotein. To identify those interactome components promoting RAS oncogenesis, we created and screened sgRNA library targeting the interactomes for genes modifying oncogenic HRAS-, NRAS-, or KRAS-mediated transformation. This analysis identified the protein EFR3A as not only a common component of all three RAS interactomes, but when inactivated, uniformly reduced the growth of cells transformed by any of the three RAS isoforms. EFR3A recruits a complex containing the druggable phosphatidylinositol (Ptdlns) 4 kinase alpha (PI4KA) to the plasma membrane to generate the Ptdlns species PI4P. We show that EFR3A sgRNA reduced multiple RAS effector signaling pathways, suggesting that EFR3A acts at the level of the oncoprotein itself. As lipids play a critical role in the membrane localization of RAS, we tested and found that EFR3A sgRNA reduced not only the occupancy of RAS at the plasma membrane, but also the nanoclustering necessary for signaling. Furthermore, the loss of oncogenic RAS signaling induced by EFR3A sgRNA was rescued by targeting PI4K to the plasma membrane. Taken together, these data support a model whereby EFR3A recruits PI4K to oncogenic RAS to promote plasma membrane localization and nonclustering, and in turn, signaling and transformation. To investigate the therapeutic potential of this new RAS enhancer, we show that EFR3A sgRNA reduced oncogenic KRAS signaling and transformed growth in a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines. Encouraged by these results we are exploring whether genetically inactivating the kinase activity of PI4KA inhibits oncogenic signaling and transformation in PDAC cell lines. If true, pharmacologically targeting PI4K may hold promise as a way to enhance the anti-neoplastic activity of drugs targeting oncogenic RAS or its effectors.

@DukeU

@DukeMedSchool

@MDAndersonNews

  • different isoforms of ras mutations exist differentially in various tumor types e.g. nras vs kras
  • the C terminal end serve as hotspots of mutations and probably isoform specific functions
  • they determined the interactomes of nras and kras and determined how many candidates are ras specific
  • they overlayed results from proteomic and CRSPR screen; EFR3a was a potential target that stuck out
  • using TCGA patients with higher EFR3a had poorer prognosis
  • EFR3a promotes Ras signaling; and required for RAS driven tumor growth (in RAS addicted tumors?)
  • EGFR3a promotes clustering of oncogenic RAS at plasma membrane

 

12:05 PM – 12:10 PM
– Discussion

12:10 PM – 12:20 PM
1086 – Downstream kinase signaling is dictated by specific KRAS mutations; Konstantin Budagyan, Jonathan Chernoff. Drexel University College of Medicine, Philadelphia, PA, Fox Chase Cancer Center, Philadelphia, PA @FoxChaseCancer

Abstract: Oncogenic KRAS mutations are common in colorectal cancer (CRC), found in ~50% of tumors, and are associated with poor prognosis and resistance to therapy. There is substantial diversity of KRAS alleles observed in CRC. Importantly, emerging clinical and experimental analysis of relatively common KRAS mutations at amino acids G12, G13, A146, and Q61 suggest that each mutation differently influences the clinical properties of a disease and response to therapy. For example, KRAS G12 mutations confer resistance to EGFR-targeted therapy, while G13D mutations do not. Although there is clinical evidence to suggest biological differences between mutant KRAS alleles, it is not yet known what drives these differences and whether they can be exploited for allele-specific therapy. We hypothesized that different KRAS mutants elicit variable alterations in downstream signaling pathways. To investigate this hypothesis, we created a novel system by which we can model KRAS mutants in isogenic mouse colon epithelial cell lines. To generate the cell lines, we developed an assay using fluorescent co-selection for CRISPR-driven genome editing. This assay involves simultaneous introduction of single-guide RNAs (sgRNAs) to two different endogenous loci resulting in double-editing events. We first introduced Cas9 and blue fluorescent protein (BFP) into mouse colon epithelial cell line containing heterozygous KRAS G12D mutation. We then used sgRNAs targeting BFP and the mutant G12D KRAS allele along with homology-directed repair (HDR) templates for a GFP gene and a KRAS mutant allele of our choice. Cells that successfully undergo HDR are GFP-positive and contain the desired KRAS mutation. Therefore, selection for GFP-positive cells allows us to identify those with phenotypically silent KRAS edits. Ultimately, this method allows us to toggle between different mutant alleles while preserving the wild-type allele, all in an isogenic background. Using this method, we have generated cell lines with endogenous heterozygous KRAS mutations commonly seen in CRC (G12D, G12V, G12C, G12R, G13D). In order to elucidate cellular signaling pathway differences between the KRAS mutants, we screened the mutated cell lines using a small-molecule library of ~160 protein kinase inhibitors. We found that there are mutation-specific differences in drug sensitivity profiles. These observations suggest that KRAS mutants drive specific cellular signaling pathways, and that further exploration of these pathways may prove to be valuable for identification of novel therapeutic opportunities in CRC.

  • Flourescent coselection of KRAS edits by CRSPR screen in a colorectal cancer line; a cell that is competent to undergo HR can undergo combination multiple KRAS
  • target only mutant allele while leaving wild type intact;
  • it was KRAS editing event in APC  +/- mouse cell line
  • this enabled a screen for kinase inhibitors that decreased tumor growth in isogenic cell lines; PKC alpha and beta 1 inhibitors, also CDK4 inhibitors inhibited cell growth
  • questions about heterogeneity in KRAS clones; they looked at off target guides and looked at effects in screens; then they used top two clones that did not have off target;  questions about 3D culture- they have not done that; Question ? dependency on AKT activity? perhaps the G12E has different downstream effectors

 

12:20 PM – 12:25 PM
– Discussion

12:25 PM – 12:35 PM
1087 – NF1 regulates the RAS-related GTPases, RRAS and RRAS2, independent of RAS activity; Jillian M. Silva, Lizzeth Canche, Frank McCormick. University of California, San Francisco, San Francisco, CA @UCSFMedicine

Abstract: Neurofibromin, which is encoded by the neurofibromatosis type 1 (NF1) gene, is a tumor suppressor that acts as a RAS-GTPase activating protein (RAS-GAP) to stimulate the intrinsic GTPase activity of RAS as well as the closely related RAS subfamily members, RRAS, RRAS2, and MRAS. This results in the conversion of the active GTP-bound form of RAS into the inactive GDP-bound state leading to the downregulation of several RAS downstream effector pathways, most notably MAPK signaling. While the region of NF1 that regulates RAS activity represents only a small fraction of the entire protein, a large extent of the NF1 structural domains and their corresponding mechanistic functions remain uncharacterized despite the fact there is a high frequency of NF1 mutations in several different types of cancer. Thus, we wanted to elucidate the underlying biochemical and signaling functions of NF1 that are unrelated to the regulation of RAS and how loss of these functions contributes to the pathogenesis of cancer. To accomplish this objective, we used CRISPR-Cas9 methods to knockout NF1 in an isogenic “RASless” MEF model system, which is devoid of the major oncogenic RAS isoforms (HRAS, KRAS, and NRAS) and reconstituted with the KRAS4b wild-type or mutant KRASG12C or KRASG12D isoform. Loss of NF1 led to elevated RAS-GTP levels, however, this increase was not as profound as the levels in KRAS-mutated cells or provided a proliferative advantage. Although ablation of NF1 resulted in sustained activation of MAPK signaling, it also unexpectedly, resulted in a robust increase in AKT phosphorylation compared to KRAS-mutated cells. Surprisingly, loss of NF1 in KRAS4b wild-type and KRAS-mutated cells potently suppressed the RAS-related GTPases, RRAS and RRAS2, with modest effects on MRAS, at both the transcript and protein levels. A Clariom™D transcriptome microarray analysis revealed a significant downregulation in the NF-κB target genes, insulin-like growth factor binding protein 2 (IGFBP2), argininosuccinate synthetase 1 (ASS1), and DUSP1, in both the NF1 knockout KRAS4b wild-type and KRAS-mutated cells. Moreover, NF1Null melanoma cells also displayed a potent suppression of RRAS and RRAS2 as well as these NF-κB transcription factors. Since RRAS and RRAS2 both contain the same NF-κB transcription factor binding sites, we hypothesize that IGFBP2, ASS1, and/or DUSP1 may contribute to the NF1-mediated regulation of these RAS-related GTPases. More importantly, this study provides the first evidence of at least one novel RAS-independent function of NF1 to regulate the RAS-related subfamily members, RRAS and RRAS2, in a manner exclusive of its RAS-GTPase activity and this may provide insight into new potential biomarkers and molecular targets for treating patients with mutations in NF1.
  • NF1 and SPRED work together to signal from RTK cKIT through RAS
  • NF1 knockout cells had higher KRAS and had increased cell proliferation
  • NF1 -/-  or SPRED loss had increased ERK phosphorylation and some increase in AKT activity compared to parental cells
  • they used isogenic cell lines devoid of all RAS isoforms and then reconstituted with specific RAS WT or mutants
  • NF1 and SPRED KO both reduce RRAS expression; in an AKT independent mannner
  • NF1 SPRED KO cells have almost no IGFBP2 protein expression and SNAIL so maybe affecting EMT?
  • this effect is independent of its RAS GTPAse activity (noncanonical)

12:35 PM – 12:40 PM
– Discussion

12:40 PM – 12:50 PM
1088 – Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling; Kali J. Dale, Martin McMahon. University of Utah, Salt Lake City, UT, Huntsman Cancer Institute, Salt Lake City, UT

Abstract: RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer. Mutationally activated RAS or BRAF signal through the MEK→ERK MAP kinase (MAPK) pathway to regulate key cancer cell hallmarks such as cell division cycle progression, reduced programmed cell death, and enhanced cell motility. Amongst the list of RAS/RAF-regulated genes are those encoding integrins, alpha-beta heterodimeric transmembrane proteins that regulate cell adhesion to the extracellular matrix. Altered integrin expression has been linked to the acquisition of more aggressive behavior by melanoma, lung, and breast cancer cells leading to diminished survival of cancer patients. We have previously documented the ability of the RAS-activated MAPK pathway to induce the expression of ITGB3 encoding integrin β3 in several different cell types. RAS/RAF-mediated induction of ITGB3 mRNA requires sustained, high-level activation of RAF→MEK→ERK signaling mediated by oncogene activation and is classified as “delayed-early”, in that it is sensitive to the protein synthesis inhibitor cycloheximide. However, to date, the regulatory mechanisms that allow for induced ITGB3 downstream of sustained, high-level activation of MAPK signaling remains obscure. We have identified over 300 DEGs, including those expressing additional cell surface proteins, that display similar regulatory characteristics as ITGB3. We use integrin β3 as a model to test our hypothesis that there is a different mechanism of regulation for delayed-early genes (DEG) compared to the canonical regulation of Immediate-Early genes. There are three regions in the chromatin upstream of the ITGB3 that become more accessible during RAF activation. We are relating the chromatin changes seen during RAF activation to active enhancer histone marks. To elucidate the essential genes of this regulation process, we are employing the use of a genome-wide CRISPR knockout screen. The work presented from this abstract will help elucidate the regulatory properties of oncogenic progression in BRAF mutated cancers that could lead to the identification of biomarkers.

12:50 PM – 12:55 PM
– Discussion

12:55 PM – 1:05 PM
1090 – Regulation of PTEN translation by PI3K signaling maintains pathway homeostasis

Radha Mukherjee, Kiran Gireesan Vanaja, Jacob A. Boyer, Juan Qiu, Xiaoping Chen, Elisa De Stanchina, Sarat Chandarlapaty, Andre Levchenko, Neal Rosen. Memorial Sloan Kettering Cancer Center, New York, NY, Yale University, West Haven, CT, Memorial Sloan Kettering Cancer Center, New York, NY, Memorial Sloan Kettering Cancer Center, New York, NY @sloan_kettering

Abstract: The PI3K pathway is a key regulator of metabolism, cell proliferation and migration and some of its components (e.g. PIK3CA and PTEN) are frequently altered in cancer by genetic events that deregulate its output. However, PI3K signaling is not usually the primary driver of these tumors and inhibitors of components of the pathway have only modest antitumor effects. We now show that both physiologic and oncogenic activation of the PI3K signaling by growth factors and an activating hotspot PIK3CA mutation respectively, cause an increase in the expression of the lipid phosphatase PTEN, thus limiting the duration of the signal and the output of the pathway in tumors. Pharmacologic and physiologic inhibition of the pathway by HER2/PI3K/AKT/mTOR inhibitors and nutrient starvation respectively reduce PTEN, thus buffering the effects of inhibition and contributing to the rebound in pathway activity that occurs in tumors. This regulation is found to be a feature of multiple types of cancer, non-cancer cell line and PDX models thereby highlighting its role as a key conserved feedback loop within the PI3K signaling network, both in vitro and in vivo. Regulation of expression is due to mTOR/4EBP1 dependent control of PTEN translation and is lost when 4EBP1 is knocked out. Translational regulation of PTEN is therefore a major homeostatic regulator of physiologic PI3K signaling and plays a role in reducing the output of oncogenic mutants that deregulate the pathway and the antitumor activity of PI3K pathway inhibitors.

  • mTOR can be a potent regulator of PTEN and therefore a major issue when developing PI3K inhibitors

1:05 PM – 1:10 PM
– Discussion

1:10 PM – 1:20 PM
1091 – BI-3406 and BI 1701963: Potent and selective SOS1::KRAS inhibitors induce regressions in combination with MEK inhibitors or irinotecan

Daniel Gerlach, Michael Gmachl, Juergen Ramharter, Jessica Teh, Szu-Chin Fu, Francesca Trapani, Dirk Kessler, Klaus Rumpel, Dana-Adriana Botesteanu, Peter Ettmayer, Heribert Arnhof, Thomas Gerstberger, Christiane Kofink, Tobias Wunberg, Christopher P. Vellano, Timothy P. Heffernan, Joseph R. Marszalek, Mark Pearson, Darryl B. McConnell, Norbert Kraut, Marco H. Hofmann. Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria, The University of Texas MD Anderson Cancer Center, Houston, TX, The University of Texas MD Anderson Cancer Center, Houston, TX, Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria

  • there is rational for developing an SOS1 inhibitor (GEF); BI3406 shows better PK and PD as a candidate
  • most sensitive cell lines to inhibitor carry KRAS mutation; NRAS or BRAF mutations are not sensititve
  • KRAS mutation defines sensitivity so they created KRAS mut isogenic cell lines
  • found best to co inhibit SOS and MEK as observed plasticity with only SOS
  • dual combination in lung NSCLC pancreatic showed enhanced efficacy compared to monotherapy
  • SOS1 inhibition plus irinotecan enhances DNA double strand breaks; no increased DNA damage in normal stroma but preferentially in tumor cells
  • these SOS1 had broad activity against KRAS mutant models;
  • phase 1 started in 2019;

@Boehringer

1:20 PM – 1:25 PM
– Discussion

1:25 PM – 1:30 PM
– Closing Remarks

Adrienne D. Cox. University of North Carolina at Chapel Hill, Chapel Hill, NC

Follow on Twitter at:

@pharma_BI

@AACR

@GenomeInstitute

@CureCancerNow

@UCLAJCCC

#AACR20

#AACR2020

#curecancernow

#pharmanews

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Lesson 7 of Cell Signaling 7 Motility: Tubulin and Tutorial Quizes for #TUBiol3373

Stephen J. Williams, Ph.D.

This lesson (lesson 7) will discuss the last type of cytoskeletal structure: microtubules and tubulin.  In addition I want to go over the last quiz answers and also introduce some new poll quizes.

I had given the lecture 7 over Canvas and each of you can download and go over the lecture but I will highlight a few slides in the lecture.

Let’s first review:

Remember that microtubules are the largest of the three cytoskeletal structures:

actin microfilaments < intermediate filaments < microtubules

This is very important to understand as the microtubules, as shown later, shuttle organelles and cellular structures like synaptic vesicles, as well as forming the centrisome and spindle fibers of mitosis.

 

 

 

 

 

 

 

 

 

 

 

 

 

Now remember the quiz question from last time

Remember that actin monomers (the G actin binds ATP)  while tubulin, the protein which makes up the microtubules binds GTP {although it is a little more complex than that as the following diagram shows}

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

See how the growth at the plus end is dependent on tubulin heterodimer GTP while when GDP is only bound to tubulin (both forms) you get a destabilization of the plus end and removal of tubulin dimers (catastrophe) if there is no source of tubulin GTP dimers (alpha tubulin GTP with beta tubulin GTP).

 

 

 

 

Also remember that like actin microfilaments you can have treadmilling (the plus end  continues growing while minus end undergoes catasrophe).  The VIDEO below describes these processes:

 

 

 

Certain SNPs and mutants of tubulin are found and can result in drastic phenotypic changes in microtubule stability and structure. Below is an article where a mutation in tubulin can result in microtubule catastrophe or destabilization of microtubule structures.

 

From: A mutation uncouples the tubulin conformational and GTPase cycles, revealing allosteric control of microtubule dynamics;, E.A. Geyer et al..; elife 2015;4:e10113

Abstract

Microtubule dynamic instability depends on the GTPase activity of the polymerizing αβ-tubulin subunits, which cycle through at least three distinct conformations as they move into and out of microtubules. How this conformational cycle contributes to microtubule growing, shrinking, and switching remains unknown. Here, we report that a buried mutation in αβ-tubulin yields microtubules with dramatically reduced shrinking rate and catastrophe frequency. The mutation causes these effects by suppressing a conformational change that normally occurs in response to GTP hydrolysis in the lattice, without detectably changing the conformation of unpolymerized αβ-tubulin. Thus, the mutation weakens the coupling between the conformational and GTPase cycles of αβ-tubulin. By showing that the mutation predominantly affects post-GTPase conformational and dynamic properties of microtubules, our data reveal that the strength of the allosteric response to GDP in the lattice dictates the frequency of catastrophe and the severity of rapid shrinking.

https://doi.org/10.7554/eLife.10113.001

 

Remember the term allosterism: change in the affinity for binding of a ligand or substrate that is caused by the binding of another ligand away from the active site (for example like 2,3 DPG effect on oxygen binding to hemoglobin

 

Cellular transport of organelles and vesicles: a function of microtubules

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Now the above figure (figure 9 in your Powerpoint) shows the movement of organelles and vesicles in two different types of cells along microtubules.

Note the magenta arrow which goes from the nucleus toward the plus end of the microtubule (at cell membrane) is referred to as anterograde transport and is movement away from center of cell to the periphery.  Retrograde transport is movement of organelles and vesicles from periphery of cell to the center of the cell.

Note that kinesin is involved in anterograde transport while dyenin is involved in retrograde transport

Also refer to the Wiki page which shows a nice cartoon of this walking down a microtubule on the right hand side of the page

https://en.wikipedia.org/wiki/Axonal_transport

 

 

 

 

 

 

 

Cilia; a cellular structure of microtubules (we will talk about cilia later)

for more information on structure of Cillia please see https://www.ncbi.nlm.nih.gov/books/NBK21698/

This is from a posting by Dr. Larry Bernstein of Yale University at https://pharmaceuticalintelligence.com/2015/11/04/cilia-and-tubulin/

 

RESEARCHERS VIDEO AND MEASURE TUBULIN TRANSPORT IN CILIA FOR THE FIRST TIME.

http://health-innovations.org/2015/01/27/researchers-image-and-measure-tubulin-transport-in-cilia/

 

 

https://michellepetersen76.files.wordpress.com/2015/01/uga-researchers-image-and-measure-tubulin-transport-in-cilia-healthinnovations1.png

 

Defective cilia can lead to a host of diseases and conditions in the human body, from rare, inherited bone malformations to blindness, male infertility, kidney disease and obesity. It is known that these tiny cell organelles become deformed and cause these diseases because of a problem related to their assembly, which requires the translocation of vast quantities of the vital cell protein tubulin. What they didn’t know was how tubulin and another cell organelle known as flagella fit into the process.

Now, a new study from University of Georgia shows the mechanism behind tubulin transport and its assembly into cilia, including the first video imagery of the process. The study was published in the Journal of Cell Biology.

Cilia are found throughout the body, so defects in cilia formation affect cells that line airways, brain ventricles or the reproductive track.  One of the main causes of male infertility is the cilia won’t function properly.

The team used total internal reflection fluorescence microscopy to analyze moving protein particles inside the cilia of Chlamydomonas reinhardtii, a green alga widely used as a model for cilia analysis.

The team exploited the natural behaviour of the organism, which is to attach by its cilia to a smooth surface, such as a microscope glass cover. This positions the cilia within the 200-nanometer reach of the total internal reflection fluorescence microscope allowing for the imaging of individual proteins as they move inside the cilia.  A video explaining the process was published along with the study.

Tubulin is transported by this process called intraflagellar transport, or IFT.  Though it has long been suspected in the field and there was indirect evidence to support the theory, this is the first time it has been shown directly, through live imaging, that IFT does function as a tubulin pump.  The team observed that about 400,000 tubulin dimers need to be transported within 60 minutes to assemble a single cilium. Being able to see tubulin moving into cilia allowed for first insights into how this transport is regulated to make sure cilia will have the correct size.

The new findings are expected to have wide implications for a variety of diseases and conditions related to cilia defects in the body.  The team state that they are on the very basic side of this research.  But because more and more diseases are being connected to cilia-related conditions, including obesity and even diabetes, the number of people working on cilia has greatly expanded over the last few years.

 

So here are the answer to last weeks polls

  1. Actin filaments are the SMALLEST of the cytoskeletal structures.  As shown in this lecture it is tubulin that binds GTP.  Actin binds ATP.
  2.  ARP2/3 or actin related proteins 2 and 3 are nucleating proteins that assist in initiating growth of branched chain micofiliment networks.  Formins are associated with unbranched actin formations.
  3.  The answer is GAPs or GTPase activating proteins.  Remember RAS in active state when GTP is bound and when you hydrolyze the GTP to GDP Ras is inactive state

 

 

 

 

 

4.  Okay so I did a type here but the best answer was acetylcholinesterase (AchE) degrading acetylcholine.  Acetylcholinesterase degrades the neurotransmitter acetylcholine into choline and acetate not as I accidentally put into acetylCoA.  The freed choline can then be taken back up into the presynaptic neuron and then, with a new acetyl group (with Coenzyme A) will form acetylcholine.

 

Synthesis of the neurotransmitter acetylcholine

 

 

 

The neuromuscular junction

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Thanks to all who took the quiz.  Remember it is for your benefit.

 

 

 

 

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Lesson 6 of Cell Signaling & Motility – Cytoskeleton II: #TUBiol3373

Stephen J. Williams, Ph.D.

In this lesson we will go over the biochemical makeup and formation of various actin containing cellular structures involved in cellular motility, structure, as well as the dynamics of muscular contraction.  The lesson had been put on your Canvas and I am emailing you the Google Docs version.  If you are having problems downloading you can download here (I believe maybe the Canvas version had problems with embedding videos properly so that is why I am sending you also by email)

Download Below

cell signaling 6 lesson 2020

After opening the powerpoint (or Google Doc) please review with the following notes which highlight some concepts as well as some reviews and reminders of past lectures.  It may be handy to also have lecture 5 handy if you need to refer to it.  In between some sections there will be polls (really multiple choice quizzes DON’T WORRY you will not be graded on them but they are for your benefit.  There will also be a section under Comments all the way at the end and at the last quiz where you can also ask questions.

Remember you can always email me or Tweet me any questions @StephenJWillia2 using the hashtag #TUBiol3373.

In addition you can also leave comments at the very bottom which can be answered.

Slide 2 of lesson 6 is a refresher of the end of our last lecture, talking about Actin Binding Regulatory Proteins.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The picture above shows a brief review of some of the structures and actin binding proteins involved in helping to form these actin filament structures (like filamin in cross linked structures, profilin which binds the actin monomers [G-actin] and helps with addition of these monomers to the leading plus end.

*** Remember G-actin (Globular Actin) is the monomer and F-actin (filamentious actin) is the polymerized actin strand [filament]

Also remember from the last lecture that G-Actin as monomer has affinity for ATP {Adenosine triphosphate} and these G-Actin-ATP will be able to polymerize to form the F-Actin form.  Also F-actin can then hydrolyze the ATP to ADP and inorganic phosphate.  At this point the actin-ADP unit looses affinity for the remaining F-Actin chain and depolymerization can occur

 

An event referred to as TREADMILLING or when the G actin units are removed from minus end and added to the plus (or growing barbed) end

Also remember that there is a critical concentration of G-Actin-ATP needed for bypassing the lag phase of nucleation before the elongation phase and the rate of addition to the plus end is faster than addition to minus end and greater than the rate of depolymerization at the minus end

Cell Structures That Involve Actin (see links for more information)

  1. filopodia
  2. parallel actin bundles
  3. actin cortex
  4. lamellipodia
  5. stress fibers
  6. microvilli
  7. contractile ring in cytokinesis

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Nucleating proteins Arp (actin related protein and Formins

Arp ====> formation of lamellipodia

Formins ====> formation of stress fibers

Process involving formins starts with a signaling event by activation of a G-protein, the GTP binding protein Rho

Rho is a subfamily member of the Ras superfamily.  The Rho family consists of cdc42, rac1, and RhoA (we will discuss at a later date).  Rho acts like G proteins, as a molecular switch.

Note that just like the Ras member of G-proteins and the Ras GTP/GDP cycle, the Rho activation, deactivation cycle also depends on GEFs [Guanine nucleotide exchange factors] and GAPs [GTPase activating proteins] and also GDIs [guanine nucleotide dissociation inhibitors which we will discuss later but involved in preventing Rho diffusion in the cell, acting as a tether].

Myosin and Motor (muscle) Function; Neuromuscular junctions, the sarcoplasmic reticulum and Ohhh the plethora of signaling events

In this section, from slides 29 to 54, we talk about myosin and the interactions between myosin and actin in formation of the contractile unit of the muscle (skeletal).

We also talk about some familiar signaling events, in particular the neuromuscular junction.

At this junction is a special type of acetylcholine receptor

Remember we talked about two types of acetylcholine receptors:

  1. muscarinic receptors – typical GPCRs that tranduce the signal via Gi or Gq depending on the muscarinic subtype
  2. nicotinic receptors – these are ligand {receptor} operated channels and when activated opens a Na+ channel which leads to depolarization

 

Now the depolarization activates another set of channels, the voltage operated calcium channels so we have two types of ion channels: Receptor {ligand} operated channels and Voltage operated channels.  These are sometimes abbreviated as ROCs and VOCs.

The unit of the myofibril on the contactile unit of the skeletal muscle is the sarcomere and upon the calcium transient, the sarcomere shortens with the two z-disks moving closer to each other as shown in the video in the lecture.

Also briefly review the introduction part on microtubules. We will finish that next week. Note that the microtubule is comprised of the protein tubulin, which is another GTP binding protein.

For other articles and more information please see

Lesson 5 Cell Signaling And Motility: Cytoskeleton & Actin: Curations and Articles of reference as supplemental information: #TUBiol3373

Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Identification of Biomarkers that are Related to the Actin Cytoskeleton

 

 

 

 

 

 

 

 

 

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New Type of Killer T-Cell

Reporter: Irina Robu, PhD

Scientists at Cardiff University have revealed a new type of killer T-cell which offers hope of a “one-size-fits-all” cancer therapy. Cancer-targeting via MR1-restricted T-cells is a thrilling new frontier, it increases the prospect of a ‘one-size-fits-all’ cancer treatment; a single type of T-cell that could be proficient of destroying numerous different types of cancers across the population.

T-cell therapies for cancer anywhere immune cells are removed, modified and returned to the patient’s blood to seek and destroy cancer cells – are the latest paradigm in cancer treatments. The most extensively-used therapy, known as CAR-T (Chimeric Antigen Receptor T-cell therapy) encompasses genetic modification of patient’s autologous T-cells to express a CAR specific for a tumor antigen, subsequent by ex vivo cell expansion and re-infusion back to the patient. The therapy is personalized to each patient, but targets only a few types of cancers.

Currently, Cardiff academics discovered T-cells equipped with a new type of T-cell receptor (TCR) which recognizes and kills most human cancer types, while ignoring healthy cells. This new TCR distinguishes when a molecule is present on the surface of a wide range of cancer cells and is able to distinguish between cancerous and healthy cells. Normal T-cells scans the surface of other cells to find anomalies and eliminate cancerous cells, yet ignores cells that contain only normal proteins.

The researchers at Cardiff was published in Nature Immunology, labels a unique TCR that can identify various types of cancer via a single HLA-like molecule called MR1 which varies in the human population. HLA differs extensively between individuals, which has previously prevented scientists from creating a single T-cell-based treatment that targets most cancers in all people. To investigate the therapeutic potential of these cells in vivo, the investigators injected T-cells able to identify MR1 into mice bearing human cancer and with a human immune system.

The Cardiff group were able to demonstrate that T-cells of melanoma patients modified to express this new TCR could destroy not only the patient’s own cancer cells, but also other patients’ cancer cells in the laboratory, irrespective of the patient’s HLA type. Experiments are under way to regulate the exact molecular mechanism by which the new TCR differentiates between healthy cells and cancer.

Source

https://www.eurekalert.org/pub_releases/2020-01/cu-don012020.php

 

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