Healthcare analytics, AI solutions for biological big data, providing an AI platform for the biotech, life sciences, medical and pharmaceutical industries, as well as for related technological approaches, i.e., curation and text analysis with machine learning and other activities related to AI applications to these industries.
Responses to the #COVID-19 outbreak from Oncologists, Cancer Societies and the NCI: Important information for cancer patients
Curator: Stephen J. Williams, Ph.D.
UPDATED 3/20/2020
Among the people who are identified at risk of coronovirus 2019 infection and complications of the virus include cancer patients undergoing chemotherapy, who in general, can be immunosuppressed, especially while patients are undergoing their treatment. This has created anxiety among many cancer patients as well as their care givers and prompted many oncologist professional groups, cancer societies, and cancer centers to formulate some sort of guidelines for both the cancer patients and the oncology professional with respect to limiting the risk of infection to coronavirus (COVID19).
This information will be periodically updated and we are working to get a Live Twitter Feed to bring oncologist and cancer patient advocacy groups together so up to date information can be communicated rapidly. Please see this page regularly for updates as new information is curated.
IN ADDITION, I will curate a listing of drugs with adverse events of immunosuppression for people who might wonder if the medications they are taking are raising their risk of infections.
From the Cancer Letter:The following is a guest editorial by American Society of Clinical Oncology (ASCO) Executive Vice President and Chief Medical Officer Richard L. Schilsky MD, FACP, FSCT, FASCO. This story is part of The Cancer Letter’s ongoing coverage of COVID-19’s impact on oncology. A full list of our coverage, as well as the latest meeting cancellations, is available here.
The worldwide spread of the coronavirus (COVID-19) presents unprecedented challenges to the cancer care delivery system.
Our patients are already dealing with a life-threatening illness and are particularly vulnerable to this viral infection, which can be even more deadly for them.Further, as restrictions in daily movement and social distancing take hold, vulnerable patients may be disconnected from friends, family or other support they need as they manage their cancer.
As providers, we rely on evidence and experience when treating patients but now we face uncertainty. There are limited data to guide us in the specific management of cancer patients confronting COVID-19 and, at present, we have no population-level guidance regarding acceptable or appropriate adjustments of treatment and practice operations that both ensure the best outcome for our patients and protect the safety of our colleagues and staff.
As normal life is dramatically changed, we are all feeling anxious about the extreme economic challenges we face, but these issues are perhaps even more difficult for our patients, many of whom are now facing interruption
As we confront this extraordinary situation, the health and safety of members, staff, and individuals with cancer—in fact, the entire cancer community—is ASCO’s highest priority.
ASCO has been actively monitoring and responding to the pandemic to ensure that accurate information is readily available to clinicians and their patients. Recognizing that this is a rapidly evolving situation and that limited oncology-specific, evidence-based information is available, we are committed to sharing what is known and acknowledging what is unknown so that the most informed decisions can be made.
To help guide oncology professionals as they deal with the impact of coronavirus on both their patients and staff, ASCO has collated questions from its members, postedresponses at asco.organd assembled a compendium of additional resources we hope will be helpful as the virus spreads and the disease unfolds. We continue to receive additional questions regarding clinical care and we are updating our FAQs on a regular basis.
We hope this information is helpful even when it merely confirms that there are no certain answers to many questions. Our answers are based on the best available information we identify in the literature, guidance from public health authorities, and input received from oncology and infectious disease experts.
For patients, we have posted a blog byDr. Merry Jennifer Markham, chair of ASCO’s Cancer Communications Committee. This can be found onCancer.Net, ASCO’s patient information website, and it provides practical guidance to help patients reduce their risk of exposure, better understand COVID-19 symptoms, and locate additional information.
This blog is available both in English and Spanish. Additional blog posts addressing patient questions will be posted as new questions are received and new information becomes available.
Find below a Tweet from Dr.Markham which includes links to her article on COVID-19 for cancer patients
JNCCN: How to Manage Cancer Care during COVID-19 Pandemic
Experts from the Seattle Cancer Care Alliance (SCCA)—a Member Institution of the National Comprehensive Cancer Network® (NCCN®)—are sharing insights and advice on how to continue providing optimal cancer care during the novel coronavirus (COVID-19) pandemic. SCCA includes the Fred Hutchinson Cancer Research Center and the University of Washington, which are located in the epicenter of the COVID-19 outbreak in the United States. The peer-reviewed article sharing best practices is available for free online-ahead-of-print via open access at JNCCN.org.
Coronavirus disease 2019 (COVID-19) Resources for the Cancer Care Community
NCCN recognizes the rapidly changing medical information relating to COVID-19 in the oncology ecosystem, but understands that a forum for sharing best practices and specific institutional responses may be helpful to others. Therefore, we are expeditiously providing documents and recommendations developed by NCCN Member Institutions or Guideline Panels as resources for oncology care providers. These resources have not been developed or reviewed by the standard NCCN processes, and are provided for information purposes only. We will post more resources as they become available so check back for additional updates.
Both the resources at cancer.gov (NCI) as well as the resources from ASCO are updated as new information is evaluated and more guidelines are formulated by members of the oncologist and cancer care community and are excellent resources for those living with cancer, and also those who either care for cancer patients or their family and relatives.
Related Resources for Patients (please click on links)
@DrMarkham Dr. Markham is Chief of Heme-Onc & gyn med onc@UF | AD Med Affairs@UFHealthCancerand has collected very good information for patients concerning #Covid19
Cancer patients on chemotherapy concerned about traveling for treatment during the #COVID19 crisis may have another option. Find out about the pros and cons of taking an oral medication. https://t.co/4djwfji5WR#CTCABlog
— CTCA, part of City of Hope (@CancerCenter) March 19, 2020
UPDATED 3/20/2020 INFORMATION FROM NCI DESIGNATED CANCER CENTERS FOR PATIENTS/PROVIDERS
The following is a listing with links of NCI Designated Comprehensive Cancer Centers and some select designated Cancer Centers* which have information on infectious risk guidance for cancer patients as well as their physicians and caregivers. There are 51 NCI Comprehensive Cancer Centers and as more cancer centers formulate guidance this list will be updated.
Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Newsletter #1 – February 2020
Welcome to the premier issue of LPBI Group News, where readers can find relevant news and updates about science, business and medical innovation. This newsletter is distributed as a service for our readers.
The Conference Forum Highlights Immuno-Oncology 360° in New York
The Conference Forum is hosting Immuno-Oncology 360°, which reports on current data and developments of immuno-oncology in the science and business communities. The summit takes place on February 26-28 at the Crowne Plaza Times Square in New York.
Please visit www.io360summit.com to register and use code LPBI20 for a 20% discount.
e-Proceedings of 15th Annual Personalized Medicine Conference at Harvard Medical School
The 15th Annual Personalized Medicine Conference at Harvard Medical School, Boston last year [November 13-14, 2019], entitled The Paradigm Evolves, explored the science, business and policy issues facing personalized medicine. In today’s world, scientists need to understand how molecular diagnostics augmented by artificial intelligence, data analytics and digital health empowers physicians and patients in their health care decisions.
Please visit for LPBI Group coverage of the meeting, including social media activities at the conference:
3D Medical BioPrinting Technology Featured in Podcast
LPBI Group leaders, Aviva Lev-Ari, Ph.D., R.N., Stephen Williams, Ph.D., and Irina Robu, Ph.D., spoke with Partners in Health and Biz, a half-hour audio podcast that reaches 40,000 listeners, about the topic of 3D Medical BioPrinting Technology: A Revolution in Medicine.
The 3D BioPrinting technology is being used to develop advanced medical practices that will help with previously difficult processes, such as delivering drugs via micro-robots, targeting specific cancer cells and even assisting in difficult eye operations.
The table of contents in this book includes: Chapter 1: 3D Bioprinting: Latest Innovations in a Forty year-old Technology. Chapter 2: LPBI Initiative on 3D BioPrinting, Chapter 3: Cardiovascular BioPrinting, Chapter 4: Medical and Surgical Repairs – Advances in R&D Research, Chapter 5: Organ on a Chip, Chapter 6: FDA Regulatory Technology Issues, Chapter 7: DNA Origami, Chapter 8: Aptamers and 3D Scaffold Binding, Chapter 9: Advances and Future Prospects, Chapter 10: BioInks and MEMS, Chapter 11: BioMedical MEMS, Chapter 12: 3D Solid Organ Printing and Chapter 13: Medical 3D Printing: Sources and Trade Groups – List of Secondary Material.
New e-Book: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS & BioInformatics, Simulations and the Genome Ontology
LPBI Group’s latest e-book entitled, Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS &BioInformatics, Simulations and the Genome Ontology, offers the reader content curation with embedded videos and audio podcasts, real-time conference e-Proceedings by LPBI’s scientists and professors and archived tweets of quotes from speakers at leading biotechnology conferences.
The book integrates in a single volume four distinct perspectives: basic science, technologies and methodologies, clinical aspects and business and legal aspects of genomics research. “The materials in this book represents the scientific frontier in Biological Sciences and Medicine related to the genomics aspects of disease onset,” said Aviva Lev-Ari, Ph.D., R.N., and founder of LPBI Group.
The book addresses:
aspects of life: the Cell, the Organ, the Human Body and Human Populations;
methodologies of genomic data analysis: Next Generation Sequencing, Gene Editing, AI, Single Cell Genomics, Evolution Biology Genomics, Simulation Modeling in Genomics, Genotypes and Phenotypes Modeling, measurement of Epigenomics effects on disease, and developments in Pharmaco-Genomics.
Additionally, artificial Intelligence in medicine is covered in Part 3 of the e-Book, which represents the frontier in this emerging field, with topics, such as the science, technologies and methodologies, clinical aspects, business and legal implications as well as the latest machine learning algorithms harnessed for medical diagnosis.
This e-book is significant because it:
contains 326 articles on topics, such as gene editing, bioinformatics and genome ontology;
incorporates 74e-Proceedings created in real time by the Book’s authors and editors
includes four collectionsof Tweets representing quotes from speakers at global leading conferences on Genomics
has 13 locationsof Videos and Audio Podcasts that serve to enrich the e-Reader’s experience.
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.
Google CFO Ruth Porat has blogged about twice battling breast cancer.
Artificial intelligence was often more accurate than radiologists in detecting breast cancer from mammograms in a study conducted by researchers using Google AI technology.
The study, published in the journal Nature, used mammograms from approximately 90,000 women in which the outcomes were known to train technology from Alphabet Inc’s DeepMind AI unit, now part of Google Health, Yahoo news reported.
The AI system was then used to analyze images from 28,000 other women and often diagnosed early cancers more accurately than the radiologists who originally interpreted the mammograms.
In another test, AI outperformed six radiologists in reading 500 mammograms. However, while the AI system found cancers the humans missed, it also failed to find cancers flagged by all six radiologists, reports The New York Times.
The researchers said the study “paves the way” for further clinical trials.
Writing in Nature, Etta D. Pisano, chief research officer at the American College of Radiology and professor in residence at Harvard Medical School, noted, “The real world is more complicated and potentially more diverse than the type of controlled research environment reported in this study.”
Ruth Porat, senior vice president and chief financial officer Alphabet, Inc., wrote in a company blog titled “Breast cancer and tech…a reason for optimism” in October about twice battling the disease herself, and the importance of her company’s application of AI to healthcare innovations.
She said that focus had already led to the development of a deep learning algorithm to help pathologists assess tissue associated with metastatic breast cancer.
“By pinpointing the location of the cancer more accurately, quickly and at a lower cost, care providers might be able to deliver better treatment for more patients,” she wrote.
Google also has created algorithms that help medical professionals diagnose lung cancer, and eye disease in people with diabetes, per the Times.
Porat acknowledged that Google’s research showed the best results occur when medical professionals and technology work together.
Any insights provided by AI must be “paired with human intelligence and placed in the hands of skilled researchers, surgeons, oncologists, radiologists and others,” she said.
Core member and chair of the faculty, Broad Institute of MIT and Harvard; director, Klarman Cell Observatory, Broad Institute of MIT and Harvard; professor of biology, MIT; investigator, Howard Hughes Medical Institute; founding co-chair, Human Cell Atlas.
millions of genome variants, tens of thousands of disease-associated genes, thousands of cell types and an almost unimaginable number of ways they can combine, we had to approximate a best starting point—choose one target, guess the cell, simplify the experiment.
In 2020, advances in polygenic risk scores, in understanding the cell and modules of action of genes through genome-wide association studies (GWAS), and in predicting the impact of combinations of interventions.
we need algorithms to make better computational predictions of experiments we have never performed in the lab or in clinical trials.
Human Cell Atlas and the International Common Disease Alliance—and in new experimental platforms: data platforms and algorithms. But we also need a broader ecosystem of partnerships in medicine that engages interaction between clinical experts and mathematicians, computer scientists and engineers
Feng Zhang, PhD
investigator, Howard Hughes Medical Institute; core member, Broad Institute of MIT and Harvard; James and Patricia Poitras Professor of Neuroscience, McGovern Institute for Brain Research, MIT.
fundamental shift in medicine away from treating symptoms of disease and toward treating disease at its genetic roots.
Gene therapy with clinical feasibility, improved delivery methods and the development of robust molecular technologies for gene editing in human cells, affordable genome sequencing has accelerated our ability to identify the genetic causes of disease.
1,000 clinical trials testing gene therapies are ongoing, and the pace of clinical development is likely to accelerate.
refine molecular technologies for gene editing, to push our understanding of gene function in health and disease forward, and to engage with all members of society
Elizabeth Jaffee, PhD
Dana and Albert “Cubby” Broccoli Professor of Oncology, Johns Hopkins School of Medicine; deputy director, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins.
a single blood test could inform individuals of the diseases they are at risk of (diabetes, cancer, heart disease, etc.) and that safe interventions will be available.
developing cancer vaccines. Vaccines targeting the causative agents of cervical and hepatocellular cancers have already proven to be effective. With these technologies and the wealth of data that will become available as precision medicine becomes more routine, new discoveries identifying the earliest genetic and inflammatory changes occurring within a cell as it transitions into a pre-cancer can be expected. With these discoveries, the opportunities to develop vaccine approaches preventing cancers development will grow.
shape how the culture of research will develop over the next 25 years, a culture that cares more about what is achieved than how it is achieved.
building a creative, inclusive and open research culture will unleash greater discoveries with greater impact.
John Nkengasong, PhD
Director, Africa Centres for Disease Control and Prevention.
To meet its health challenges by 2050, the continent will have to be innovative in order to leapfrog toward solutions in public health.
Precision medicine will need to take center stage in a new public health order— whereby a more precise and targeted approach to screening, diagnosis, treatment and, potentially, cure is based on each patient’s unique genetic and biologic make-up.
Eric Topol, MD
Executive vice-president, Scripps Research Institute; founder and director, Scripps Research Translational Institute.
In 2045, a planetary health infrastructure based on deep, longitudinal, multimodal human data, ideally collected from and accessible to as many as possible of the 9+ billion people projected to then inhabit the Earth.
enhanced capabilities to perform functions that are not feasible now.
AI machines’ ability to ingest and process biomedical text at scale—such as the corpus of the up-to-date medical literature—will be used routinely by physicians and patients.
the concept of a learning health system will be redefined by AI.
Linda Partridge, PhD
Professor, Max Planck Institute for Biology of Ageing.
Geroprotective drugs, which target the underlying molecular mechanisms of ageing, are coming over the scientific and clinical horizons, and may help to prevent the most intractable age-related disease, dementia.
Trevor Mundel, MD
President of Global Health, Bill & Melinda Gates Foundation.
finding new ways to share clinical data that are as open as possible and as closed as necessary.
moving beyond drug donations toward a new era of corporate social responsibility that encourages biotechnology and pharmaceutical companies to offer their best minds and their most promising platforms.
working with governments and multilateral organizations much earlier in the product life cycle to finance the introduction of new interventions and to ensure the sustainable development of the health systems that will deliver them.
deliver on the promise of global health equity.
Josep Tabernero, MD, PhD
Vall d’Hebron Institute of Oncology (VHIO); president, European Society for Medical Oncology (2018–2019).
genomic-driven analysis will continue to broaden the impact of personalized medicine in healthcare globally.
Precision medicine will continue to deliver its new paradigm in cancer care and reach more patients.
Immunotherapy will deliver on its promise to dismantle cancer’s armory across tumor types.
AI will help guide the development of individually matched
genetic patient screenings
the promise of liquid biopsy policing of disease?
Pardis Sabeti, PhD
Professor, Harvard University & Harvard T.H. Chan School of Public Health and Broad Institute of MIT and Harvard; investigator, Howard Hughes Medical Institute.
the development and integration of tools into an early-warning system embedded into healthcare systems around the world could revolutionize infectious disease detection and response.
But this will only happen with a commitment from the global community.
Els Toreele, PhD
Executive director, Médecins Sans Frontières Access Campaign
we need a paradigm shift such that medicines are no longer lucrative market commodities but are global public health goods—available to all those who need them.
This will require members of the scientific community to go beyond their role as researchers and actively engage in R&D policy reform mandating health research in the public interest and ensuring that the results of their work benefit many more people.
The global research community can lead the way toward public-interest driven health innovation, by undertaking collaborative open science and piloting not-for-profit R&D strategies that positively impact people’s lives globally.
New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)
New Mutant KRAS Inhibitors Are Showing Promise in Cancer Clinical Trials: Hope For the Once ‘Undruggable’ Target
Press release from FDA: FDA approves RAS inhibitor for metastatic pancreatic cancer, a cancer subtype with few successful therapies. Initial clinical results were very positive.
FDA Approves First in Class Targeted Therapy for Metastatic Pancreatic Cancer
For Immediate Release:
The U.S. Food and Drug Administration today approved Rasonque (daraxonrasib), a RAS inhibitor for the most common form of pancreatic cancer—delivering a new treatment option to patients with advanced pancreatic cancer months ahead of schedule.
This action demonstrates the agency’s commitment to moving with urgency, reducing unnecessary delays and advancing innovative treatments that can make a meaningful difference in the lives of American patients and their families.
Rasonque, a tablet taken once daily, targets multiple forms of a protein called RAS, a key driver of tumor growth in most patients with pancreatic adenocarcinoma, which arises from cells lining the ducts of the pancreas.
“Today’s approval provides a critical new option for patients facing an extraordinarily difficult and historically hard-to-treat cancer. It is our fundamental duty to deliver more cures and meaningful treatments to patients as quickly as possible,” said Acting FDA Commissioner Kyle Diamantas, J.D. “I am immensely proud of the dedicated FDA scientists whose fast, thorough review and relentless commitment made this groundbreaking milestone a reality.”
The approval is for the treatment of adults with metastatic pancreatic adenocarcinoma who have received at least one prior systemic therapy or are not candidates for multiagent systemic therapy.
Approximately 90% to 95% of the 67,000 new cases of pancreatic cancer diagnosed in the United States each year are pancreatic adenocarcinoma, according to the National Cancer Institute. Despite representing roughly 3.2% of all cancer diagnoses, pancreatic adenocarcinoma accounts for a disproportionately high share of cancer deaths, owing to its typically late detection, aggressive disease course, and historically limited treatment options.
In a randomized, open-label, multicenter clinical trial involving 500 adults with previously treated metastatic pancreatic adenocarcinoma, Rasonque improved median overall survival to 13.2 months compared to 6.7 months for standard chemotherapy.
“This drug showed unprecedented results in an area of high unmet need,” said Angelo de Claro, M.D., director of the FDA’s Oncology Center of Excellence. “The approval was granted 6.5 months before the user fee deadline, demonstrating the FDA’s commitment to accelerating the approval of new cancer treatments for patients with serious and life-threatening conditions.”
🔵 The Phase 3 Krascendo-1 trial enrolled patients with previously treated KRAS G12C-mutated non-small cell lung cancer (NSCLC).
🔵 Roche reported no new safety findings of concern, with most treatment-related side effects described as manageable and reversible.
🔵 Detailed efficacy data have not yet been released and will be presented at a future medical meeting.
The KRAS G12C market has evolved rapidly since the first approval in 2021.
Until now, Amgen and Bristol Myers Squibb have defined this treatment landscape.
Roche’s positive head-to-head Phase 3 results suggest the competitive landscape may be expanding.
The next question isn’t simply whether divarasib works.
💡 It is how much better it performs once the full clinical dataset is presented, and what that could mean for physician adoption, future treatment guidelines, and competition in targeted lung cancer.
Roche KRAS drug succeeds in head-to-head lung cancer study
The Phase 3 trial compared Roche’s divarasib with treatments sold by Amgen and Bristol Myers in patients with non-small cell lung cancer.
Published July 2, 2026
By Kristin Jensen
Exterior view of the building housing the headquarters of Roche France, French subsidiary of the Swiss pharmaceutical group F. Hoffmann-La Roche, Jan 15, 2024. The upped its customer analytics game by reengineering its data strategy. Getty Images
Dive Brief:
Roche’s experimental medicine divarasib helped patients with a certain type of lung cancer live longer than two approved treatments in a new study.
The Phase 3 trial compared divarasib with Amgen’s Lumakra and Bristol Myers Squibb’s Krazati in patients with KRAS G12C non-small cell lung cancer who had already received previous treatment. Researchers found significant improvements in both overall survival and progression-free survival for the group taking divarasib, Roche said Thursday.
Roche did not release detailed data, saying the results would be presented at an upcoming medical meeting. There were no new safety findings of concern and most treatment-related side effects were “manageable and reversible,” the company said.
Dive Insight:
Roche is trying to break into a relatively new market for medicines targeting a genetic mutation known as KRAS G12C that occurs in about 14% of non-small cell lung cancer cases. When working properly, the KRAS gene helps regulate cell growth, acting as an on-and-off switch. The G12C mutation locks the switch to “on,” leading to continuous cell growth that drives tumors.
Amgen won the first approval for a KRAS-targeting medicine in 2021. The Food and Drug Administration cleared Krazati for sale the next year. The promise of the medications caught the eye of Bristol Myers Squibb, which announced its $4.8 billion acquisition of Krazati’s developer, Mirati Therapeutics, in 2023.
Now, Roche wants to carve out part of the market for itself. The company’s head-to-head study, titled Krascendo-1, “should establish divarasib as a new standard of care for previously treated lung cancer patients with this genetically defined tumor subtype,” Roche’s chief medical officer, Levi Garraway, said in the company’s statement.
The second-line treatment study is a boost for Roche, but the bigger market opportunity is in patients who have not yet received therapy, Jefferies analyst Michael Leuchten wrote in a note to clients. Roche is testing those patients in the “Krascendo-2” trial, which compares a regimen of divarasib plus Merck & Co.’s blockbuster cancer drug Keytruda against Keytruda plus chemotherapy.
UPDATED 10/2/2021
A Newly Identified Mutant RAS G12C GTPase Activating Protein (GAP) may lead to discovery of new class of RAS inhibitors
From the journal Science in Filling in the GAPs in understanding RAS: A newly identified regulator increases the efficacy of a new class of targeted anti-RAS drugs.
According to canonical thinking, mutationally activated RAS (constitutive activation) is insensitive to GTPase-activating proteins (GAPs). GAPs accelerated the conversion of GTP bound to RAS to GDP, a critical step in the inactivation of RAS signaling cycle. However a small molecule has just been FDA approved, sotorasib, in cancer cells by binding only the GDP bound KRAS G12C mutant. A few non small cell lung cancers (NSCLC) are resistant to another such inhibitor, adagrasib. In the same Science issue, another paper by Li et. al. explains the potential that a GAP, RGS3, may play in this conundrum and demonstrates that certain other inhibitors of the RAS cycle, mainly the GEF (guanine exchange factor) SOS1, in combination with MEK inhibitors may circumvent this resistance.
Inhibitors of mutant KRAS
The KRASG12C (Gly12→Cys) mutant is refractory to canonical GAPs, p120 RASGAP and neurofibromin, but not RGS3, which promotes GTP hydrolysis. The resulting GDP-bound KRASG12C is an anticancer drug target. Combination with inhibitors of SOS1 or with inhibitors of downstream signaling may further improve efficacy.
There is now considerable and increasing evidence for a causal role of aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases act as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. We assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.
Box 1
Ras superfamily of small GTPases
The human Ras superfamily comprised of over 150 members which is divided into five major branches on the basis of sequence and functional similarities1, 2. In addition to the three Ras isoforms, other members of the Ras family with important roles in cancer include Rheb and Ral proteins. The ~20 kDa core G domain (corresponding to Ras residues 4–166) is conserved among all Ras superfamily proteins and is involved in GTP binding and hydrolysis148. This domain is comprised of five conserved guanine nucleotide consensus sequence elements (Ras residue numbering) involved in binding phosphate/Mg2+ (PM) or the guanine base (G). The switch I (Ras residues 30–38) and II (59–76) regions change in conformation during GDP-GTP cycling and contribute to preferential effector binding to the GTP-bound state and the core effector domain (E; Ras residues 32–40). Ras and Rho family proteins have additional C-terminal hypervariable (HV) sequences that commonly terminate with a CAAX motif that signals for farnesyl or geranylgeranyl isoprenoid addition to the cysteine residue, proteolytic removal of the AAX residues and carboxylmethylation of the prenylated cysteine. Some are modified additionally by a palmitate fatty acid to cysteine residues in the HV sequence that contributes to membrane association. Rab proteins also contain a C-terminal HV region that terminates with cysteine-containing motifs that are modified by addition of geranylgeranyl lipids, with some undergoing carboxylmethylation. Arf family proteins are characterized by an N-terminal extension involved in membrane interaction, with some cotranslationally modified by addition of a myristate fatty acid. Ran is not lipid modified but contains a C-terminal extension that is essential for function. Rho proteins are characterized by an up to 13 amino acid “Rho insert” sequence positioned between Ras residues 122 and 123 involved in effector regulation.
The GDP-GTP cycle
Ras superfamily proteins possess intrinsic guanine nucleotide exchange and GTP hydrolysis activities. However, these activities are too low to allow efficient and rapid cycling between their active GTP-bound and inactive GDP-bound states. GEFs and GAPs accelerate and regulate these intrinsic activities. Members of the different branches of the superfamily are regulated by GEFs and GAPs with structurally distinct catalytic domains3, 4, 149–152. Here we have utilized the Rho family as an example to illustrate the complexity of this process, where multiple GEFs and GAPs may regulate one specific GTPase. For the 20 human Rho GTPases there are 83 GEFs and 67 GAPs and a subset of Rho GTPases are not likely regulated by GEFs and GAPs (e.g., Rnd3/RhoE). Rho GTPases are activated by distinct RhoGEF families. Dbl family RhoGEFs (68) possesses a tandem Dbl homology (DH) catalytic and pleckstrin homology (PH) regulatory domain topology. DOCK family RhoGEFs (11) are characterized by two regions of high sequence conservation that are designated Dock-homology region regulatory DHR-1 and catalytic DHR-2 domains. Two other RhoGEFs have been described (SWAP70 and SLAT) contain a PH but no DH domain (2) and smgGDS (1) is an unusual GEF in that it functions as a GEF for some Rho as well as non-Rho family GTPases. At least 24 Dbl RhoGEFs have been reported to activate RhoA151. Rho (and Rab) GTPases are also controlled by a third class of regulatory proteins, Rho dissociation inhibitors (RhoGDI) (of which there are 3) whose main function involves regulation of Rho GTPase membrane association by masking the isoprenoid group.
So GEFs like SOS 1,2,3 are responsible for activation of the G Protien RAS upon Receptor Tyrosine Kinase (RTK) activation by a multitude of growth factors. The GAPs are responsible for termination of the activated RAS cycle and now RAS, in its normal unmuted version, can now be activated again. The G12C mutant keeps RAS in its activated state as GAPs cannot fascilitate the hydroylsis of GTP.
Li et al. (in this issue) theorized that there must be cellular factors that stimulate formation of GDP-bound KRAS G12C, enabling its vulnerability to KRAS G12C inhibitors, and discovered a regulator of G protein signaling 3 (RGS3), which had GAP activity previously for heterotrimeric G proteins GαI and Gαq with unexpected GAP activity for the KRAS G12C mutant. They also found the fully activated GTP bound KRAS G12C is dependent on RTK-mediated activation of SOS1. This suggested that SOS1 inhibitors with MEK inhibitors could be effective against mutant KRAS.
The paper by Li is summarized below:
The G protein signaling regulator RGS3 enhances the GTPase activity of KRAS
Recently reported to be effective in patients with lung cancer, KRASG12C inhibitors bind to the inactive, or guanosine diphosphate (GDP)–bound, state of the oncoprotein and require guanosine triphosphate (GTP) hydrolysis for inhibition. However, KRAS mutations prevent the catalytic arginine of GTPase-activating proteins (GAPs) from enhancing an otherwise slow hydrolysis rate. If KRAS mutants are indeed insensitive to GAPs, it is unclear how KRASG12C hydrolyzes sufficient GTP to allow inactive state–selective inhibition. Here, we show that RGS3, a GAP previously known for regulating G protein–coupled receptors, can also enhance the GTPase activity of mutant and wild-type KRAS proteins. Our study reveals an unexpected mechanism that inactivates KRAS and explains the vulnerability to emerging clinically effective therapeutics.
UPDATED 09/26/2021
The KRAS G12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients
Source: Hallin J, Engstrom LD, Hargis L, Calinisan A, Aranda R, Briere DM, Sudhakar N, Bowcut V, Baer BR, Ballard JA, Burkard MR, Fell JB, Fischer JP, Vigers GP, Xue Y, Gatto S, Fernandez-Banet J, Pavlicek A, Velastagui K, Chao RC, Barton J, Pierobon M, Baldelli E, Patricoin EF 3rd, Cassidy DP, Marx MA, Rybkin II, Johnson ML, Ou SI, Lito P, Papadopoulos KP, Jänne PA, Olson P, Christensen JG. The KRASG12C Inhibitor MRTX849 Provides Insight toward Therapeutic Susceptibility of KRAS-Mutant Cancers in Mouse Models and Patients. Cancer Discov. 2020 Jan;10(1):54-71. doi: 10.1158/2159-8290.CD-19-1167. Epub 2019 Oct 28. PMID: 31658955; PMCID: PMC6954325.
Abstract
Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE: The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.
Introduction
KRAS is one of the most frequently mutated oncogenes in cancer; however, efforts to directly target KRAS have been largely unsuccessful due to its high affinity for GTP/GDP and the lack of a clear binding pocket (1–4). More recently, compounds were identified that covalently bind to KRASG12C at the cysteine 12 residue, lock the protein in its inactive GDP-bound conformation, inhibit KRAS-dependent signaling, and elicit antitumor responses in tumor models (5–7). Advances on early findings demonstrated that the binding pocket under the switch II region was exploitable for drug discovery, culminating in the identification of more potent KRASG12C inhibitors with improved physiochemical properties that are now entering clinical trials. The identification of KRASG12C inhibitors provides a renewed opportunity to develop a more comprehensive understanding of the role of KRAS as a driver oncogene and to explore the clinical utility of direct KRAS inhibition.
KRASG12C mutations are present in lung and colon adenocarcinomas as well as smaller fractions of other cancers. The genetic context of KRASG12C alteration can vary significantly among tumors and is predicted to affect response to KRAS inhibition. KRAS mutations are often enriched in tumors due to amplification of mutant or loss of wild-type allele (8, 9). In addition, KRAS mutations often co-occur with other key genetic alterations including TP53 and CDKN2A in multiple cancers, KEAP1 and/or STK11 in lung adenocarcinoma, or APC and PIK3CA in colon cancer (3, 8–12). Whether differences in KRAS-mutant allele fraction or co-occurrence with other mutations influence response to KRAS blockade is not yet well understood. In addition, due to the critical importance of the RAS pathway in normal cellular function, there is extensive pathway isoform redundancy and a comprehensive regulatory network in normal cells to ensure tight control of temporal pathway signaling. RAS pathway negative feedback signaling is mediated by ERK1/2 and receptor tyrosine kinases (RTK) as well as by ERK pathway target genes including dual-specificity phosphatases (DUSP) and Sprouty (SPRY) proteins (13–17). One important clinically relevant example is provided by the reactivation of ERK signaling observed following treatment of BRAFV600E-mutant cancers with selective BRAF inhibitors (18–20). The observed intertumoral heterogeneity and extensive feedback signaling network in KRAS-mutant cancers may necessitate strategies to more comprehensively block oncogenic signal transduction and deepen the antitumor response in concert with KRAS blockade (15, 21, 22).
Potential strategies to augment the response to KRASG12C inhibitor treatment are evident at multiple nodes of the signaling pathway regulatory machinery. RAS proteins are small GTPases that normally cycle between an active, GTP-bound state and an inactive, GDP-bound state. RAS proteins are loaded with GTP through guanine nucleotide exchange factors (e.g., SOS1) which are activated by upstream RTKs, triggering subsequent interaction with effector proteins that activate RAS-dependent signaling. RAS proteins hydrolyze GTP to GDP through their intrinsic GTPase activity, which is dramatically enhanced by GTPase-activating proteins (GAP). Mutations at codons 12 and 13 in RAS proteins impair GAP-stimulated GTP hydrolysis, leaving RAS predominantly in the GTP-bound, active state.
Potent covalent KRASG12C inhibitors described to date bind only GDP-bound KRAS (5–7). Although codon 12 and 13 mutations decrease the fraction of GDP-bound KRAS, recent biochemical analyses revealed that KRASG12C exhibits the highest intrinsic GTP hydrolysis rate and highest nucleotide exchange rate among KRAS mutants (23). Furthermore, the nucleotide-bound state of KRASG12C can be shifted toward the GDP-bound state by pharmacologically modulating upstream signaling with RTK inhibitors that increase the activity of KRASG12C inhibitors (7, 22, 24). Likewise, SHP2 is a phosphatase that positively transduces RTK signaling to KRAS. Accordingly, SHP2 inhibitors are active in cancers driven by KRAS mutations that are dependent on nucleotide cycling, including KRASG12C (25–27).
MRTX849 is among the first KRASG12C inhibitors to advance to clinical trials. The comprehensive and durable inhibition of KRASG12C by MRTX849 provides a unique opportunity to understand the extent to which KRAS functions as an oncogenic driver. In addition, the observation that the response to blockade of KRAS is markedly different in vitro and in vivo indicates that evaluation of the consequences of KRAS blockade in in vivo model systems is critical to understand the role of KRAS-driven tumor progression. The demonstration of partial responses in patients with lung and colon adenocarcinomas treated with MRTX849 in clinical trials indicates that results observed in tumor models extend to KRASG12C-positive human cancers. Our comprehensive molecular characterization of multiple tumor models at baseline and during response to KRAS inhibition has provided further insight toward the contextual role of KRAS mutation in the setting of genetic and tumoral heterogeneity. Finally, further interrogation of these genetic alterations and signaling pathways utilizing functional genomics strategies including CRISPR and combination approaches uncovered regulatory nodes that sensitize tumors to KRAS inhibition when cotargeted.
Introduction
KRAS is one of the most frequently mutated oncogenes in cancer; however, efforts to directly target KRAS have been largely unsuccessful due to its high affinity for GTP/GDP and the lack of a clear binding pocket (1–4). More recently, compounds were identified that covalently bind to KRASG12C at the cysteine 12 residue, lock the protein in its inactive GDP-bound conformation, inhibit KRAS-dependent signaling, and elicit antitumor responses in tumor models (5–7). Advances on early findings demonstrated that the binding pocket under the switch II region was exploitable for drug discovery, culminating in the identification of more potent KRASG12C inhibitors with improved physiochemical properties that are now entering clinical trials. The identification of KRASG12C inhibitors provides a renewed opportunity to develop a more comprehensive understanding of the role of KRAS as a driver oncogene and to explore the clinical utility of direct KRAS inhibition.
KRASG12C mutations are present in lung and colon adenocarcinomas as well as smaller fractions of other cancers. The genetic context of KRASG12C alteration can vary significantly among tumors and is predicted to affect response to KRAS inhibition. KRAS mutations are often enriched in tumors due to amplification of mutant or loss of wild-type allele (8, 9). In addition, KRAS mutations often co-occur with other key genetic alterations including TP53 and CDKN2A in multiple cancers, KEAP1 and/or STK11 in lung adenocarcinoma, or APC and PIK3CA in colon cancer (3, 8–12). Whether differences in KRAS-mutant allele fraction or co-occurrence with other mutations influence response to KRAS blockade is not yet well understood. In addition, due to the critical importance of the RAS pathway in normal cellular function, there is extensive pathway isoform redundancy and a comprehensive regulatory network in normal cells to ensure tight control of temporal pathway signaling. RAS pathway negative feedback signaling is mediated by ERK1/2 and receptor tyrosine kinases (RTK) as well as by ERK pathway target genes including dual-specificity phosphatases (DUSP) and Sprouty (SPRY) proteins (13–17). One important clinically relevant example is provided by the reactivation of ERK signaling observed following treatment of BRAFV600E-mutant cancers with selective BRAF inhibitors (18–20). The observed intertumoral heterogeneity and extensive feedback signaling network in KRAS-mutant cancers may necessitate strategies to more comprehensively block oncogenic signal transduction and deepen the antitumor response in concert with KRAS blockade (15, 21, 22).
Potential strategies to augment the response to KRASG12C inhibitor treatment are evident at multiple nodes of the signaling pathway regulatory machinery. RAS proteins are small GTPases that normally cycle between an active, GTP-bound state and an inactive, GDP-bound state. RAS proteins are loaded with GTP through guanine nucleotide exchange factors (e.g., SOS1) which are activated by upstream RTKs, triggering subsequent interaction with effector proteins that activate RAS-dependent signaling. RAS proteins hydrolyze GTP to GDP through their intrinsic GTPase activity, which is dramatically enhanced by GTPase-activating proteins (GAP). Mutations at codons 12 and 13 in RAS proteins impair GAP-stimulated GTP hydrolysis, leaving RAS predominantly in the GTP-bound, active state.
Potent covalent KRASG12C inhibitors described to date bind only GDP-bound KRAS (5–7). Although codon 12 and 13 mutations decrease the fraction of GDP-bound KRAS, recent biochemical analyses revealed that KRASG12C exhibits the highest intrinsic GTP hydrolysis rate and highest nucleotide exchange rate among KRAS mutants (23). Furthermore, the nucleotide-bound state of KRASG12C can be shifted toward the GDP-bound state by pharmacologically modulating upstream signaling with RTK inhibitors that increase the activity of KRASG12C inhibitors (7, 22, 24). Likewise, SHP2 is a phosphatase that positively transduces RTK signaling to KRAS. Accordingly, SHP2 inhibitors are active in cancers driven by KRAS mutations that are dependent on nucleotide cycling, including KRASG12C (25–27).
MRTX849 is among the first KRASG12C inhibitors to advance to clinical trials. The comprehensive and durable inhibition of KRASG12C by MRTX849 provides a unique opportunity to understand the extent to which KRAS functions as an oncogenic driver. In addition, the observation that the response to blockade of KRAS is markedly different in vitro and in vivo indicates that evaluation of the consequences of KRAS blockade in in vivo model systems is critical to understand the role of KRAS-driven tumor progression. The demonstration of partial responses in patients with lung and colon adenocarcinomas treated with MRTX849 in clinical trials indicates that results observed in tumor models extend to KRASG12C-positive human cancers. Our comprehensive molecular characterization of multiple tumor models at baseline and during response to KRAS inhibition has provided further insight toward the contextual role of KRAS mutation in the setting of genetic and tumoral heterogeneity. Finally, further interrogation of these genetic alterations and signaling pathways utilizing functional genomics strategies including CRISPR and combination approaches uncovered regulatory nodes that sensitize tumors to KRAS inhibition when cotargeted.
Results
MRTX849 Is a Potent and Selective Inhibitor of KRASG12C, KRAS-Dependent Signal Transduction, and Cell Viability In Vitro
A structure-based drug design approach, including optimization for favorable drug-like properties, led to the discovery of MRTX849 as a potent, covalent KRASG12C inhibitor (Fig. 1A; Supplementary Table S1). An LC/MS-based KRASG12C protein modification assay revealed that MRTX849 demonstrated much greater modification of KRASG12C when preloaded with GDP compared with GTP (Supplementary Table S2), supporting that MRTX849 binds to and stabilizes the inactive GDP-bound form of KRASG12C. Indeed, introducing a comutation that impairs the GTPase activity of KRASG12C (24) attenuated the inhibitory activity of MRTX1257, a close analogue of MRTX849 (Supplementary Fig. S1A). Secondary mutations that modulate the nucleotide exchange function of KRASG12C also affected inhibition by MRTX1257, supporting that the MRTX compound series is dependent on KRASG12C nucleotide cycling.
MRTX849 is a potent, covalent KRASG12C inhibitor in vitro. A, Structure of MRTX849. B, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated from 1 hours to 72 hours with MRTX849 at 100 nmol/L. C, Immunoblot protein Western blot analyses of KRAS pathway targets in MIA PaCa-2 cells treated for 24 hours with MRTX849 over a 13-point dose response. D, Left y-axis shows active RAS ELISA assay to determine the reduction in RAS-GTP abundance following MRTX849 treatment in MIA PaCa-2 cells for 24 hours. The vehicle value was normalized to 1 by dividing all average values by the vehicle value. Right y-axis shows quantitation of KRAS band shift by MRTX849 treatment in MIA PaCa-2 cells for 24 hours as assessed by Western blot and densitometry. E, In-cell Western blot assay to evaluate modulation of pERK in MIA PaCa-2 cells grown in standard tissue-culture conditions treated with MRTX849 over a time course. F, CellTiter-Glo assay to evaluate cell viability performed on seven KRASG12C-mutant cell lines and three non–KRASG12C-mutant cell lines grown in 2-D tissue-culture conditions in a 3-day assay (left plot) or 3-D conditions using 96-well, ULA plates in a 12-day assay (right plot).
We next determined the cellular activity of MRTX849 utilizing the KRASG12C-mutant H358 lung and MIA PaCa-2 pancreatic cancer cell lines. In both models, MRTX849 demonstrated an upward electrophoretic mobility shift of KRASG12C protein band migration by immunoblot, indicative of covalent modification of KRASG12C. A maximal mobility shift was observed by 1 hour, was maintained through 72 hours (Fig 1B; Supplementary Fig. S1B), and was evident at concentrations as low as 2 nmol/L with near-maximal modification observed at 15.6 nmol/L (Fig. 1C; Supplementary Fig. S1C). Comparable inhibition of active RAS was observed as determined by a RAF RAS-binding domain capture ELISA assay (Fig. 1D; 1D). MRTX849 also inhibited KRAS-dependent signaling targets including ERK1/2 phosphorylation (pERK; Thr202/Tyr204 ERK1), S6 phosphorylation (pS6; RSK-dependent Ser235/236), and expression of the ERK-regulated DUSP6, each with IC50 values in the single-digit nanomolar range in both cell lines (Fig. 1B and C; Supplementary Fig. S1B and S1C). The evaluation of pERK over a time course of 48 hours indicated maximal inhibition was observed at 24 hours (Fig. 1E; Supplementary Fig. S1E). Treatment with the des-acrylamide version of MRTX849, which is unable to covalently bind to KRASG12C, did not demonstrate significant inhibition of ERK phosphorylation (Supplementary Fig. S1F). The H358 cell line was selected for determination of MRTX849 cysteine selectivity utilizing an LC/MS-based proteomics approach able to detect approximately 6,000 cysteine-containing peptides. After treatment for 3 hours, decreased KRASG12C Cys12-free peptide was detected with treated-to-control ratios of 0.029 and 0.008 determined at 1 and 10 μmol/L, respectively, indicating near-complete engagement of the intended target (Supplementary Table S3). In contrast, the only other peptides identified were from lysine-tRNA ligase (KARS) at Cys209 near the detection limit, indicating a high degree of selectivity toward KRASCYS12.
To evaluate the breadth of MRTX849 activity, its effect on cell viability was determined across a panel of 17 KRASG12C-mutant and 3 non–KRASG12C-mutant cancer cell lines using 2-D (3-day, adherent cells) and 3-D (12-day, spheroids) cell-growth conditions. MRTX849 potently inhibited cell growth in the vast majority of KRASG12C-mutant cell lines with IC50 values ranging between 10 and 973 nmol/L in the 2-D format and between 0.2 and 1,042 nmol/L in the 3-D format (Supplementary Table S4; Fig. 1F). In agreement with prior KRASG12C inhibitor studies (5), MRTX849 demonstrated improved potency in the 3-D assay format, as all but one KRASG12C-mutant cell line exhibited an IC50 value below 100 nmol/L. Although MRTX849 was broadly effective in inhibiting viability of KRASG12C-mutant cell lines, IC50 values varied across the cell panel by 100-fold, suggesting a differential degree of sensitivity to treatment. All three non–KRASG12C-mutant cell lines tested demonstrated IC50 values greater than 1 μmol/L in 2-D conditions and greater than 3 μmol/L in 3-D conditions, suggesting the effect of MRTX849 on cell viability was dependent on the presence of KRASG12C.
To determine whether the difference in sensitivity across the cell panel correlated with the ability of MRTX849 to bind to KRAS or inhibit KRAS-dependent signal transduction, seven KRASG12C-mutant cancer cell lines were selected from the panel for further evaluation. In each cell line, MRTX849 demonstrated a very similar concentration-dependent electrophoretic mobility shift (IC50) for KRASG12C protein migration, suggesting that the ability to bind to and modify KRASG12C does not readily account for differences in response in viability studies (Fig. 1B and C; Supplementary Figs. S1B and S1C and S2A and S2B). The effect of MRTX849 on selected phosphoproteins implicated in mediating KRAS-dependent signaling was also evaluated across the cell panel by immunoblot and/or reverse-phase protein array (RPPA) following treatment for 6 or 24 hours. Notably, the concentration–response relationship and maximal effect of MRTX849 on inhibition of ERK and S6S235/236 phosphorylation varied across the cell panel (Supplementary Fig. S2A and S2C; Supplementary Table S7). MRTX849 demonstrated only partial inhibition of pERK in KYSE-410 and SW1573 cells and a minimal effect on pS6S235/236 in SW1573, H2030, and KYSE-410 cells (Supplementary Fig. S2A and S2C). Each of these cell lines were among those that exhibited a submaximal response to MRTX849 in both 2-D and 3-D viability assays (Fig. 1F). Although KRAS is implicated in mediating signal transduction through the PI3K and mTOR pathways, there was minimal evidence of a significant and/or durable effect of MRTX849 on AKT (S473, T308) or 4E-BP1 (T37/T46, S65, T70) phosphorylation at any time point in any cell lines evaluated (Supplementary Fig. S2D). However, MRTX849 demonstrated concentration-dependent partial inhibition of the mTOR-dependent signaling targets p70 S6 kinase (T412) and/or pS6 (S240/44) in the H358, MIA PaCa-2,H2122, and H1373 cell lines, each of which exhibited a maximal response to treatment. Together, these data suggest that maximizing inhibition of KRAS-dependent ERK and S6 signaling may be required to elicit a robust response in tumor-cell viability assays.
MRTX849 Treatment In Vivo Leads to Dose-Dependent KRASG12C Modification, KRAS Pathway Inhibition, and Antitumor Efficacy
Studies were conducted to evaluate MRTX849 antitumor activity along with its pharmacokinetic and pharmacodynamic properties in vivo, both to understand the clinical utility of this agent and to provide insight toward response to treatment. MRTX849 demonstrated moderate plasma clearance and prolonged half-life following oral administration (Supplementary Table S1; Supplementary Fig. S3). To evaluate the pharmacodynamic response to MRTX849 and to correlate drug exposure with target inhibition, MRTX849 was administered via oral gavage over a range of dose levels to H358 xenograft–bearing mice, and plasma and tumors were collected at defined time points. The fraction of covalently modified KRASG12C protein was proportional to the plasma concentration of MRTX849 (Fig. 2A). When evaluated over time after a single oral dose at 30 mg/kg, the modified fraction of KRASG12C was 74% at 6 hours after dose and gradually decreased to 47% by 72 hours (Fig. 2B). This extended pharmacodynamic effect, despite declining levels of MRTX849 in plasma, was consistent with the irreversible inhibition of KRASG12C by MRTX849 and the relatively long half-life for the KRASG12C protein (∼24–48 hours; Supplementary Table S5). The modification of KRASG12C was maximized after repeated daily dosing for 3 days at 30 mg/kg (Fig. 2B), and higher dose levels did not demonstrate additional KRASG12C modification in multiple tumor models (data not shown). The maximum level of modification of approximately 80%, despite increasing dose and plasma levels of MRTX849, suggests that accurate measurement of complete inhibition of KRASG12C utilizing LC/MS may not be attainable, potentially due to a pool of active, noncycling, or unfolded KRASG12C protein in tumors. Together, these studies demonstrated a dose-dependent increase in covalent modification of KRASG12C by MRTX849 and that the majority of targetable KRAS was covalently modified by MRTX849 over a repeated administration schedule at dose levels at or exceeding 30 mg/kg.
MRTX849 modifies KRASG12C and inhibits KRAS signaling and tumor growth in vivo. A, MRTX849 was administered orally as a single dose to mice bearing established H358 xenografts (average tumor volume ∼350 mm3) at 10, 30, and 100 mg/kg. KRAS modification and MRTX849 plasma concentration data from n = 3 mice are shown as mean ± SD. KRASG12C modification was statistically significant versus vehicle control using the two-tailed Student t test. **, P < 0.01. B, MRTX849 was administered orally as a single dose or daily (QD) for 3 days to mice bearing established H358 xenografts (average tumor volume ∼350 mm3) at 30 mg/kg. Plasma was collected at 0.5, 2, 6, 24, 48, and 72 hours after administration of the last dose, and tumors were collected at 6, 24, 48, and 72 hours after dose. KRASG12C modification and MRTX849 plasma concentration data are shown from n = 3 mice as mean ± SD. Induction of modified KRASG12C protein at all time points was determined to be statistically significant versus vehicle control using two-way ANOVA. In addition, induction of modified KRASG12C protein at 72 hours in day 1 samples and 48 and 72 hours in day 3 samples was statistically significant versus the 6-hour time point. Brackets indicate P < 0.05 as compared with left-most sample. C, MRTX849 was administered as in A. Tumors were collected 6 hours after dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed by immunoblot and quantified by densitometric analysis. Relative fluorescence intensity of pERK1/2 and pS6 was normalized by dividing pERK1/2 and pS6 by total ERK1/2 and total S6, respectively. Vehicle-treated tumors were normalized to 1 by dividing all average values by the vehicle value. Average pERK1/2 and pS6 values were divided by the average value in vehicle-treated tumors. Data shown represent the average of 2 to 3 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity was determined to be statistically significant versus vehicle control using the two-tailed Student t test. Brackets indicate P < 0.05 compared with left-most sample. D, MRTX849 was administered as in B. Tumors were collected at 6, 24, 48, or 72 hours after administration of the last dose, and total and phosphorylated ERK1/2 and total and phosphorylated S6 were analyzed as in C. Data shown represent the average of 3 to 4 tumors per treatment group plus SD. Reduction of pS6 relative fluorescence intensity on day 3 was determined to be statistically significant versus vehicle control using two-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. E, MRTX849 was administered via daily oral gavage at the doses indicated to mice bearing established MIA PaCa-2 xenografts. Dosing was initiated when tumors were approximately 350 to 400 mm3.MRTX849 was administered to mice daily until day 16. Data are shown as mean tumor volume ± SEM. Tumor volumes at day 16 were determined to be statistically significant versus vehicle control via two-tailed Student t test. **, P < 0.01; *, P < 0.05.
To evaluate the effect of MRTX849 on KRAS-dependent signal transduction in vivo, a single dose of MRTX849 at 10, 30, or 100 mg/kg was administered to H358 tumor–bearing mice. Dose-dependent inhibition of ERK1/2 and pS6S235/36 phosphorylation was observed at 6 hours after dose based on immunoblot and densitometric analysis (Fig. 2C). MRTX849 also demonstrated marked inhibition of ERK1/2 and S6S235/36 phosphorylation after one or three daily doses at 6 or 24 hours, and levels gradually recovered by 72 hours after the final dose (Fig. 2D). pERK1/2 and pS6S235/36 were further evaluated in formalin-fixed, paraffin-embedded sections from vehicle-treated and MRTX849-treated xenografts in four tumor models utilizing IHC methods coupled with image analysis algorithms. These studies demonstrated increased pERK1/2 and pS6 in nontumor/stromal cells following MRTX849 administration, indicating that immunoblotting studies with bulk tumor lysate likely underrepresent the degree of pathway inhibition in tumor cells, whereas IHC-based evaluation may more accurately reflect both the degree and spatial impact of pathway inhibition. Maximal inhibition was observed for both ERK and S6S235/36 phosphorylation after a single dose at the 6-hour time point, with a rebound in signaling evident 24 hours after single dose in each model (Supplementary Fig. S4). Marked inhibition of ERK phosphorylation was observed at 6 hours after administration, with 89%, 94%, and 94% inhibition observed compared with vehicle controls in MIA PaCa-2, H1373, and H2122 tumors, respectively (H358 pERK not quantifiable). This indicates that dose levels at or exceeding 30 mg/kg dose maximized inhibition of ERK phosphorylation in multiple models (Supplementary Fig. S4A and S4B). Inhibition of S6 phosphorylation at 6 hours was more variable, with percent inhibition values of 76%, 50%, 86%, and 56% observed in MIA PaCa-2, H1373, H358, and H2122 tumors, respectively (Supplementary Fig. S4B). Together, these data indicate that consistent acute (6 hours) inhibition of KRAS-dependent ERK phosphorylation was maximized in all evaluated models, whereas inhibition of S6S235/36 was more variable, presumably due to varying degrees of KRAS-independent activation of this pathway in different tumor models.
MIA PaCa-2 and H358 were selected as MRTX849-responsive tumor models, thereby enabling a high-resolution understanding of dose–response relationships. Significant, dose-dependent, antitumor activity was observed at the 3, 10, 30, and 100 mg/kg dose levels in the MIA PaCa-2 model (Fig. 2E). Evidence of rapid tumor regression was observed at the earliest post-treatment tumor measurement, and animals in the 30 and 100 mg/kg cohorts exhibited evidence of a complete response at study day 15. Dosing was stopped at study day 16, and all 4 mice in the 100 mg/kg cohort and 2 of 7 mice in the 30 mg/kg cohort remained tumor-free through study day 70 (Supplementary Fig. S5A). In a second MIA PaCa-2 study, dose-dependent antitumor efficacy was observed at the 5, 10, and 20 mg/kg dose levels, and 2 of 5 mice at the 20 mg/kg dose level exhibited complete tumor regression (Supplementary Fig. S5B). Significant dose-dependent antitumor efficacy was also observed in the H358 model, including 61% and 79% tumor regression at the 30 and 100 mg/kg dose levels, respectively, at day 22 (Supplementary Fig. S5C). MRTX849 was well tolerated, and no effect on body weight was observed at all dose levels evaluated (Supplementary Fig. S5D). These studies indicated that MRTX849 demonstrated dose-dependent antitumor efficacy over a well-tolerated dose range and that the maximally efficacious dose of MRTX849 is between 30 and 100 mg/kg/day.
MRTX849 Demonstrates Broad-Spectrum Tumor Regression in KRASG12C Cell Line and Patient-Derived Xenograft Models
To evaluate the breadth of antitumor activity across genetically and histologically heterogeneous KRASG12C-mutant cancer models, MRTX849 was evaluated at a fixed dose of 100 mg/kg/day (a dose projected to demonstrate near-maximal target inhibition in most models) in a panel of human KRASG12C-mutant cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) models. MRTX849 demonstrated tumor regression exceeding 30% volume reduction from baseline in 17 of 26 models (65%) at approximately 3 weeks of treatment (Fig. 3A; Supplementary Table S6). By comparison, MRTX849 did not exhibit significant antitumor activity at 100 mg/kg in three non–KRASG12C-mutant models (Fig. 3A; Supplementary Table S6). Together, these results indicate that KRASG12C-mutant tumors are broadly dependent upon mutant KRAS for tumor-cell growth and survival and that MRTX849 elicits antitumor activity through a KRASG12C-dependent mechanism.
Antitumor activity of MRTX849 in KRASG12C-mutant and non–KRASG12C-mutant human tumor xenograft models. A, MRTX849 was administered via oral gavage at 100 mg/kg every day to mice bearing the CDX or PDX model indicated. Dosing was initiated when tumors were, on average, approximately 250 to 400 mm3. MRTX849 was formulated as a free base and resuspended as a solution in 10% Captisol and 50 mmol/L citrate buffer, pH 5.0. The percent change from baseline control was calculated at days 19 to 22 for most models. Statistical significance was determined for each model and is shown in Supplementary Table S6. Status of mutations and alterations in key genes is shown below each model. MAF (%), percent KRASG12C-mutant allele fraction by RNA-seq; CNV, copy-number variation; * denotes very high CDK4 expression by RNA-seq and possible amplification. HER family status was determined by averaging EGFR, ERBB2, and ERBB3 RNA-seq expression for CDX (CCLE) or PDX (Crown huBase) models. Positive HER family calls denote greater than the median expression of the models tested. CDX and PDX model HER family calls were determined independently. B, Tumor-growth inhibition plots from representative xenograft models that were categorized as sensitive, partially sensitive, or treatment refractory.
Although MRTX849 exhibited marked antitumor responses in the majority of models tested, a response pattern ranging from delayed tumor growth to complete regression was observed across the xenograft panel. The response to treatment was categorized as sensitive, partially sensitive, or treatment refractory (Fig. 3B). Rank order and Pearson statistical analyses were performed to evaluate the correlation between in vitro potency (IC50 in 2-D or 3-D viability assays) and antitumor response in vivo (% regression or progression on day 22), and a significant correlation between response in cell lines compared with tumor models was not observed (Supplementary Fig. S6A and S6B). Thus, we focused on a comprehensive analysis of correlates with MRTX849 tumor response in vivo, including tumor histology, co-occurring genetic alterations, as well as baseline or drug-induced changes in expression of KRAS-related genes [RNA sequencing (RNA-seq)] and/or protein signaling networks (RPPA in 18 models, ref. 28; Supplementary Fig. S7). No individual genetic alteration, including but not limited to KRAS-mutant allele frequency, TP53, STK11, or CDKN2A, predicted the antitumor activity of MRTX849. Interestingly, baseline gene and/or protein expression of selected members of the HER family of RTKs and of regulators of early cell-cycle transition did exhibit a trend with the degree of antitumor response, suggesting these pathways may influence the response to KRAS inhibitors (Supplementary Fig. S7A). Together, these data indicate that there are no individual binary biomarkers that clearly predict therapeutic response and that the molecular complexity and heterogeneity present in distinct KRAS-mutated tumors may contribute to the response to target blockade.
MRTX849 Antitumor Activity Translates to RECIST Responses in Patients with Cancer
A 45-year-old female former smoker diagnosed with stage IV lung adenocarcinoma and refractory to multiple lines of therapy including carboplatin/pemetrexed/pembrolizumab, docetaxel, and investigational treatment with binimetinib and palbociclib was enrolled onto the MRTX849-001 phase Ib clinical trial with two bilateral lung lesions and mediastinal lymph node as target lesions. Targeted next-generation sequencing (NGS) demonstrated a KRASG12C mutation (c.34G>T). In addition, loss-of-function KEAP1 (K97M) and STK11 (E223*) mutations were detected and are predicted to be deleterious to their respective proteins. The patient was administered MRTX849 (600 mg twice a day) and had marked clinical improvement within 2 weeks, including complete resolution of baseline cough and oxygen dependency. A RECIST-defined partial response of 33% reduction of target lesions was observed at cycle 3 day 1 (45 days), and the patient continues on study (Fig. 4A).
Activity of MRTX849 in patients with lung and colon cancers. A, Pretreatment and 6-week scans of a heavily pretreated patient with a KRASG12C mutation–positive lung adenocarcinoma indicating 33% reduction of target lesions. Patient continues on study. The top plots show a coronal view, and bottom plots an axial view of CT chest images prior to MRTX849 treatment (left) and after two cycles of MRTX849 treatment (right). B, Baseline, 6-week (Cycle 2), and 12-week (Cycle 4) scans of a patient with a KRASG12C mutation–positive colon adenocarcinoma. Partial response (PR) was confirmed at Cycle 4, and patient continues on study. Four lesions (TL1–4) are shown with axial views of CT images prior to MRTX849 treatment (top), after two cycles of MRTX849 treatment (center), and after four cycles of MRTX849 treatment (bottom).
A 47-year-old female never-smoker with metastatic adenocarcinoma of the left colon who exhibited progressive disease after receiving multiple lines of systemic therapy, including FOLFOX plus bevacizumab, single-agent capecitabine, FOLFIRI plus bevacizumab, and an investigational antibody–drug conjugate, was enrolled into the MRTX849-001 phase Ib clinical trial. This patient had extensive metastases involving the liver, peritoneum, ovaries, and lymph nodes. Targeted NGS identified a KRASG12C mutation. The patient was administered MRTX849 (600 mg twice a day) and demonstrated marked clinical improvement within 3 weeks and a visible decrease in size of her umbilical Sister Mary Joseph nodule. Her carcinoembryonic antigen levels decreased from 77 ng/mLat baseline to 11 ng/mL at cycle 2 day 1 and 3 ng/mL by cycle 3 day 1 (normal range, 0–5 ng/mL). A RECIST-defined partial response with a 37% reduction of target lesions and complete response of a nontarget lesion was observed at cycle 3 day 1(day 42). Confirmatory CT scans were conducted at cycle 5, day 1 (day 84) and indicated a confirmed RECIST partial response with further reduction of target lesions at −47% from baseline (Fig. 4B). The patient remains on treatment through Cycle 6.
Temporal Effects of MRTX849 on KRAS-Dependent Signaling and Feedback Pathways and Relationship to Antitumor Activity Following Repeat Dosing in Xenograft Models
A comprehensive analysis was conducted to evaluate MRTX849-induced temporal molecular changes to further interrogate mechanisms of drug response across sensitive and partially sensitive models. To evaluate temporal changes in global gene expression, xenograft-bearing mice were administered vehicle or 100 mg/kg MRTX849, and RNA-seq was performed on tumors at 6 and 24 hours after treatment. Gene expression was evaluated at day 1 and day 5 for the sensitive models MIA PaCa-2 and H1373 to ensure sufficient tissue availability from regressing tumors, or at day 7 in the partially sensitive models H358, H2122, and H2030 to coordinate with tumor stasis plateau. The top differentially expressed gene set enrichment analysis (GSEA) hallmark gene sets, regardless of tumor response, in all five models were several KRAS-annotated gene sets confirming MRTX849 selectively inhibits multiple genes directly related to KRAS signaling. MYC, mTOR, cell cycle, and apoptosis/BCL2 pathway gene sets were also strongly differentially expressed, confirming MRTX849 broadly affected multiple well-established, KRAS-regulated pathways, several of which have proved difficult to directly inhibit with previous targeted therapies (Fig. 5A and B; Supplementary Fig. S8A—S8D). The marked impact of MRTX849 on a large number of genes that regulate cell cycle and apoptosis provides further insight into molecular mechanisms which mediate its antitumor activity.
MRTX849 treatment in vivo regulates KRAS-dependent oncogenic signaling and feedback-inhibitory pathways. A, Volcano plots displaying differentially expressed genes in xenograft tumors 24 hours after oral administration of vehicle or 100 mg/kg MRTX849 in a representative MRTX849-sensitive (H1373) and MRTX849-partially sensitive (H358) model. Significance denoted in the legend (Padj < 0.01). B, GSEA heat maps depicting hallmark signature pathways differentially regulated in at least one model 24 hours following oral administration of a single 100 mg/kg MRTX849 dose compared with vehicle. Normalized enrichment score shown in all models 6 or 24 hours after a single dose (QD × 1) or 5 (QD × 5) or 7 (QD × 7) days dosing. C, Genes that feedback-inhibit MAP kinase signaling are downregulated following MRTX849 treatment in all five cell line xenografts assessed by RNA-seq. TPM, Transcripts Per Kilobase Million.
Targeted RNA-seq analysis was performed on genes implicated in the temporal regulation of external signaling inputs and feedback pathways which collectively temper signaling flux through the RAS–RAF–MEK–ERK MAP kinase (MAPK) pathway including DUSP, SPRY, and PHLDA family genes (13, 18). These MAPK pathway–negative regulators were each ranked among the most strongly decreased genes following MRTX849 treatment, providing evidence that ERK-dependent transcriptional output is blocked and that pathways involved in reactivation of RTK- and ERK-dependent signaling were activated (Fig. 5C; Supplementary Fig. S4A).
On the basis of the observation of dynamic changes in transcriptional programs linked to KRAS pathway reactivation, IHC plus quantitative imaging of tumor cell–specific pERK and pS6 was evaluated over a range of time points. In the sensitive MIA PaCa-2 and H1373 tumor models, treatment with MRTX849 (100 mg/kg) demonstrated ≥90% inhibition of ERK phosphorylation at 6 and 24 hours on both days 1 and 5 (Supplementary Fig. S4). In contrast, in the partially sensitive H358 and H2122 models, robust inhibition of ERK phosphorylation was observed at 6 hours after a single dose; however, marked recovery of ERK phosphorylation was observed at 24 hours after single dose and at both 6 and 24 hours following 7 days of repeat-dose administration. Because DUSP, SPRY, and ETV family transcripts remain downregulated through 5 to 7 days in all models, it is evident that other independent factors contribute to temporal reactivation of ERK (Fig. 5C). Similar to what was observed with single-dose administration, the effect of MRTX849 on pS6 was variable over time and did not track with the antitumor activity of MRTX849. Together, these results suggest that the extent and duration of inhibition of pERK may track with the magnitude of antitumor efficacy of KRASG12C inhibitors and that further evaluation of the role of S6 is required to understand if it plays a role in drug sensitivity.
The effect of MRTX849 on cell proliferation and apoptosis was characterized by IHC analysis of Ki-67 or cleaved caspase-3 after a single dose or repeat administration. The fraction of Ki-67–positive cells was significantly reduced in tumors after repeat administration in all four models tested, further supporting a broadly operative antiproliferative mechanism, independent of the magnitude of MRTX849 antitumor response (Supplementary Fig. S4). Induction of apoptosis as determined by cleaved caspase-3 immunostaining was also evident on day 1 of treatment (6 and/or 24 hours after treatment) in the sensitive H358, MIA PaCa-2, and H1373 models (79%–100% maximal regression) but not in the partially sensitive H2122 model (Supplementary Fig. S4). An expanded RPPA-based pathway analysis of several models also indicated a correlation between antitumor activity of MRTX849 and decreased survivin (statistically significant at days 5/7 in 7 models evaluated; Supplementary Fig. S7B) and a trend toward increased cleaved caspase-3 induction (day 1, P = 0.08, 16 models), supporting the induction of apoptosis as a key mediator of a cytoreductive antitumor response (Supplementary Fig. S7C). Interestingly, the magnitude of reduction of MYC and cyclin B1 protein levels at days 5/7 also closely correlated with MRTX849 antitumor activity, consistent with their roles as critical regulators of KRAS-mediated cell growth and survival pathways (Supplementary Fig. S7B). Collectively, these data support that durable inhibition of ERK activity and maximal inhibition of ERK-regulated outputs including MYC and E2F-mediated transcription are associated with induction of apoptosis and maximal response to MRTX849 treatment.
CRISPR/Cas9 Screen Identifies Vulnerabilities and Modifiers of Response to MRTX849 in KRASG12C-Mutant Cancer Cell Lines In Vitro and In Vivo
The correlative analysis of genomic or proteomic markers with response to MRTX849 in the defined panel of models provided only limited insight toward mechanism of therapeutic response or resistance. Therefore, we directly interrogated the role of selected genes in mediating therapeutic response utilizing a focused CRISPR/Cas9 knockout screen targeting approximately 400 genes including many genes involved in KRAS signaling. This was conducted in H358 and H2122 cells in vitro and in H2122 xenografts in vivo in the presence and absence of MRTX849 treatment (Supplementary Fig. S9A–S9F). In MRTX849-anchored screens in vitro, single guide RNAs (sgRNA) that target RAS signaling pathway genes including MYC, SHP2 (H2122), mTOR pathway (MTOR and RPS6), and cell-cycle genes (CDK1, CDK2, CDK4/6, and RB1) were identified to affect cell fitness. sgRNAs that target KEAP1 and CBL were enriched in the H2122 model, demonstrating cell-specific genetic routes toward improved fitness through loss of classic tumor-suppressor genes, including in the context of MRTX849 treatment. KRAS sgRNA dropout was less pronounced in the MRTX849-treated cells compared with DMSO control–treated cells, as would be expected with redundant depletion of the drug target (Supplementary Fig. S9C and S9D). To evaluate whether a distinct KRAS dependence or modulation of MRTX849 therapeutic response was observed in vitro versus in vivo, xenograft-bearing mice bearing H2122 cells (∼250 mm3) transduced with the sgRNA library were orally administered vehicle or MRTX849 for 2 weeks (Supplementary Fig. S9A, S9E, and S9F). In MRTX849-treated xenografts, sgRNAs targeting cell cycle, SHP2, MYC, and mTOR pathway genes remained among the top depleted sgRNAs, demonstrating that inhibition of these targets in vivo, in the context of KRAS inhibition, leads to further tumor-growth inhibition over and above the effects of KRAS inhibition alone (Supplementary Fig. S9E and S9F). sgRNAs targeting the tumor suppressor KEAP1 were enriched in MRTX849-treated xenografts, suggesting loss of KEAP1 may represent a mechanism of intrinsic or acquired resistance. Interestingly, NRAS was one of the top enriched genes in the vehicle-treated xenografts, suggesting NRAS functions as a tumor suppressor in this context; however, enrichment was not as pronounced in the MRTX849-treated xenografts, suggesting NRAS may compensate for KRAS in the context of KRAS inhibition (Supplementary Fig. S9F). Collectively, these data demonstrate the importance of selected proteins that regulate RTK- and RAS-dependent signaling and cell-cycle transition in mediating the oncogenic effects of mutant KRAS, and also provide a catalog of potentially druggable vulnerabilities that complement KRAS blockade.
Cancer Therapeutic Combination Screen to Identify Rational and Clinically Tractable Strategies to Address Feedback and Resistance Pathways
To further interrogate pathways that mediate the antitumor response to MRTX849 and to identify combinations capable of enhancing response to MRTX849, a combination screen was conducted in vitro using a focused library of small-molecule inhibitors across a panel of cell lines (Supplementary Fig. S10A and S10B; Supplementary Table S8). Approximately 70 compounds targeting relevant pathways (RTKs, MAPK/ERK, PI3K, mTOR, cell cycle) were tested in a 3- or 7-day viability assay, and synergistic combinations were identified and ranked. Multiple hits from this screen were then identified for additional evaluation in combination studies with MRTX849, including the HER family inhibitor afatinib, the CDK4/6 inhibitor palbociclib, the SHP2 inhibitor RMC-4550, and mTOR pathway inhibitors.
Combination Strategies That Target Upstream Signaling Pathways Implicated in Extrinsic Regulation of KRAS Nucleotide Cycling and Feedback/Bypass Pathways
MRTX849 in combination with HER family inhibitors synergistically inhibited tumor-cell viability in the majority of cell lines evaluated and were the top hit in the combination screen in vitro (Supplementary Fig. S10). Cell lines with the highest (top 50th percentile) average composite baseline RNA expression values of selected HER family members exhibited the highest synergy scores to these combinations (Supplementary Fig. S11A). Afatinib was selected as a prototype HER family inhibitor based on its broad in vitro combination activity. Combination studies were conducted with MRTX849 and afatinib in five tumor models that were partially sensitive or treatment refractory to single-agent MRTX849. The MRTX849 and afatinib combination demonstrated significantly greater antitumor efficacy compared with either single agent in all five models evaluated, including multiple models exhibiting complete or near-complete responses to the combination (Fig. 6A; Supplementary Fig. S11B).
HER family and SHP2 inhibitor combinations further inhibit KRAS signaling and exhibit increased antitumor responses. A, MRTX849 at 100 mg/kg, afatinib at 12.5 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or KYSE-410 cell line xenografts (n = 5). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.01. B, Quantification of KRAS mobility shift and pERK in H2122 cells treated for 24 hours with MRTX849 (0.1–73 nmol/L), afatinib (200 nmol/L), or the combination assessed by Western blot densitometry. C, MRTX849 at 100 mg/kg, afatinib at 12.5 mg/kg, or the combination was administered once or daily for 7 days via oral gavage to mice bearing H2122 cell line xenografts (n = 3/group). Tumors were harvested at 6 and 24 hours following the final dose. Tumor sections were stained for pERK and pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK or pS6 staining intensity was determined to be statistically significant relative to vehicle or either single agent using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. D, Quantitation of KRAS band shift and pERK after 24-hour treatment with MRTX849 (0.1–73 nmol/L), RMC-4550 (1 μmol/L), or the combination in H358 cells assessed by Western blot densitometry. E, MRTX849 at 100 mg/kg, RMC-4550 at 30 mg/kg, or the combination was administered daily via oral gavage to mice bearing the KYSE-410 or H358 cell line xenografts (n = 5/group). Combination treatment led to a statistically significant reduction in tumor growth compared with either single agent on the last day of dosing. *, Padj < 0.05. F, MRTX849 at 100 mg/kg, RMC-4550 at 30 mg/kg, or the combination was administered via oral gavage to mice bearing KYSE-410 cell line xenografts (n = 3/group), and tumors were harvested at 6 and 24 hours post-dose. Tumor sections were stained with pERK or pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK staining intensity was determined to be statistically significant relative to RMC-4550 alone using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample.
To evaluate whether afatinib affected covalent modification of KRASG12C by MRTX849, partially sensitive H2122 cells were treated with increasing concentrations of MRTX849 alone or in the presence of afatinib (200 nmol/L, IC90), and the mobility shift in KRAS protein was densitometrically determined from immunoblots. A clear shift in the concentration response to MRTX849 was apparent in the presence of afatinib, indicating that the combination increased the fraction of modified KRASG12C consistent with the putative role of HER family receptors in extrinsic regulation of KRASG12C GTP loading (Fig. 6B). The concentration–response relationship for inhibition of ERK phosphorylation was also clearly shifted in the presence of afatinib. To further evaluate the effect of the combination on KRAS-dependent signaling, four cell lines (H2030, H2122, H358, and KYSE-410) were treated with a range of MRTX849 concentrations in the presence or absence of afatinib for 6 or 24 hours, and key signaling molecules were evaluated by RPPA. Afatinib demonstrated clear inhibition of EGFR (pY1068) and HER2 (pY1248) activity and partial inhibition of ERK, AKT (S473), and p70S6K phosphorylation at both time points (Supplementary Fig. S11C). The effect of afatinib on S6 (S235/236, S240/244) and p90 RSK (S380) phosphorylation was variable and exhibited only minimal inhibition in most of the cell lines evaluated. The combination of afatinib and MRTX849 demonstrated markedly enhanced concentration-dependent inhibition and/or a greater magnitude of effect on ERK, RSK, p70 S6K, and S6 (S235/236) phosphorylation compared with MRTX849 alone at both 6 and 24 hours. Of note, neither afatinib nor MRTX849 alone inhibited S6 phosphorylation at the S240/244 site regulated by mTOR/S6K, whereas the combination demonstrated marked inhibition at 24 hours.
In vivo, the combination also exhibited a trend toward increased pERK and pS6 (S235/236) inhibition in the partially sensitive H2122 model in combination groups as determined by quantitation of immunostaining after 1- or 7-day administration (Fig. 6C). Similar results were observed in the MRTX849-refractory KYSE-410 model, and the combination also increased the number of apoptotic cells in this model (Supplementary Fig. S12A–S12C). Collectively, these data indicate that upstream baseline HER family activation may limit the ability of MRTX849 to achieve robust inhibition of the ERK and mTOR–S6 signaling pathways. Accordingly, the combination of afatinib and MRTX849 can limit feedback reactivation of ERK and demonstrate complementary inhibition of AKT–mTOR–S6 signaling, resulting in significantly improved antitumor activity.
SHP2 inhibition has been shown to inhibit the growth of cells that harbor KRASG12C mutations, and this effect is likely mediated, in part, by decreasing KRAS GTP loading (25–27). To evaluate whether SHP2 inhibition enhanced covalent modification of KRASG12C by MRTX849, H358 and H2122 cells were incubated with increasing MRTX849 concentrations with or without the SHP2 inhibitor RMC-4550. In both cell lines, cotreatment with RMC-4550 (1 μmol/L, IC90) demonstrated a MRTX849 concentration-dependent increase in KRASG12C protein modification and a concomitant decrease in ERK phosphorylation compared with MRTX849 alone (Fig. 6D; Supplementary Fig. S13A). RPPA analysis of KRAS-dependent signaling was conducted at 6 or 24 hours after treatment in three cell lines (H358, H2030, H2122) over a range of MRTX849 concentrations in the presence or absence of RMC-4550. RMC-4550 demonstrated robust inhibition of ERK phosphorylation and partial inhibition of p90 RSK (S380) and p70 S6K (T412) at both time points (Supplementary Fig. S13B). The combination of RMC-4550 and MRTX849 demonstrated incrementally increased concentration-dependent inhibition of ERK and RSK phosphorylation in all cell lines at both 6 and 24 hours and markedly improved inhibition of S6 (S235/236) phosphorylation compared with MRTX849 alone in H2122 and H358 cells at 24 hours. In addition, the combination demonstrated near-complete inactivation of KRAS in MRTX849-refractory KYSE-410 xenografts as determined using an active RAS ELISA assay, and this was significant compared with single agents (Supplementary Fig. S13C). On the basis of these findings, combination studies were conducted with MRTX849 and RMC-4550 in six KRASG12C-mutated tumor models in vivo, and the combination demonstrated significantly greater antitumor efficacy compared with either single agent in 4 of 6 models evaluated (Fig. 6E; Supplementary Fig. S13D). Consistent with the in vitro data, the combination also demonstrated a significant decrease in ERK phosphorylation compared with either single agent in the KYSE-410 model as determined by quantitation of tumor-cell immunostaining on day 1 at 6 and 24 hours and day 7 at 6 hours after dose (Fig. 6F). Together, these data indicate that EGFR family and SHP2 blockade can augment the antitumor activity of KRASG12C inhibitors through enhancing covalent target modification and establishing a more comprehensive blockade of KRAS-dependent signaling.
Combinations That Inhibit Bypass Pathways Downstream of KRAS and Exhibit Increased Antitumor Activity in Xenograft Models
KRAS is implicated in regulation of the oncogenic S6 protein translation pathway through both ERK-dependent activation of RSK, which phosphorylates S6 at Ser235/236, and cross-talk with the PI3K and mTOR pathway that additionally phosphorylates S6 at Ser240/244 (29). However, the S6 pathway can also be activated independently of mutated KRAS in tumor cells through hyperactivated RTK signaling, PI3K activation, or STK11 mutations, each of which converge on mTOR-mediated activation of S6. In the in vitro combination screen, mTOR inhibitors demonstrated synergy in a subset of evaluated cell lines (Supplementary Fig. S14A). To further evaluate the effect of the combination on KRAS and mTOR pathway–dependent signaling, four cell lines were treated with MRTX849 in the presence or absence of the selective ATP-competitive mTOR inhibitor vistusertib (1 μmol/L), for 6 or 24 hours, and several signaling molecules were evaluated by RPPA. Vistusertib demonstrated clear and robust inhibition of several components of the PI3K–mTOR signaling pathway including AKT (S473), p70 S6K (T412), S6 (pS235/236, S240/244), and 4E-BP1 (S65, T70) phosphorylation in each cell line at both time points consistent with its mechanism of action (Supplementary Fig. S14B). MRTX849 alone did not affect 4E-BP1 or S6 (S240/244) activity, and it exhibited a variable and cell line–dependent effect on p70 S6K and S6 (pS235/236) phosphorylation in these cell lines. Vistusertib also demonstrated marked induction of ERK phosphorylation, often several-fold over vehicle control, at both time points in all four cell lines, consistent with prior reports (30). The combination of vistusertib and MRTX849 demonstrated a comparable level of inhibition of ERK phosphorylation compared with single-agent MRTX849, indicating that the activation of ERK signaling by vistusertib was impeded by the combination of the two agents. In addition, MRTX849 combined with vistusertib further inhibited p70 S6K and AKT S473 phosphorylation compared with either single agent. Near-complete inhibition of S6 (S235/236, S240/244) phosphorylation at limit of detection was observed for the combination in each cell line at evaluated time points.
Consequently, a cohort of tumor models was identified, and the combination of MRTX849 with the selective mTOR inhibitor vistusertib demonstrated marked tumor regression and significantly improved antitumor activity compared with either single agent in all six models evaluated (Fig. 7A; Supplementary Fig. S14C). MRTX849 in combination with a second, differentiated mTOR inhibitor, everolimus, which inhibits TORC1 but not TORC2, in the H2030 xenograft model also demonstrated a striking combination effect (Supplementary Fig. S14D). In the KRASG12C, STK11-mutant H2030 model, MRTX849 demonstrated marked inhibition of ERK phosphorylation through 24 hours, but exhibited only partial inhibition of pS6235/36 at 6 hours after dose, on days 1 and 7 (Fig. 7B and C). Vistusertib demonstrated marked inhibition of pS6235/36 at 6 hours after treatment with evidence of recovery by 24 hours. The combination of vistusertib and MRTX849 did not have a further effect on ERK phosphorylation but demonstrated a significant reduction in pS6235/36 on day 1 at 24 hours compared with vistusertib alone and a trend toward reduced pS6235/36 on both day 1 and day 7 at 6 hours compared with either single agent (Fig. 7B; Supplementary Fig. S14E). Together, these data indicate that MRTX849 and mTOR inhibitor combination demonstrates complementary inhibition of the ERK and mTOR–S6 signaling pathways, resulting in broad antitumor activity in KRASG12C-mutant tumor models.
CDK4/6 and mTOR combinations suppress independently hyperactivated downstream pathways and exhibit increased antitumor responses. A, MRTX849 at 100 mg/kg, vistusertib at 15 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or H2030 cell line xenografts (n = 5/group). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.05. B, MRTX849 at 100 mg/kg, vistusertib at 15 mg/kg, or the combination was administered once or daily for 7 days via oral gavage to mice bearing H2030 cell line xenografts (n = 3/group). Tumors were harvested at 6 and 24 hours following the final dose. Tumor sections were stained with pERK and pS6 via IHC methods. Quantitation of images shown by H-score in tumor tissue. Reduction of pERK or pS6 staining intensity was determined to be statistically significant relative to vehicle or either single agent using one-way ANOVA. Brackets indicate P < 0.05 compared with left-most sample. C, Protein Western blot analysis of KRAS pathway targets in H2030 xenografts treated with MRTX849 (100 mg/kg), vistusertib (15 mg/kg), or the combination, 6 or 24 hours after a single dose. D, Protein Western blot analysis of KRAS pathway and cell-cycle targets in H2122 cells treated for 24 hours with MRTX849, palbociclib, or the combination. E, Normalized RNA-seq gene-expression data on E2F targets in H2122 xenografts treated with MRTX849, palbociclib, or the combination, 6 and 24 hours after a single daily dose or seven daily doses. F, MRTX849 at 100 mg/kg, palbociclib at 130 mg/kg, or the combination was administered daily via oral gavage to mice bearing the H2122 or SW1573 cell line xenografts (n = 5). Combination treatment led to a statistically significant decrease in tumor growth compared with either single-agent treatment. *, Padj < 0.05.
Signaling through KRAS is known to mediate cell proliferation, at least in part, through the regulation of the cyclin D family and triggering RB/E2F-dependent entry of cells into cell cycle. Loss-of-function mutations and homozygous deletions in the cell-cycle tumor suppressor CDKN2A (p16) are coincident in a subset of KRAS-mutant non–small cell lung cancer (NSCLC) and hyperactivate CDK4/6-dependent RB phosphorylation and cell-cycle transition. In the CDKN2A-null H2122 and SW1573 cell lines in vitro, MRTX849 demonstrated concentration-dependent partial inhibition of RB phosphorylation (pRB pS807/811) and concurrent increase in p27 in H2122 cells, but not SW1573 cells, at 24 hours (Fig. 7D; Supplementary Fig. S15A). MRTX849 in combination with the CDK4/6 inhibitor palbociclib (1 μmol/L) demonstrated near-complete inhibition of pRB in both H2122 and SW1573 cells and further induced p27 in H2122 cells. Interestingly, pS6 (S235/236) was also much more effectively suppressed by the combination in both H2122 and SW1573 cells, which is consistent with a recent report (31). RNA expression of target genes and RPPA analysis of target protein signaling events were also used as a readout of cell-cycle inhibition in the H2122 tumor model in vivo, and the combination of MRTX849 and palbociclib significantly inhibited E2F1 and selected E2F family target genes, induced p27 protein expression to a greater degree compared with either single agent, and further reduced the number of Ki-67–positive cells after 7 days of administration (Fig. 7E; Supplementary Fig. S15B and S15C). In addition, the combination demonstrated a significant decrease in pRB (S780) compared with either single agent after 7 days of administration in SW1573 tumors in vivo (Supplementary Fig. S15D). This combination also induced tumor regression in five tumor xenograft models that was significant compared with either single-agent control (Fig. 7F; Supplementary Fig. S15E). Although not significant, a trend was noted in which models with CDKN2A homozygous deletion exhibited an increased antitumor response to the combination of MRTX849 and CDK4/6 inhibition compared with models lacking evidence of genetic dysregulation of key cell-cycle genes (Supplementary Fig. S15F and S15G).
Discussion
The identification of MRTX849 as a highly selective KRASG12C inhibitor capable of near-complete inhibition of KRAS in vivo provides a renewed opportunity to better understand the role of this mutation as an oncogenic driver in various cancers and to guide rational clinical trial design. The lack of a significant correlation between sensitivity to MRTX849 antitumor activity in in vitro versus in vivo model systems made it necessary to further study KRAS oncogene dependence in tumor models in vivo, a more clinically relevant setting. The demonstration that MRTX849 exhibited significant antitumor efficacy in all evaluated KRASG12C-mutated cancer models and demonstrated marked regression in the majority (65%) confirms that this mutation is a broadly operative oncogenic driver and that MRTX849 represents a compelling therapeutic opportunity. This evidence of activity extended to patients, as demonstrated by RECIST partial responses in 2 patients enrolled in a phase I clinical trial of MRTX849. Collectively however, these data also illustrate that the degree of dependence of cancer cells on the presence of a KRASG12C mutation for growth and survival can vary across tumors and that co-occurring genetic alterations observed in KRAS-mutated cancers may influence response to direct targeted therapy. The further observation that KRAS mutations occur across different cancers and that no single co-occurring genetic alteration predicted response to treatment illustrates the genetic heterogeneity of KRAS-driven cancers. Findings in the present studies are consistent with other functional genomics or therapeutic strategies to block KRAS function across panels of cell lines or models which demonstrated a highly significant response of KRAS-mutant cells to target knockdown, a heterogeneous magnitude of response, and no clear co-occurring aberrations that predict resistance to target blockade (5, 32, 33). Interestingly, despite the implication that certain mutations that co-occur with KRAS including TP53, STK11, and KEAP1 may limit therapeutic response in KRASG12C-positive lung cancers, none of these mutations correlated with response or resistance in the cell-line panel. In addition, the partial response we reported in the patient with lung adenocarcinoma was observed in a patient harboring deleterious comutations in both STK11 and KEAP1. Together, these data further illustrate the heterogeneity and complexity of KRAS-mutated cancers and suggest that no binary co-occurring genetic event may be predictive of therapeutic response.
Temporal and dose–response analysis indicated maximal modification of KRASG12C and durable inhibition of KRAS-dependent signaling was important in maximizing therapeutic response. The recovery of ERK signaling and the inability to inhibit mTOR–S6 signaling despite continued treatment were each associated with transient or submaximal response to MRTX849. ERK1/2 is implicated in direct phosphorylation and negative feedback regulation of EGFR (T669), FGFR1 (S777), and SOS1, and each of these targets may facilitate KRASG12C-independent resetting of ERK signaling flux (34–36). The rapid and remarkable suppression of ERK pathway–regulated transcripts such as DUSP and SPRY/SPRED family members by MRTX849 in all models evaluated is consistent with that observed for RAF inhibitors and is implicated in reactivation of ERK and RTK signaling (18, 19). The dual-specificity phosphatases DUSP4 and 6 were strongly suppressed by MRTX849 and are implicated in dephosphorylating and inactivating ERK1/2 (14, 18, 37), whereas SPRY family members are implicated in the negative regulation of RTKs and adaptor proteins (e.g., GRB2), and may participate in modifying RAS family nucleotide exchange and effector binding (e.g., RAF1; ref. 38). Although suppression of DUSP and SPRY/SPRED was broadly observed in all models, the magnitude of signaling reactivation and response to MRTX849 varied across models. This suggests some tumor models harbor additional factors that bypass KRAS dependence or affect RAS pathway signaling flux, such as expression or activation of selected RTKs (e.g., ERBB2 amplification in the KYSE-410 model) or STK11 loss-of-function mutations, and may be primed for feedback reactivation of RAS-dependent signaling and/or limit the degree of signaling inhibition by MRTX849. This phenomenon was observed for BRAFV600E-mutant colon cancer (but not melanoma) which exhibits high baseline EGFR expression, is primed for rapid feedback activation of this RTK, and is resistant to single-agent inhibition but highly responsive to cotargeting BRAF (and/or MEK) and EGFR (20). In addition, blockade of BRAF or MEK1/2 resulted in feedback-mediated activation of the PI3K–mTOR signaling pathway in concert with the coactivation of upstream RTKs (e.g., EGFR), resulting in bypass of ERK pathway dependence and therapeutic resistance (17, 20, 39). The observations that baseline expression of HER family RTKs trended with MRTX849 antitumor activity and that CRISPR-based drug-anchored screens implicated EGFR, SHP2, and mTOR–S6 pathways as cotargetable vulnerabilities both support the hypothesis that these targets act as conditional response modifiers.
Activation of RTK signaling in the context of KRASG12C-mutant cancer was predicted to limit MRTX849 therapeutic response both by enhancing extrinsic regulation of GTPase activity and initiating KRAS-independent ERK and mTOR–S6 pathway activation. Therefore, HER family and SHP2 inhibition were employed as strategies to either block the critical RTK family in KRAS-mutant cells or block collective RTK signaling downstream, respectively. As MRTX849 binds only GDP-KRASG12C, both HER family and SHP2 inhibition each enhanced KRASG12C modification by MRTX849 and significantly improved antitumor activity. This observation is consistent with the putative role of activated RTKs in the engagement of SHP2 to mediate SOS1-dependent RAS GTP loading and to diminish RAS GAP activity, each of which converge on enhanced RAS activation state (40). The afatinib combination demonstrated a clear and marked inhibition of both the ERK–RSK and AKT–mTOR–S6 signaling pathways, whereas the SHP2 inhibitor combination demonstrated a clear impact on ERK–RSK signaling and a relatively less prominent impact on mTOR–S6 signaling. Although afatinib may more effectively address mTOR–S6 bypass signaling, SHP2 inhibition should be an effective combinatorial strategy to combat other RTKs outside of the HER family, such as FGFRs or MET, that could affect KRAS dependence. To further address bypass signaling mediated by RTK activation or STK11 mutations, each of which activate the mTOR–S6 signaling pathway independently of KRAS, mTOR inhibition in combination with MRTX849 was also evaluated. MRTX849 in combination with vistusertib, in fact, demonstrated significantly improved antitumor activity in vivo compared with either single agent in all six tumor models evaluated, regardless of STK11 mutational status. Consistent with the mechanism of action of vistusertib, comprehensive inhibition of AKT–mTOR–S6 signaling was observed for vistusertib alone and near-complete inhibition of pS6S235–36 and pS6240–44 was observed in combination. In addition, the marked feedback reactivation of ERK by vistusertib was relieved by the combination. The induction of ERK activity has been observed in tumor cells following mTORC1 inhibition by rapalogs or ATP-competitive inhibitors and has been implicated in limiting antitumor activity of this class of agents (30, 41, 42), supporting the suppression of ERK signaling by MRTX849 as a key mechanism of response to the combination. Notably, all three combination strategies converge on more comprehensive inhibition of KRAS-dependent signaling, converging on ERK and S6 activity. In addition, although the inhibition of the AKT–mTOR–S6 pathway did not correlate with model response to MRTX849 (potentially due to tumor heterogeneity), the observations that both MTOR and RPS6 drop out in drug-anchored CRISPR screens and that effective combination strategies more comprehensively block this pathway illustrate its likely importance in maximizing therapeutic response in KRAS-mutated cancers.
Cell-cycle dysregulation due to genetic alterations in cell-cycle regulators identified additional factors that could modify the therapeutic response to MRTX849. In addition, CDKN2A, RB1, CDK4, and CDK6 were all identified as gene targets that affected cell fitness in CRISPR screens. Genetic alterations including homozygous deletion of CDKN2A or amplification of CDK4 or CCND1 comprise up to 20% of KRAS-mutated NSCLC (43). Combination studies with MRTX849 and palbociclib in vivo demonstrated more comprehensive inhibition of RB and E2F family target genes and increased antitumor activity compared with either single agent in NSCLC models. In addition, these studies indicated that the combination resulted in more effective inhibition of S6 (S235/236) phosphorylation, establishing a previously unappreciated connection between cell-cycle blockade and protein translation pathways. Notably, this combination was especially effective in CDKN2A-deleted models, suggesting that this combination strategy may be primarily beneficial in a molecularly defined subset of patients characterized by decoupling of cell-cycle regulation from KRAS.
Collectively, models exhibiting a cytoreductive response to single-agent MRTX849 demonstrated a more comprehensive and durable inhibition of KRAS-dependent signaling and induction of an apoptotic response. These data suggest that maintaining durable inhibition of KRAS-dependent signaling below a defined threshold is required to elicit tumor regression. The elucidation of mechanisms that limit the therapeutic response to single-agent KRAS inhibition has provided insight toward strategies to enhance therapeutic activity in KRAS-mutant tumors. Of the 35% of models (9/26) that did not exhibit durable regression with single-agent MRTX849 treatment, five models (KYSE410, SW1573, H2122, H2030, and LU6405) were selected for rational combination studies, and at least one combination demonstrated significant improvement in antitumor efficacy and elicited a >50% tumor regression in all five models evaluated. These results suggest that essentially all KRASG12C-mutated cancers can derive clinical benefit from direct KRAS inhibitor–directed therapy either alone or in combination. Furthermore, rational pathway-centric combination regimens directed at hallmark signaling nodes may be directed to genetically defined patient subsets. For example, KRAS-mutated NSCLC exhibits mutually exclusive, co-occurring genetic alterations in STK11 and CDKN2A (43). The present data suggest that KRASG12C/STK11-mutated NSCLC could be readily addressed by combining a KRASG12C inhibitor with an RTK or mTOR inhibitor, whereas KRASG12C/CDKN2A-mutated NSCLC could be more effectively addressed by combination with a CDK4/6 inhibitor. Collectively, the present studies support the broad utility of covalent KRASG12C inhibitors in treating KRASG12C-mutated cancers and provide defining strategies to identify patients likely to benefit from single-agent therapy or rationally directed combinations.
UPDATED 02/07/2021
The November 1st issue of Science highlights a series of findings which give cancer researchers some hope in finally winning a thirty year war with the discovery of drugs that target KRAS, one of the most commonly mutated oncogenes (25% of cancers), and thought to be a major driver of tumorigenesis. Once considered an undruggable target, mainly because of the smooth surface with no obvious pockets to fit a drug in, as well as the plethora of failed attempts to develop such an inhibitor, new findings with recently developed candidates, highlighted in this article and other curated within, are finally giving hope to researchers and oncologists who have been hoping for a clinically successful inhibitor of this once considered elusive target.
Figure 1Mechanism of Action of ARS853 showing that the inhibitors may not need bind to the active conformation of KRAS for efficacy
Abstract: Two recent studies evaluated a small molecule that specifically binds to and inactivates the KRAS G12C mutant. The new findings argue that the perception that mutant KRAS is persistently frozen in its active GTP-bound form may not be accurate.
Although the development of the KRASG12C-specific inhibitor, compound 12 (Ostrem et al., 2013), was groundbreaking, subsequent studies found that the potency of compound 12 in cellular assays was limited (Lito et al., 2016, Patricelli et al., 2016). A search for more-effective analogs led to the development of ARS853 (Patricelli et al., 2016), which exhibited a 600-fold increase of its reaction rate in vitro over compound 12 and cellular activities in the low micromolar range.
A Summary and more in-depth curation of the Science article is given below:
Cancer researchers are making progress toward a goal that has eluded them for more than 30 years: shrinking tumors by shutting off a protein called KRAS that drives growth in many cancer types. A new type of drug aimed at KRAS made tumors disappear in mice and shrank tumors in lung cancer patients, two companies report in papers published this week. It’s not yet clear whether the drugs will extend patients’ lives, but the results are generating a wave of excitement. And one company, Amgen, reports an unexpected bonus: Its drug also appears to stimulate the immune system to attack tumors, suggesting it could be even more powerful if paired with widely available immunotherapy treatments.
In 2013, Dr. Kevan Shokat’s lab at UCSF discovered a small molecule that could fit in the groove of the KRAS mutant G12C. The G12C as well as the G12D is a common mutation found in KRAS in cancers. KRAS p.G12C mutations predominate in NSCLC comprising 11%–16% of lung adenocarcinomas (45%–50% of mutant KRAS is p.G12C) (Campbell et al., 2016; Jordan et al., 2017), as well as 1%–4% of pancreatic and colorectal adenocarcinomas, respectively (Bailey et al., 2016; Giannakis et al., 2016). This inhibitor was effective in shrinking, in mouse studies conducted by Wellspring Biosciences, implanted tumors containing this mutant KRAS.
Amgen press release on AMG510 Clinical Trial at ASCO 2019
THOUSAND OAKS, Calif., June 3, 2019 /PRNewswire/ — Amgen (NASDAQ: AMGN) today announced the first clinical results from a Phase 1 study evaluating investigational AMG 510, the first KRASG12C inhibitor to reach the clinical stage. In the trial, there were no dose-limiting toxicities at tested dose levels. AMG 510 showed anti-tumor activity when administered as a monotherapy in patients with locally-advanced or metastatic KRASG12C mutant solid tumors. These data are being presented during an oral session at the 55th Annual Meeting of the American Society of Clinical Oncology (ASCO) in Chicago.
“KRAS has been a target of active exploration in cancer research since it was identified as one of the first oncogenes more than 30 years ago, but it remained undruggable due to a lack of traditional small molecule binding pockets on the protein. AMG 510 seeks to crack the KRAS code by exploiting a previously hidden groove on the protein surface,” said David M. Reese, M.D., executive vice president of Research and Development at Amgen. “By irreversibly binding to cysteine 12 on the mutated KRAS protein, AMG 510 is designed to lock it into an inactive state. With high selectivity for KRASG12C, we believe investigational AMG 510 has high potential as both a monotherapy and in combination with other targeted and immune therapies.”
The Phase 1, first-in-human, open-label multicenter study enrolled 35 patients with various tumor types (14 non-small cell lung cancer [NSCLC], 19 colorectal cancer [CRC] and two other). Eligible patients were heavily pretreated with at least two or more prior lines of treatment, consistent with their tumor type and stage of disease.
Canon, J., Rex, K., Saiki, A.Y. et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature 575, 217–223 (2019) doi:10.1038/s41586-019-1694-1
Besides blocking tumor growth, AMG510 appears to stimulate T cells to attack the tumor, thus potentially supplying a two pronged attack to the tumor, inhibiting oncogenic RAS and stimulating anti-tumor immunity.
Nearly four decades have passed since researchers first identified the RAS gene family, which includes HRAS, NRAS and KRAS. RAS is the most frequently mutated family of oncogenes – or potentially cancerous genes – in human cancers.1,2 While research efforts have been able to identify and develop treatments for other driver gene mutations that contribute to cancer growth, success with treating KRAS, the most frequently mutated variant of the RAS family, has remained elusive.2 But now there is hope.
Amgen, a leading biotechnology company, has taken on one of the toughest challenges of the last 40 years in cancer research.3 Chemical biologist Kevan Shokat’s lab at the University of California, San Francisco, identified a small molecule that could slip into a groove on a KRAS mutation called G12C in 2013.4 Building on their own research strategies and this new insight, scientists at Amgen used structural biology and medicinal chemistry to identify an adjacent groove, and by November 2017, made the initial decision to advance the molecule that would become investigational sotorasib.5
KRAS G12C is the most common KRAS mutation in NSCLC.6,7 In the U.S., about 13% of patients with NSCLC harbor the KRAS G12C mutation.8 There is a high unmet need and poor outcomes in the second-line treatment of KRAS G12C-driven non-small cell lung cancer (NSCLC) and, currently, there are no KRASG12C targeted therapies approved.2
According to Amgen’s head of research and development David Reese, “the company’s scientists had an idea some time ago that the future of oncology would be led by the marriage of immuno-oncology and precision therapy. We wanted to go after high value targets, and RAS proteins are one of them.”
At the same time that scientists discovered investigational sotorasib, the team was undertaking a project to map out every step it takes to progress a potential new treatment from an idea in a lab to being made available for patients. The goal was to shrink timelines and eliminate gaps to develop drugs more rapidly in order to reach patients with serious illnesses like NSCLC as quickly as possible.
Because of this effort to rapidly accelerate the speed of innovation, sotorasib entered clinical trials in humans less than 12 months.5 Sotorasib was the first investigational KRASG12C inhibitor to enter the clinic, and is now being studied in the broadest clinical program exploring 10 combinations with global investigational sites spanning five continents.9 In a little more than two years, the sotorasib clinical program has established a clinical data set of more than 700 patients studied across 13 tumor types.9
The investigational treatment was recently submitted to the FDA for review and was granted Breakthrough Therapy designation, a distinction designed to expedite the development and review of drugs.5 It was also accepted into the FDA’s Real-Time Oncology Review pilot program, which aims to explore a more efficient review process.5
To learn more about Amgen and how the speed of innovation is bringing new oncology treatments to patients with high unmet needs, visit Amgen.com/KnowKRAS.
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1 Ryan MB, et al. Nat Rev Clin Oncol. 2018;15:709-720.
2 Cox AD, et al. Nat Rev Drug Discov. 2014;13:828-851.
3 Kim D, et al. Cell. 2020. doi:10.1016/j.cell.2020.09.044.
Campbell, J.D., Alexandrov, A., Kim, J., Wala, J., Berger, A.H., Pedamallu, C.S., Shukla, S.A., Guo, G., Brooks, A.N., Murray, B.A., et al.; Cancer Genome Atlas Research Network (2016). Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat. Genet.48, 607–616
Jordan, E.J., Kim, H.R., Arcila, M.E., Barron, D., Chakravarty, D., Gao, J., Chang, M.T., Ni, A., Kundra, R., Jonsson, P., et al. (2017). Prospective comprehensive molecular characterization of lung adenocarcinomas for efficient patient matching to approved and emerging therapies. Cancer Discov. 7, 596–609.
Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.M., Gingras, M.C., Miller, D.K., Christ, A.N., Bruxner, T.J., Quinn, M.C., et al.; Australian Pancreatic Cancer Genome Initiative (2016). Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52.
Giannakis, M., Mu, X.J., Shukla, S.A., Qian, Z.R., Cohen, O., Nishihara, R., Bahl, S., Cao, Y., Amin-Mansour, A., Yamauchi, M., et al. (2016). Genomic correlates of immune-cell infiltrates in colorectal carcinoma. Cell Rep. 15, 857–865.
Efficiency of PARP inhibitors beyond BRCA mutations
Reporter
Irina Robu, PhD
PARP inhibitors are a group of pharmacological inhibitors of the enzyme poly ADP ribose polymerase, which are developed for multiple indications but most visible is the treatment of cancer. Several forms of cancer are extra dependent on PARP than regular cells, making PARP an striking target for cancer therapy. PARP inhibitors seem to improve progression-free survival in women with recurrent platinum-sensitive cancer. In addition to their use in cancer therapy, PARP inhibitors can be a potential treatment for acute life-threatening diseases, such as stroke and myocardial infarction and neurodegenerative diseases.
With this knowledge in hand, Lee Kraus, director of the Green Center for Reproductive Biology Sciences at UT Southwestern his team identified a potential biomarker, DDX21 protein, which is required for the production of ribosomes in nucleoli. Nonetheless, DDX21 in the nucleolus requires PARP-1, which is targeted by existing PARP inhibitors. The use of these drugs, blocks DDX21, hence inhibiting ribosome production which as result means that enhanced DDX21 levels in the nucleolus could regulate cancers that might be the most responsive to PARP inhibitors.
Their data published in the journal Molecular Cell explains why breast cancer patients can be responsive to PARP inhibitors, even though they do not carry BRCA mutation. It is well known that the PARP inhibitors currently on the market such as AstraZeneca’s Lynparza, Clovis’ Rubraca and GSK’s Zejula work by disturbing PARP proteins that help repair damaged DNA in cell, hence steering cancer cells onto a path of annihilation. Since cancer cells are addicted to ribosomes to grow and make proteins to support cell division, inhibiting PARP proteins can slow down the growth of the cell.
Kraus’s group is currently working to design clinical trials with UT Southwestern oncologists to see if their hypothesis works. At the same time, they founded Ribon Therapeutics which is the first industrial biotech program going after PARP7, a protein also similarly activated by stress and cellular response mechanisms.
An Intelligent DNA Nanorobot to Fight Cancer by Targeting HER2 Expression
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
3.2.9 An Intelligent DNA Nanorobot to Fight Cancer by Targeting HER2 Expression, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
HER2 is an important prognostic biomarker for 20–30% of breast cancers, which is the most common cancer in women. Overexpression of the HER2 receptor stimulates breast cells to proliferate and differentiate uncontrollably, thereby enhancing the malignancy of breast cancer and resulting in a poor prognosis for affected individuals. Current therapies to suppress the overexpression of HER2 in breast cancer mainly involve treatment with HER2-specific monoclonal antibodies. However, these monoclonal anti-HER2 antibodies have severe side effects in clinical trials, such as diarrhea, abnormal liver function, and drug resistance. Removing HER2 from the plasma membrane or inhibiting the gene expression of HER2 is a promising alternative that could limit the malignancy of HER2-positive cancer cells.
DNA origami is an emerging field of DNA-based nanotechnology and intelligent DNA nanorobots show great promise in working as a drug delivery system in healthcare. Different DNA-based nanorobots have been developed as affordable and facile therapeutic drugs. In particular, many studies reported that a tetrahedral framework nucleic acid (tFNA) could serve as a promising DNA nanocarrier for many antitumor drugs, owing to its high biocompatibility and biosecurity. For example, tFNA was reported to effectively deliver paclitaxel or doxorubicin to cancer cells for reversing drug resistance, small interfering RNAs (siRNAs) have been modified into tFNA for targeted drug delivery. Moreover, the production and storage of tFNA are not complicated, and they can be quickly degraded in lysosomes by cells. Since both free HApt and tFNA can be diverted into lysosomes, so, combining the HApt and tFNA as a novel DNA nanorobot (namely, HApt-tFNA) can be an effective strategy to improve its delivery and therapeutic efficacy in treating HER2-positive breast cancer.
Researchers reported that a DNA framework-based intelligent DNA nanorobot for selective lysosomal degradation of tumor-specific proteins on cancer cells. An anti-HER2 aptamer (HApt) was site-specifically anchored on a tetrahedral framework nucleic acid (tFNA). This DNA nanorobot (HApt-tFNA) could target HER2-positive breast cancer cells and specifically induce the lysosomal degradation of the membrane protein HER2. An injection of the DNA nanorobot into a mouse model revealed that the presence of tFNA enhanced the stability and prolonged the blood circulation time of HApt, and HApt-tFNA could therefore drive HER2 into lysosomal degradation with a higher efficiency. The formation of the HER2-HApt-tFNA complexes resulted in the HER2-mediated endocytosis and digestion in lysosomes, which effectively reduced the amount of HER2 on the cell surfaces. An increased HER2 digestion through HApt-tFNA further induced cell apoptosis and arrested cell growth. Hence, this novel DNA nanorobot sheds new light on targeted protein degradation for precision breast cancer therapy.
It was previously reported that tFNA was degraded by lysosomes and could enhance cell autophagy. Results indicated that free Cy5-HApt and Cy5-HApt-tFNA could enter the lysosomes; thus, tFNA can be regarded as an efficient nanocarrier to transmit HApt into the target organelle. The DNA nanorobot composed of HApt and tFNA showed a higher stability and a more effective performance than free HApt against HER2-positive breast cancer cells. The PI3K/AKT pathway was inhibited when membrane-bound HER2 decreased in SK-BR-3 cells under the action of HApt-tFNA. The research findings suggest that tFNA can enhance the anticancer effects of HApt on SK-BR-3 cells; while HApt-tFNA can bind to HER2 specifically, the compounded HER2-HApt-tFNA complexes can then be transferred and degraded in lysosomes. After these processes, the accumulation of HER2 in the plasma membrane would decrease, which could also influence the downstream PI3K/AKT signaling pathway that is associated with cell growth and death.
However, some limitations need to be noted when interpreting the findings: (i) the cytotoxicity of the nanorobot on HER2-positive cancer cells was weak, and the anticancer effects between conventional monoclonal antibodies and HApt-tFNA was not compared; (ii) the differences in delivery efficiency between tFNA and other nanocarriers need to be confirmed; and (iii) the confirmation of anticancer effects of HApt-tFNA on tumors within animals remains challenging. Despite these limitations, the present study provided novel evidence of the biological effects of tFNA when combined with HApt. Although the stability and the anticancer effects of HApt-tFNA may require further improvement before clinical application, this study initiates a promising step toward the development of nanomedicines with novel and intelligent DNA nanorobots for tumor treatment.
Single-cell RNA-seq helps in finding intra-tumoral heterogeneity in pancreatic cancer
Reporter and Curator: Dr. Sudipta Saha, Ph.D.
4.3.6 Single-cell RNA-seq helps in finding intra-tumoral heterogeneity in pancreatic cancer, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 4: Single Cell Genomics
Pancreatic cancer is a significant cause of cancer mortality; therefore, the development of early diagnostic strategies and effective treatment is essential. Improvements in imaging technology, as well as use of biomarkers are changing the way that pancreas cancer is diagnosed and staged. Although progress in treatment for pancreas cancer has been incremental, development of combination therapies involving both chemotherapeutic and biologic agents is ongoing.
Cancer is an evolutionary disease, containing the hallmarks of an asexually reproducing unicellular organism subject to evolutionary paradigms. Pancreatic ductal adenocarcinoma (PDAC) is a particularly robust example of this phenomenon. Genomic features indicate that pancreatic cancer cells are selected for fitness advantages when encountering the geographic and resource-depleted constraints of the microenvironment. Phenotypic adaptations to these pressures help disseminated cells to survive in secondary sites, a major clinical problem for patients with this disease.
The immune system varies in cell types, states, and locations. The complex networks, interactions, and responses of immune cells produce diverse cellular ecosystems composed of multiple cell types, accompanied by genetic diversity in antigen receptors. Within this ecosystem, innate and adaptive immune cells maintain and protect tissue function, integrity, and homeostasis upon changes in functional demands and diverse insults. Characterizing this inherent complexity requires studies at single-cell resolution. Recent advances such as massively parallel single-cell RNA sequencing and sophisticated computational methods are catalyzing a revolution in our understanding of immunology.
PDAC is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. In the present study to comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, single-cell RNA-seq (scRNA-seq) was employed to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases. The diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, were identified in PDAC.
The researchers found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, it was found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, the findings provided a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.
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