An Aspirin a Day: New Pharmacological Developments and Cancer Chemoprevention.

Chemoprevention refers to the use of natural or synthetic agents to reverse, suppress, or prevent the progression or recurrence of cancer. A large body of preclinical and clinical data suggest the ability of aspirin to prevent precursor lesions and cancers, but much of the clinical data are inferential and based on descriptive epidemiology, case control, and cohort studies or studies designed to answer other questions (e.g., cardiovascular mortality). Multiple pharmacological, clinical, and epidemiologic studies suggest that aspirin can prevent certain cancers but may also cause other effects depending on the tissue or disease and organ site in question. The best-known biological targets of aspirin are cyclooxygenases, which drive a wide variety of functions, including hemostasis, inflammation, and immune modulation. Newly recognized molecular and cellular interactions suggest additional modifiable functional targets, and the existence of consensus molecular cancer subtypes suggests that aspirin may have differential effects based on tumor heterogeneity. This review focuses on new pharmacological developments and innovations in biopharmacology that clarify the potential role of aspirin in cancer chemoprevention.

Of vascular defense, hemostasis, cancer, and platelet biology: an evolutionary perspective.

We have established considerable expertise in studying the role of platelets in cancer biology. From this expertise, we were keen to recognize the numerous venous-, arterial-, microvascular-, and macrovascular thrombotic events and immunologic disorders are caused by severe, acute-respiratory-syndrome coronavirus 2 (SARS-CoV-2) infections. With this offering, we explore the evolutionary connections that place platelets at the center of hemostasis, immunity, and adaptive phylogeny. Coevolutionary changes have also occurred in vertebrate viruses and their vertebrate hosts that reflect their respective evolutionary interactions. As mammals adapted from aquatic to terrestrial life and the heavy blood loss associated with placentalization-based live birth, platelets evolved phylogenetically from thrombocytes toward higher megakaryocyte-blebbing-based production rates and the lack of nuclei. With no nuclei and robust RNA synthesis, this adaptation may have influenced viral replication to become less efficient after virus particles are engulfed. Human platelets express numerous receptors that bind viral particles, which developed from archetypal origins to initiate aggregation and exocytic-release of thrombo-, immuno-, angiogenic-, growth-, and repair-stimulatory granule contents. Whether by direct, evolutionary, selective pressure, or not, these responses may help to contain virus spread, attract immune cells for eradication, and stimulate angiogenesis, growth, and wound repair after viral damage. Because mammalian and marsupial platelets became smaller and more plate-like their biophysical properties improved in function, which facilitated distribution near vessel walls in fluid-shear fields. This adaptation increased the probability that platelets could then interact with and engulf shedding virus particles. Platelets also generate circulating microvesicles that increase membrane surface-area encounters and mark viral targets. In order to match virus-production rates, billions of platelets are generated and turned over per day to continually provide active defenses and adaptation to suppress the spectrum of evolving threats like SARS-CoV-2.

Sequential Administration of XPO1 and ATR Inhibitors Enhances Therapeutic Response in TP53-mutated Colorectal Cancer.

BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

Methylation-eQTL analysis in cancer research.

MOTIVATION: DNA methylation is a key epigenetic factor regulating gene expression. While promoter methylation has been well studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to discover and characterize gene-level relationships between methylation and expression. RESULTS: We introduce a novel sequential penalized regression approach to identify methylation-expression quantitative trait loci (methyl-eQTLs), a term that we have coined to represent, for each gene and tissue type, a sparse set of CpG loci best explaining gene expression and accompanying weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than current commonly used gene-level methylation summaries. The methyl-eQTLs identified by our approach can be used to construct gene-level methylation summaries that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation. AVAILABILITY AND IMPLEMENTATION: We produce an R Shiny app (https://rstudio-prd-c1.pmacs.upenn.edu/methyl-eQTL/) that interactively presents methyl-eQTL results for colorectal, breast and pancreatic cancer. The source R code for this work is provided in the Supplementary Material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Bioactive lipid metabolism in platelet "first responder" and cancer biology.

Platelets can serve as "first responders" in cancer and metastasis. This is partly due to bioactive lipid metabolism that drives both platelet and cancer biology. The two primary eicosanoid metabolites that maintain platelet rapid response homeostasis are prostacyclin made by endothelial cells that inhibits platelet function, which is counterbalanced by thromboxane produced by platelets during activation, aggregation, and platelet recruitment. Both of these arachidonic acid metabolites are inherently unstable due to their chemical structure. Tumor cells by contrast predominantly make more chemically stable prostaglandin E2, which is the primary bioactive lipid associated with inflammation and oncogenesis. Pharmacological, clinical, and epidemiologic studies demonstrate that non-steroidal anti-inflammatory drugs (NSAIDs), which target cyclooxygenases, can help prevent cancer. Much of the molecular and biological impact of these drugs is generally accepted in the field. Cyclooxygenases catalyze the rate-limiting production of substrate used by all synthase molecules, including those that produce prostaglandins along with prostacyclin and thromboxane. Additional eicosanoid metabolites include lipoxygenases, leukotrienes, and resolvins that can also influence platelets, inflammation, and carcinogenesis. Our knowledge base and technology are now progressing toward identifying newer molecular and cellular interactions that are leading to revealing additional targets. This review endeavors to summarize new developments in the field.

Signet ring cell colorectal cancer: genomic insights into a rare subpopulation of colorectal adenocarcinoma.

BACKGROUND: Signet ring cell carcinoma (SRCC) is a rare subtype of colorectal cancer (CRC). The aim of this study was to characterise the genomic alterations and outcomes of SRCC. METHODS: Medical records of metastatic CRC (mCRC) patients whose tumours were evaluated by NGS analysis were reviewed. SC-mCRC were classified into two groups: SRCC (>50% signet ring cells) and adenocarcinoma (AC) with SC component (≤50% signet ring cells). RESULTS: Six hundred and sixty-five mCRC patients were included. Of the 93 mCRC cases with SC features, 63 had slides for review. Of those 63 cases, 35 were confirmed SRCC, and 28 were AC with SC component. Compared with AC group, KRAS and PIK3CA mutations (mts) were found in only 11% (OR: 0.13) and 3% (OR: 0.15) of SRCC cases, respectively. In contrast to the 44% rate of APC mts in AC group, only 3% of SRCC patients had APC mts (OR = 0.04). CONCLUSIONS: SRCC has distinct molecular features, including low rates of KRAS, PIK3CA and APC mts. Further study to identify activation pathways and potential therapeutic targets are needed.

Back to the Colorectal Cancer Consensus Molecular Subtype Future.

PURPOSE OF REVIEW: This review seeks to provide an informed prospective on the advances in molecular profiling and analysis of colorectal cancer (CRC). The goal is to provide a historical context and current summary on how advances in gene and protein sequencing technology along with computer capabilities led to our current bioinformatic advances in the field. RECENT FINDINGS: An explosion of knowledge has occurred regarding genetic, epigenetic, and biochemical alterations associated with the evolution of colorectal cancer. This has led to the realization that CRC is a heterogeneous disease with molecular alterations often dictating natural history, response to treatment, and outcome. The consensus molecular subtypes (CMS) classification classifies CRC into four molecular subtypes with distinct biological characteristics, which may form the basis for clinical stratification and subtype-based targeted intervention. This review summarizes new developments of a field moving "Back to the Future." CRC molecular subtyping will better identify key subtype specific therapeutic targets and responses to therapy.

Real-Time Interrogation of Aspirin Reactivity, Biochemistry, and Biodistribution by Hyperpolarized Magnetic Resonance Spectroscopy.

Hyperpolarized magnetic resonance spectroscopy enables quantitative, non-radioactive, real-time measurement of imaging probe biodistribution and metabolism in vivo. Here, we investigate and report on the development and characterization of hyperpolarized acetylsalicylic acid (aspirin) and its use as a nuclear magnetic resonance (NMR) probe. Aspirin derivatives were synthesized with single- and double-13 C labels and hyperpolarized by dynamic nuclear polarization with 4.7 % and 3 % polarization, respectively. The longitudinal relaxation constants (T1 ) for the labeled acetyl and carboxyl carbonyls were approximately 30 seconds, supporting in vivo imaging and spectroscopy applications. In vitro hydrolysis, transacetylation, and albumin binding of hyperpolarized aspirin were readily monitored in real time by 13 C-NMR spectroscopy. Hyperpolarized, double-labeled aspirin was well tolerated in mice and could be observed by both 13 C-MR imaging and 13 C-NMR spectroscopy in vivo.

The somatic mutation landscape of premalignant colorectal adenoma.

OBJECTIVE: There are few studies which characterised the molecular alterations in premalignant colorectal adenomas. Our major goal was to establish colorectal adenoma genome atlas and identify molecular markers of progression from colorectal adenoma to adenocarcinoma. DESIGN: Whole-exome sequencing and targeted sequencing were carried out in 149 adenoma samples and paired blood from patients with conventional adenoma or sessile serrated adenoma to characterise the somatic mutation landscape for premalignant colorectal lesions. The identified somatic mutations were compared with those in colorectal cancer (CRC) samples from The Cancer Genome Atlas. A supervised random forest model was employed to identify gene panels differentiating adenoma from CRC. RESULTS: Similar somatic mutation frequencies, but distinctive driver mutations, were observed in sessile serrated adenomas and conventional adenomas. The final model included 20 genes and was able to separate the somatic mutation profile of colorectal adenoma and adenocarcinoma with an area under the curve of 0.941. CONCLUSION: The findings of this project hold potential to better identify patients with adenoma who may be candidates for targeted surveillance programmes and preventive interventions to reduce the incidence of CRC.

Platelets, circulating tumor cells, and the circulome.

Platelets are cytoplasmic fragments generated by megakaryocytes in the bone marrow and do not possess a nucleus. They contribute to the "Circulome" consisting of all circulating cells, factors and macromolecules such as cfDNA. Their primary function is to recognize vascular lesions and initiate thrombus formation that ceases bleeding. This distinctive characteristic of platelets also contributes to cancer and its progression. The ability of platelets to recognize and interact with other cells and neighboring platelets enables them to interact with tumor cells in the circulation. Receptor recognition and factor mediated crosstalk between tumor cells and platelets stimulate platelet activation, release of factors, and aggregation that promotes tumor cell survival and cancer progression. This review describes platelet: (i) contributions to the "Circulome" (ii) their importance as diagnostic tools in predicting cancer risk and (iii) therapies targeting platelet activation in inhibiting tumor progression and metastasis.

Platelet "first responders" in wound response, cancer, and metastasis.

Platelets serve as "first responders" during normal wounding and homeostasis. Arising from bone marrow stem cell lineage megakaryocytes, anucleate platelets can influence inflammation and immune regulation. Biophysically, platelets are optimized due to size and discoid morphology to distribute near vessel walls, monitor vascular integrity, and initiate quick responses to vascular lesions. Adhesion receptors linked to a highly reactive filopodia-generating cytoskeleton maximizes their vascular surface contact allowing rapid response capabilities. Functionally, platelets normally initiate rapid clotting, vasoconstriction, inflammation, and wound biology that leads to sterilization, tissue repair, and resolution. Platelets also are among the first to sense, phagocytize, decorate, or react to pathogens in the circulation. These platelet first responder properties are commandeered during chronic inflammation, cancer progression, and metastasis. Leaky or inflammatory reaction blood vessel genesis during carcinogenesis provides opportunities for platelet invasion into tumors. Cancer is thought of as a non-healing or chronic wound that can be actively aided by platelet mitogenic properties to stimulate tumor growth. This growth ultimately outstrips circulatory support leads to angiogenesis and intravasation of tumor cells into the blood stream. Circulating tumor cells reengage additional platelets, which facilitates tumor cell adhesion, arrest and extravasation, and metastasis. This process, along with the hypercoagulable states associated with malignancy, is amplified by IL6 production in tumors that stimulate liver thrombopoietin production and elevates circulating platelet numbers by thrombopoiesis in the bone marrow. These complex interactions and the "first responder" role of platelets during diverse physiologic stresses provide a useful therapeutic target that deserves further exploration.

The potential role of platelets in the consensus molecular subtypes of colorectal cancer.

The consensus molecular subtypes (CMS) in colorectal cancer (CRC) represent distinct molecular subcategories of disease as reflected by comprehensive molecular profiling. The four CMS subtypes represent unique biology. CMS1 represents high immune infiltration. CMS2 demonstrates upregulation of canonical pathways such as WNT signaling. Widespread metabolic changes are seen in CMS3. CMS4 represents a mesenchymal phenotype with hallmark features including complement activation, matrix remodeling, angiogenesis, epithelial-mesechymal transition (EMT), integrin upregulation and stromal infiltration. In contrast to this new paradigm, a number of observations regarding CRC remain disconnected. Cancers are associated with thrombocytosis. Venous thromboembolic events are more likely in malignancy and may signify worse prognosis. Aspirin, an anti-platelet agent, has been linked in large observational studies to decrease incidence of adenocarcinoma and less advanced presentations of cancer, in particular CRC. Inflammatory bowel disease is a risk factor for CRC. Gross markers to recognize the immunothrombotic link such as the platelet to lymphocyte ratio are associated with poorer outcomes in many cancers. Platelets are increasingly recognized for their dual roles in coordinating the immune response in addition to hemostasis. Here, we explore how these different but related observations coalesce. Platelets, as first responders to pathogens and injury, form the link between hemostasis and immunity. We outline how platelets contribute to tumorigenesis and how some disconnected ideas may be linked through inflammation. CMS4 through its shared mechanisms has predicted platelet activation as a hallmark feature. We demonstrate a platelet gene expression signature that predicts platelet presence within CMS4 tumors.

Beyond COX-1: the effects of aspirin on platelet biology and potential mechanisms of chemoprevention.

After more than a century, aspirin remains one of the most commonly used drugs in western medicine. Although mainly used for its anti-thrombotic, anti-pyretic, and analgesic properties, a multitude of clinical studies have provided convincing evidence that regular, low-dose aspirin use dramatically lowers the risk of cancer. These observations coincide with recent studies showing a functional relationship between platelets and tumors, suggesting that aspirin's chemopreventive properties may result, in part, from direct modulation of platelet biology and biochemistry. Here, we present a review of the biochemistry and pharmacology of aspirin with particular emphasis on its cyclooxygenase-dependent and cyclooxygenase-independent effects in platelets. We also correlate the results of proteomic-based studies of aspirin acetylation in eukaryotic cells with recent developments in platelet proteomics to identify non-cyclooxygenase targets of aspirin-mediated acetylation in platelets that may play a role in its chemopreventive mechanism.

Right Versus Left Colon Cancer Biology: Integrating the Consensus Molecular Subtypes.

Although clinical management of colon cancer generally has not accounted for the primary tumor site, left-sided and right-sided colon cancers harbor different clinical and biologic characteristics. Right-sided colon cancers are more likely to have genome-wide hypermethylation via the CpG island methylator phenotype (CIMP), hypermutated state via microsatellite instability, and BRAF mutation. There are also differential exposures to potential carcinogenic toxins and microbiota in the right and left colon. Gene expression analyses further shed light on distinct biologic subtypes of colorectal cancers (CRCs), with 4 consensus molecular subtypes (CMSs) identified. Importantly, these subtypes are differentially distributed between right- and left-sided CRCs, with greater proportions of the "microsatellite unstable/immune" CMS1 and the "metabolic" CMS3 subtypes found in right-sided colon cancers. This review summarizes important biologic distinctions between right- and left-sided CRCs that likely impact prognosis and may predict for differential responses to biologic therapy. Given the inferior prognosis of stage III-IV right-sided CRCs and emerging data suggesting that anti-epidermal growth factor receptor antibody therapy is associated with worse survival in right-sided stage IV CRCs compared with left-sided cancers, these biologic differences between right- and left-sided CRCs provide critical context and may provide opportunities to personalize therapy.

Platelets and cancer: a casual or causal relationship: revisited.

Human platelets arise as subcellular fragments of megakaryocytes in bone marrow. The physiologic demand, presence of disease such as cancer, or drug effects can regulate the production circulating platelets. Platelet biology is essential to hemostasis, vascular integrity, angiogenesis, inflammation, innate immunity, wound healing, and cancer biology. The most critical biological platelet response is serving as "First Responders" during the wounding process. The exposure of extracellular matrix proteins and intracellular components occurs after wounding. Numerous platelet receptors recognize matrix proteins that trigger platelet activation, adhesion, aggregation, and stabilization. Once activated, platelets change shape and degranulate to release growth factors and bioactive lipids into the blood stream. This cyclic process recruits and aggregates platelets along with thrombogenesis. This process facilitates wound closure or can recognize circulating pathologic bodies. Cancer cell entry into the blood stream triggers platelet-mediated recognition and is amplified by cell surface receptors, cellular products, extracellular factors, and immune cells. In some cases, these interactions suppress immune recognition and elimination of cancer cells or promote arrest at the endothelium, or entrapment in the microvasculature, and survival. This supports survival and spread of cancer cells and the establishment of secondary lesions to serve as important targets for prevention and therapy.

Nuclear orphan receptor NR4A2 modulates fatty acid oxidation pathways in colorectal cancer.

Although cancer cells have traditionally been thought to rely on the glycolytic pathway to generate ATP, recent studies suggest that cancer cells can shift to the fatty acid oxidation pathway as an alternative energy source. All of the factors that induce and regulate this adaptive shift in metabolism are not known. Cyclooxygenase-2-derived prostaglandin E(2) (PGE(2)) is produced at high levels in colon cancer, and multiple lines of evidence from human-, animal-, and cell line-based studies indicate that PGE(2) plays a pro-oncogenic role in colorectal cancer progression. We have shown previously that exposure of colon cancer cells to PGE(2) promotes cell survival, in part by inducing the expression of the nuclear orphan receptor NR4A2. Here, we report that PGE(2)-induced NR4A2 increased fatty acid oxidation by inducing the expression of multiple proteins in the fatty acid oxidation pathway. NR4A2 was found to bind directly to Nur77-binding response elements located within the regulatory region of these genes. Nur77-binding response element binding also resulted in the recruitment of transcriptional coactivators and induction of gene expression. Collectively, our findings suggest that NR4A2 plays a key role as a transcriptional integration point between the eicosanoid and fatty acid metabolic pathways. Thus, PGE(2) is a potential regulator of the adaptive shift to energy utilization via fatty acid oxidation that has been observed in several types of cancer.

Novel Markers for Liquid Biopsies in Cancer Management: Circulating Platelets and Extracellular Vesicles.

Although radiologic imaging and histologic assessment of tumor tissues are classic approaches for diagnosis and monitoring of treatment response, they have many limitations. These include challenges in distinguishing benign from malignant masses, difficult access to the tumor, high cost of the procedures, and tumor heterogeneity. In this setting, liquid biopsy has emerged as a potential alternative for both diagnostic and monitoring purposes. The approaches to liquid biopsy include cell-free DNA/circulating tumor DNA, long and micro noncoding RNAs, proteins/peptides, carbohydrates/lectins, lipids, and metabolites. Other approaches include detection and analysis of circulating tumor cells, extracellular vesicles, and tumor-activated platelets. Ultimately, reliable use of liquid biopsies requires bioinformatics and statistical integration of multiple datasets to achieve approval in a Clinical Laboratory Improvement Amendments setting. This review provides a balanced and critical assessment of recent discoveries regarding tumor-derived biomarkers in liquid biopsies along with the potential and pitfalls for cancer detection and longitudinal monitoring.

Precision Combination Therapies Based on Recurrent Oncogenic Coalterations.

Cancer cells depend on multiple driver alterations whose oncogenic effects can be suppressed by drug combinations. Here, we provide a comprehensive resource of precision combination therapies tailored to oncogenic coalterations that are recurrent across patient cohorts. To generate the resource, we developed Recurrent Features Leveraged for Combination Therapy (REFLECT), which integrates machine learning and cancer informatics algorithms. Using multiomic data, the method maps recurrent coalteration signatures in patient cohorts to combination therapies. We validated the REFLECT pipeline using data from patient-derived xenografts, in vitro drug screens, and a combination therapy clinical trial. These validations demonstrate that REFLECT-selected combination therapies have significantly improved efficacy, synergy, and survival outcomes. In patient cohorts with immunotherapy response markers, DNA repair aberrations, and HER2 activation, we have identified therapeutically actionable and recurrent coalteration signatures. REFLECT provides a resource and framework to design combination therapies tailored to tumor cohorts in data-driven clinical trials and preclinical studies. SIGNIFICANCE: We developed the predictive bioinformatics platform REFLECT and a multiomics- based precision combination therapy resource. The REFLECT-selected therapies lead to significant improvements in efficacy and patient survival in preclinical and clinical settings. Use of REFLECT can optimize therapeutic benefit through selection of drug combinations tailored to molecular signatures of tumors. See related commentary by Pugh and Haibe-Kains, p. 1416. This article is highlighted in the In This Issue feature, p. 1397.

Autologous humanized mouse models to study combination and single-agent immunotherapy for colorectal cancer patient-derived xenografts.

Designing studies of immunotherapy is limited due to a lack of pre-clinical models that reliably predict effective immunotherapy responses. To address this gap, we developed humanized mouse models of colorectal cancer (CRC) incorporating patient-derived xenografts (PDX) with human peripheral blood mononuclear cells (PBMC). Humanized mice with CRC PDXs were generated via engraftment of autologous (isolated from the same patients as the PDXs) or allogeneic (isolated from healthy donors) PBMCs. Human T cells were detected in mouse blood, tissues, and infiltrated the implanted PDXs. The inclusion of anti-PD-1 therapy revealed that tumor responses in autologous but not allogeneic models were more comparable to that of patients. An overall non-specific graft-vs-tumor effect occurred in allogeneic models and negatively correlated with that seen in patients. In contrast, autologous humanized mice more accurately correlated with treatment outcomes by engaging pre-existing tumor specific T-cell populations. As autologous T cells appear to be the major drivers of tumor response thus, autologous humanized mice may serve as models at predicting treatment outcomes in pre-clinical settings for therapies reliant on pre-existing tumor specific T-cell populations.

Targeting RAS Mutant Colorectal Cancer with Dual Inhibition of MEK and CDK4/6.

KRAS and NRAS mutations occur in 45% of colorectal cancers, with combined MAPK pathway and CDK4/6 inhibition identified as a potential therapeutic strategy. In the current study, this combinatorial treatment approach was evaluated in a co-clinical trial in patient-derived xenografts (PDX), and safety was established in a clinical trial of binimetinib and palbociclib in patients with metastatic colorectal cancer with RAS mutations. Across 18 PDX models undergoing dual inhibition of MEK and CDK4/6, 60% of tumors regressed, meeting the co-clinical trial primary endpoint. Prolonged duration of response occurred predominantly in TP53 wild-type models. Clinical evaluation of binimetinib and palbociclib in a safety lead-in confirmed safety and provided preliminary evidence of activity. Prolonged treatment in PDX models resulted in feedback activation of receptor tyrosine kinases and acquired resistance, which was reversed with a SHP2 inhibitor. These results highlight the clinical potential of this combination in colorectal cancer, along with the utility of PDX-based co-clinical trial platforms for drug development. SIGNIFICANCE: This co-clinical trial of combined MEK-CDK4/6 inhibition in RAS mutant colorectal cancer demonstrates therapeutic efficacy in patient-derived xenografts and safety in patients, identifies biomarkers of response, and uncovers targetable mechanisms of resistance.