Abstract Background: Sotorasib, a KRAS G12C inhibitor, plus panitumumab is approved for use in chemorefractory metastatic colorectal cancer (mCRC) . Yet, early effects on the tumor microenvironment (TME) in clinical samples are not well understood. In preclinical models, sotorasib potently suppresses MAPK signaling and promotes a pro-inflammatory TME characterized by CD8+ T cell infiltration and increased IFN-γ activity, implicating adaptive immunity in tumor control. We investigated if these pharmacodynamic and immunologic effects are observed clinically in patients (pts) treated with sotorasib plus panitumumab. Methods: Paired biopsies before and after 3-4 weeks of sotorasib plus panitumumab treatment were available from 12 pts in Cohort A of CodeBreaK 101 (NCT04185883). We performed whole-transcriptome RNA sequencing (n=12), pERK immunohistochemistry (n=8), spatial analysis of H&E-stained sections using digital pathology (n=10), and single-cell spatial transcriptomics (n=2). Analyses were primarily descriptive, and Wilcoxon signed-rank tests and summary statistics were used to characterize differences between paired biopsies. Results: Transcriptomic profiling revealed robust suppression of MAPK signaling, shown by an approximate 4-fold reduction in median MAPK Pathway Activity Score with consistent downregulation of cell cycle-related genes. Key MAPK targets were downregulated, including ETV4 (−6.9-fold), EPHA2 (−2.8-fold), and DUSP6 (−2.0-fold), while EPHA4 was largely unchanged. pERK immunohistochemistry showed an approximately 1.8-fold reduction in H-score and a 2.0-fold decrease in proportion of pERK+ tumor nuclei among profiled pts, consistent with effective MAPK pathway inhibition. Immune activation was reflected by a 1.9-fold increase in the Tumor Inflammation Signature score and upregulation of chemokines and antigen-presentation genes (CXCL9, CCL5, HLA-DPA1). Digital pathology analysis of H&E images showed increased immune cell clustering in the TME, mirroring immune remodeling observed in RNA-seq data. Single-cell spatial confirmed presence of CXCR6+ CD8+ T cells and macrophage derived CXCL10 in the tumor and associated stroma suggesting cytotoxic T cell recruitment and activation. Comprehensive pathway analysis showed upregulation of PI3K-AKT, HER2, and TGFβ signaling in some pts, suggesting compensatory survival mechanisms and a potential rationale for combinatorial strategies. Conclusion: Paired biopsy analysis revealed potent MAPK inhibition and immune remodeling, providing mechanistic insight into the role of the immune TME in early response to sotorasib plus panitumumab and underscoring opportunities for rational combination strategies in mCRC. Citation Format: David Hong, Lata Mukundan, Abraham Anderson, Emily Chan, Dorothy French, Linnea Haeggblom, Daniel Lu, Toshiki Masuishi, Rona Yaeger, Yuliya Katlinskaya. Immune and pharmacodynamic effects of sotorasib plus panitumumab in KRAS G12C-mutant colorectal cancer: Paired biopsy results from CodeBreaK 101 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2929.

Abstract KRAS is the most frequently mutated oncogene in solid tumors. Covalent KRAS G12C-selective inhibitors have been approved for G12C-mutant non-small cell lung cancer (NSCLC). Despite their clinical success, the durability of response and emergence of resistance have tempered the efficacy of KRAS-targeted therapies, leading to the exploration of combination strategies to enhance clinical outcomes. AMG 410, currently in Phase 1, is a reversible pan-KRAS inhibitor capable of targeting KRAS mutants (e.g., G12D, G12V, and G12C) and wild-type amplification. These altered KRAS alleles are prevalent in multiple solid tumor indications, particularly colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), and NSCLC. Preclinically, AMG 410 demonstrated significant tumor growth inhibition as a single agent in multiple cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Guided by reverse translation findings from the G12C-selective inhibitor sotorasib, we evaluated AMG 410 combination approaches in preclinical models to proactively mitigate potential primary and acquired resistance mechanisms. A large panel of diverse KRAS-mutant cancer cell lines representing three indications (NSCLC, CRC, and PDAC) was screened in combination with inhibitors of the RAS signaling pathway, DNA damage repair pathways, cell cycle regulators, and select chemotherapy agents. Robust synergistic effects on cell viability were observed with pan-HER kinase and PI3K/mTOR inhibitors. To further explore resistance mechanisms, an AMG 410-anchored genome-wide CRISPR screen was performed. The Hippo pathway and YAP1 were identified as key modifiers of response to KRAS inhibition. Co-treatment with YAP/TEAD inhibitors demonstrated strong synergy in multiple KRAS-mutant cell lines. To assess whether these observations translated to improved efficacy in vivo, rational combinations were evaluated in tumor xenograft models. Consistent with the in vitro synergy observed with pan-HER inhibition, the combination of AMG 410 with panitumumab resulted in tumor regression in a CRC PDX model. Additionally, enhanced anti-tumor activity was observed when AMG 410 was combined with chemotherapy agents in CRC and PDAC xenografts. Building on the potential benefit of clinical KRAS G12C immune-oncology combinations, treatment with AMG 410 and PD-1 blockade in a KRAS G12D syngeneic CRC model led to tumor regression and significantly enhanced survival. Finally, co-treatment with a TEAD inhibitor enhanced durability of response to AMG 410 in vivo. Taken together, these findings support the clinical investigation of AMG 410 combination strategies to extend the therapeutic benefit of KRAS inhibition across diverse KRAS-mutant cancers. Citation Format: Ying-Chu Chen, Tao Osgood, Kevin Gaida, Gilbert Diaz, Chun Su, Elissa Swearingen, Daniel Lu, Deanna Mohn, Anne Y. Saiki, Monica Leavitt, Upendra P. Dahal, Ryan P. Wurz, Brian A. Lanman, Jason DeVoss, Karen Rex, Paul E. Hughes, Rati Verma. Preclinical combination approaches with the pan-KRAS inhibitor AMG 410 in KRAS-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5866.

MET-exon-14 skipping mutations (METΔex14) are oncogenic drivers in ~3-5% of lung cancer, which can be targeted with MET tyrosine kinase inhibitors (TKIs). The emergence of resistance limits long-term responses and is commonly mediated by secondary genomic alterations. We devised a preclinical strategy to study MET-TKI resistance using a dose-escalation approach on METΔex14-dependent cancer cells to generate MET-TKI-refractory isogenic counterparts. These cell lines were profiled for clinically relevant changes that we functionally evaluated for MET-TKI resistance. Targeted sequencing of resistant clones identified acquired mutations in SPOP and MGA, both antagonists of MYC activity. SPOP- and MGA-mutant clones exhibited higher MYC levels and transcriptomic signatures of MYC activation. Expression of MYC rescued tumor growth in the presence of TKI, while MYC depletion mimicked the cytotoxic effect of TKI treatment, indicating that MYC activity is coupled to MET signaling and MYC is required to mediate drug resistance. Analysis of clinical METΔex14-positive lung cancers revealed several cases where acquired MYC pathway alterations mediated resistance to MET-TKIs. These findings collectively converge on MYC as key in the progression of MET TKI-resistant cancers, and our in vitro data support the strategy of co-targeting MYC and MET to yield more prolonged responses in patients with METΔex14-positive lung cancer.

MET fusions (MET-F) are oncogenic drivers that remain poorly characterized. Analysis of 56 MET-F-positive tumors from an institutional cohort of 91,119 patients (79,864 DNA sequencing plus 11,255 RNA sequencing) uncovered two forms of MET-F pathobiology. The first group featured 5' partners with homodimerization domains fused in-frame with the MET tyrosine kinase domain, primarily originated from translocations, frequently excluded MET exon 14, mediated oncogenesis through cytoplasmic aggregation and constitutive activation, and were markedly sensitive to MET tyrosine kinase inhibitors (TKI) in preclinical models and patients with lung cancer. The second group lacked partner homodimerization motifs and retained MET transmembrane and extracellular domains. Their pathogenesis involved intrachromosomal rearrangements, resulting in partner selection for promoter hijacking and fusion allele amplification. Membrane-bound fusions were enriched in gliomas with receptor tyrosine kinase co-alterations. We provide a framework to comprehend the heterogeneous landscape of MET-Fs, supporting that fusion oncogenicity and MET TKI sensitivity are determined by structural topology and pathogenomic context. SIGNIFICANCE:MET fusions are primary drivers of tumor growth in multiple tumor types - lung cancer and gliomas - and can be effectively targeted with either type I (crizotinib, capmatinib, tepotinib, and savolitinib) or type II (cabozantinib) MET TKIs, with best responses in tumors harboring fusions with partner homodimerization.

The hepatocyte growth factor receptor (MET) is a receptor tyrosine kinase (RTK) that mediates the activity of a variety of downstream pathways upon its activation. These pathways regulate various physiological processes within the cell, including growth, survival, proliferation, and motility. Under normal physiological conditions, this allows MET to regulate various development and regenerative processes; however, mutations resulting in aberrant MET activity and the consequent dysregulation of downstream signaling can contribute to cellular pathophysiology. Mutations within MET have been identified in a variety of cancers and have been shown to mediate tumorigenesis by increasing RTK activity and downstream signaling. In lung cancer specifically, a number of patients have been identified as possessing MET alterations, commonly receptor amplification (METamp) or splice site mutations resulting in loss of exon 14 (METex14). Due to MET’s role in mediating oncogenesis, it has become an attractive clinical target and has led to the development of various targeted therapies, including MET tyrosine kinase inhibitors (TKIs). Unfortunately, these TKIs have demonstrated limited clinical efficacy, as patients often present with either primary or acquired resistance to these therapies. Mechanisms of resistance vary but often occur through off-target or bypass mechanisms that render downstream signaling pathways insensitive to MET inhibition. This review provides an overview of the therapeutic landscape for MET-positive cancers and explores the various mechanisms that contribute to therapeutic resistance in these cases.

Regulatory T (Treg) cells have long been recognized as modulators of immunological tolerance and homeostasis. Previously, we used scRNA-seq to reveal significant Treg heterogeneity in response to IL-2-induced activation. Herein, we leveraged enrichment analyses, as well as bulk and single nucleus multi-omics in splenic and lung Tregs, to uncover and confirm the importance of transcription factors (TFs) and chromatin remodeling in Treg activation. Multiple bZIP TF motifs showed increased chromatin accessibility post IL-2 treatment, with correlated transcriptional changes resembling Th1 and Th2 molecular phenotypes, further confirmed by spatial ATAC-seq. By combining gene perturbation and CUT&RUN assays before and after Treg stimulation, we show that bZIP TFs, such as BATF and BACH1, are critical to IL-2-induced Treg activation, coordinating epigenetic and transcriptional changes that selectively drive T-helper phenotypes and metabolic pathways. ### Competing Interest Statement The authors have declared no competing interest.

Heart failure is caused in part by cardiac remodeling processes that include the death of cardiac myocytes and their replacement by cardiac fibroblasts. Here, we hypothesize that cardiac fibroblasts may harbor epigenetic contexts in which heart disease-associated non-coding SNPs perturb gene expression relevant to disease. To test this, we utilized male primary cardiac fibroblasts to generate high-resolution Hi-C data and integrate it with functional genomic information to annotate and link putative distal regulatory elements in heart disease-associated loci to gene promoters. We identify several target genes with established roles in cardiac fibrosis and/or heart disease (GJA1, TBC1D32, CXCL12, IL6R, and FURIN). We perform Perturb-seq in immortalized male cardiac fibroblasts to knock out putative regulatory elements, confirming regulatory relationships involving GJA1, CXCL12, and FURIN. Our results demonstrate that multi-omic approaches can delineate pathophysiologically relevant regulatory circuits connecting protein-coding genes to non-coding genetic variants associated with disease.

BackgroundLung cancer is the leading cause of cancer related death worldwide, mainly due to the late stage of disease at the time of diagnosis. Non-invasive biomarkers are needed to supplement existing screening methods to enable earlier detection and increased patient survival. This is critical to EGFR-driven lung adenocarcinoma as it commonly occurs in individuals who have never smoked and do not qualify for current screening protocols.MethodsIn this study, we performed mass spectrometry analysis of the secretome of cultured lung cells representing different stages of mutant EGFR driven transformation, from normal to fully malignant. Identified secreted proteins specific to the malignant state were validated using orthogonal methods and their clinical activity assessed in lung adenocarcinoma patient cohorts.ResultsWe quantified 1020 secreted proteins, which were compared for differential expression between stages of transformation. We validated differentially expressed proteins at the transcriptional level in clinical tumor specimens, association with patient survival, and absolute concentration to yield three biomarker candidates: MDK, GDF15, and SPINT2. These candidates were validated using ELISA and increased levels were associated with poor patient survival specifically in EGFR mutant lung adenocarcinoma patients.ConclusionsOur study provides insight into changes in secreted proteins during EGFR driven lung adenocarcinoma transformation that may play a role in the processes that promote tumor progression. The specific candidates identified can harnessed for biomarker use to identify high risk individuals for early detection screening programs and disease management for this molecular subgroup of lung adenocarcinoma patients.

Membrane-associated ring-CH-type finger 6 (MARCH6), also designated as TEB4 or RNF176, is an E3 ligase that is embedded in membranes of the endoplasmic reticulum where it ubiquitinates many substrate proteins to consign them to proteasome-mediated degradation. In recent years, MARCH6 has been identified as a key regulator of several metabolic pathways, including cholesterol and lipid droplet homeostasis, protein quality control, ferroptosis, and tumorigenesis. Despite its importance, there are currently no specific antibodies to detect and monitor MARCH6 levels in cultured cells and animals. Here, we address this deficiency by generating a monoclonal antibody that specifically detects MARCH6 in cultured cells of insect, mouse, hamster, and human origin, as well as in mouse tissues, with minimal cross-reactivity against other proteins. We then used this antibody to assess two properties of MARCH6. First, analysis of mouse tissues with this antibody revealed that the liver contained the highest levels of March6. Second, analysis of five different cell lines with this antibody showed that endogenous levels of MARCH6 are unchanged as the cellular content of cholesterol is varied. This reagent promises to be a useful tool in interrogating additional signaling roles of MARCH6.

The rapid evolution of SARS-CoV-2 variants highlights the need for new therapies to prevent disease spread. SARS-CoV-2, like SARS-CoV-1, uses the human cell surface protein angiotensin-converting enzyme 2 (ACE2) as its native receptor. Here, we design and characterize a mutant ACE2 that enables rapid affinity purification of a dimeric protein by altering the active site to prevent autoproteolytic digestion of a C-terminal His10 epitope tag. In cultured cells, mutant ACE2 competitively inhibits lentiviral vectors pseudotyped with spike from multiple SARS-CoV-2 variants, and infectious SARS-CoV-2. Moreover, the protein can be nebulized and retains virus-binding properties. We developed a system for delivery of aerosolized ACE2 to K18-hACE2 mice and demonstrate protection by our modified ACE2 when delivered as a prophylactic agent. These results show proof-of-concept for an aerosolized delivery method to evaluate anti-SARS-CoV-2 agents in vivo and suggest a new tool in the ongoing fight against SARS-CoV-2 and other ACE2-dependent viruses.

Heart failure is caused in part by cardiac remodeling processes that include the death of cardiac myocytes and their replacement by cardiac fibroblasts. We hypothesized that these two cell types may harbor epigenetic contexts in which heart disease-associated non-coding SNPs perturb gene expression relevant to disease. Accordingly, we generated high-resolution Hi-C data layered with functional genomic information to annotate and link putative distal regulatory elements in heart disease-associated loci to gene promoters. Our analysis identified several target genes with established roles in cardiac fibrosis and/or heart disease (GJA1, TBC1D32, CXCL12, IL6R, and FURIN). Perturb-seq in cardiac fibroblasts after knocking out putative regulatory elements confirmed regulatory relationships involving GJA1, CXCL12, and FURIN, in addition to changes in transcriptomic signatures associated with fibroblasts in heart failure. Our results demonstrate how integrative multi-omic approaches can delineate pathophysiologically relevant regulatory circuits that connect protein-coding genes to non-coding genetic variants associated with disease. ### Competing Interest Statement D L, IE, JC, HZ, BP, BA, and CL are Amgen employees and RG, JY, TY, YA, SW, BA, CL, and YH are or were Amgene employees when performing experiments and data analysis. DL, IE, JC, HZ, BP, BA, CL, RG, JY, TY, YA, SW, BA, CL, and YH all own Amgen stocks.

Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.

Abstract KRAS inhibitors are a novel, recently approved class of small molecules that target cancers with KRAS mutations, and understanding molecular mechanisms by which these inhibitors stimulate anti-tumor immunity will improve patient outcomes. In this study, we utilized a multi-omic approach combining single-cell RNA-seq, spatial transcriptomics, and flow cytometry to define cell signatures and signaling events associated with response in a murine model expressing KRAS-G12C tumors. Treatment with KRAS inhibitors but not MEK inhibitors drove neoplastic differentiation toward cell states with increased oxidative stress and extracellular matrix remodeling capacity. These changes were accompanied by spatially-defined Notch and Integrin signaling patterns between neoplastic, stromal, and classical dendritic cell (cDC) niches, leading to accelerated cDC maturation and CD8 effector T-cell activation. Our multi-omic design distills the complex cellular consequences and intercellular communication patterns in the tumor microenvironment following KRAS inhibition and provides a framework for contextualizing and validating disease-relevant signatures to guide therapeutic strategy.

Necroptosis is a type of programmed cell death which is characterized by membrane permeabilization and is known to be associated with strong inflammatory response due to the release of intracellular components based on compromised membrane integrity. Necroptosis is accompanied by key lipid and membrane remodeling and this process is yet to be elucidated. Given this critical role of membranes during necroptosis, little research has focused on understanding the roles of lipids in the membrane-related changes during this process. To identify novel lipid players of necroptosis, we employed untargeted lipidomics and showed that saturated very long chain fatty acids (VLCFAs) accumulated via activated biosynthesis in necroptosis. Depleting the levels of VLCFAs resulted in reduced necroptotic cell death and membrane permeabilization whereas increasing VLCFA levels induced membrane permeabilization, showing that VLCFAs are functionally involved in necroptosis. In this talk, I will discuss the biochemical mechanisms that result in the accumulation of lipids and the functional consequences of these accumulations in this process. Specifically, I will present recent studies from our lab on the mechanisms of fatty acylation events by VCLFAs that contribute to the membrane permeabilization during necroptosis and the sterol-regulatory element binding protein mediated lipid accumulation in this process. Broadly, the talk will focus on new mechanisms of lipid accumulation and function in cell death.