Mechanical cues are essential for the regulation of cell and tissue physiology. Hence, it has become an utmost necessity for cell biologists to account for those mechanical parameters when ...investigating biological processes and they need devices to manipulate cells accordingly. Here, we report a simple mechanical cell-stretching system that can generate uniaxial cyclic mechanical stretch on cells in tissue culture. This system is based upon a low-cost battery-powered uniaxial cyclic mechanical stretcher exclusively built out of LEGO
parts combined with a stretchable poly(dimethylsiloxane) tissue culture plate in order to grow and stretch cells. We characterize the system and show that it can be used in a wide variety of downstream applications, including immunofluorescence, western blotting and biochemical assays. We also illustrate how this system can be useful in a study as we investigated the behavior of integrin adhesion complexes upon cell stretching. We therefore present a cost-effective, multipurpose cell-stretching system that should help to increase understanding of mechanical signaling.This article has an associated First Person interview with the first author of the paper.
8529 Background: KRAS G12C inhibitors such as adagrasib and sotorasib have shown clinical promise in targeting KRAS G12C -mutated lung cancers; however, most patients develop primary or secondary ...resistance. A biomarker of response to KRAS G12C inhibitor is needed for better patient stratification and to understand the resistance mechanism. Methods: We analyzed transcriptional correlates of adagrasib treatment outcome in 68 patients in the KRYSTAL-1 trial, a phase 1/2 clinical trial of adagrasib monotherapy in the second line and beyond treatment for NSCLC. We also treated KRAS-mutated lung cancer mouse models and organoids with a KRAS inhibitor for the long term and characterized the resistant tumors’ transcriptional profile to identify resistance mechanisms. We also performed serial gene expression analysis of Kras G12D mutated lung organoids undergoing squamous transformation to identify transcription factor involved in the resistance process. Results: In patients with lung adenocarcinoma with KRAS G12C and STK11/ LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRAS G12C and Kras G12D lung cancer mouse models and organoids, treated with KRAS inhibitors adagrasib and MRTX1133, respectively, reveal that tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. We identify TP63 to be a transcription factor whose expression correlated with squamous transformation. The analysis of lineage plasticity program, adagrasib resistant tumors, and p63 regulon revealed KRT6A to be a common biomarker whose expression correlated with overall survival in the KRYSTAL-1 cohort. Conclusions: KRAS G12C mutated lung adenocarcinoma patients with a higher expression of squamous cell carcinoma gene expression signature respond poorly to adagrasib treatment. Expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer.
RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 61
. Small molecule ...inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer
. Nevertheless, KRAS
mutations account for only around 15% of KRAS-mutated cancers
, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRAS
). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRAS
cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).
KRASG12C inhibitors (adagrasib and sotorasib) have shown clinical promise in targeting KRASG12C-mutated lung cancers; however, most patients eventually develop resistance. In lung patients with ...adenocarcinoma with KRASG12C and STK11/LKB1 co-mutations, we find an enrichment of the squamous cell carcinoma gene signature in pre-treatment biopsies correlates with a poor response to adagrasib. Studies of Lkb1-deficient KRASG12C and KrasG12D lung cancer mouse models and organoids treated with KRAS inhibitors reveal tumors invoke a lineage plasticity program, adeno-to-squamous transition (AST), that enables resistance to KRAS inhibition. Transcriptomic and epigenomic analyses reveal ΔNp63 drives AST and modulates response to KRAS inhibition. We identify an intermediate high-plastic cell state marked by expression of an AST plasticity signature and Krt6a. Notably, expression of the AST plasticity signature and KRT6A at baseline correlates with poor adagrasib responses. These data indicate the role of AST in KRAS inhibitor resistance and provide predictive biomarkers for KRAS-targeted therapies in lung cancer.
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•High SCC signature at baseline correlates with poor adagrasib response in KL NSCLC•AST drives resistance to KRAS inhibition in GEMMs and organoid models•ELF5-ΔNp63 axis governs AST process and drug response•AST plasticity signature and KRT6A enrichment predict poor adagrasib responses
Tong et al. show that high squamous cell carcinoma (SCC) signature at baseline correlates with poor adagrasib response in patients with KRAS/LKB1-mutant NSCLC. Integrative GEMM and organoid studies demonstrate that adeno-to-squamous transition (AST) drives KRAS inhibitor resistance involving ELF5-mediated epigenetic regulation of ΔNp63. AST plasticity signature and KRT6A predict poor adagrasib response.
Approximately 8% to 10% of pancreatic ductal adenocarcinomas (PDAC) do not harbor mutations in KRAS. Understanding the unique molecular and clinical features of this subset of pancreatic cancer is ...important to guide patient stratification for clinical trials of molecularly targeted agents.
We analyzed a single-institution cohort of 795 exocrine pancreatic cancer cases (including 785 PDAC cases) with a targeted multigene sequencing panel and identified 73 patients (9.2%) with KRAS wild-type (WT) pancreatic cancer.
Overall, 43.8% (32/73) of KRAS WT cases had evidence of an alternative driver of the MAPK pathway, including BRAF mutations and in-frame deletions and receptor tyrosine kinase fusions. Conversely, 56.2% of cases did not harbor a clear MAPK driver alteration, but 29.3% of these MAPK-negative KRAS WT cases (12/41) demonstrated activating alterations in other oncogenic drivers, such as GNAS, MYC, PIK3CA, and CTNNB1. We demonstrate potent efficacy of pan-RAF and MEK inhibition in patient-derived organoid models carrying BRAF in-frame deletions. Moreover, we demonstrate durable clinical benefit of targeted therapy in a patient harboring a KRAS WT tumor with a ROS1 fusion. Clinically, patients with KRAS WT tumors were significantly younger in age of onset (median age: 62.6 vs. 65.7 years; P = 0.037). SMAD4 mutations were associated with a particularly poor prognosis in KRAS WT cases.
This study defines the genomic underpinnings of KRAS WT pancreatic cancer and highlights potential therapeutic avenues for future investigation in molecularly directed clinical trials. See related commentary by Kato et al., p. 4527.
Cancer-associated mutations in the guanosine triphosphatase (GTPase) RHOA are found at different locations from the mutational hotspots in the structurally and biochemically related RAS. Tyr
-to-Cys ...(Y42C) and Leu
-to-Val (L57V) substitutions are the two most prevalent RHOA mutations in diffuse gastric cancer (DGC). RHOA
exhibits a gain-of-function phenotype and is an oncogenic driver in DGC. Here, we determined how RHOA
promotes DGC growth. In mouse gastric organoids with deletion of
, which encodes the cell adhesion protein E-cadherin, the expression of RHOA
, but not of wild-type RHOA, induced an abnormal morphology similar to that of patient-derived DGC organoids. RHOA
also exhibited a gain-of-function phenotype and promoted F-actin stress fiber formation and cell migration. RHOA
retained interaction with effectors but exhibited impaired RHOA-intrinsic and GAP-catalyzed GTP hydrolysis, which favored formation of the active GTP-bound state. Introduction of missense mutations at KRAS residues analogous to Tyr
and Leu
in RHOA did not activate KRAS oncogenic potential, indicating distinct functional effects in otherwise highly related GTPases. Both RHOA mutants stimulated the transcriptional co-activator YAP1 through actin dynamics to promote DGC progression; however, RHOA
additionally did so by activating the kinases IGF1R and PAK1, distinct from the FAK-mediated mechanism induced by RHOA
. Our results reveal that RHOA
and RHOA
drive the development of DGC through distinct biochemical and signaling mechanisms.
Abstract
KRAS G12C inhibitors (G12Ci), sotorasib and adagrasib, have demonstrated antitumor activity in patients with KRAS G12C mutant (mt) non-small cell lung cancer (NSCLC), and sotorasib has ...recently received FDA approval. It has been shown that simultaneous targeting of multiple nodes in the RAS/RAF/MEK/ERK (MAPK) pathway may be optimal for durable response. Furthermore, acquired mutations in the MAPK pathway occur clinically upon progression on adagrasib. Therefore, clinical combinations with G12Ci are needed. VS-6766 is a unique dual RAF/MEK inhibitor. In contrast to MEK-only inhibitors (MEKi), VS-6766 is a potent allosteric inhibitor of MEK kinase activity and induces a dominant negative RAF/MEK complex preventing phosphorylation of MEK by BRAF and CRAF. The combination of VS-6766 with the focal adhesion kinase (FAK) inhibitor defactinib with an intermittent schedule has shown clinical activity for patients with KRAS G12V and KRAS G12C mt NSCLC with a manageable safety profile relative to MEKi. Here, we tested whether combination of G12Ci with VS-6766 for vertical blockade of RAS, RAF and MEK might yield superior pathway blockade and antitumor efficacy. In 3D proliferation assays, VS-6766 was synergistic with both sotorasib and adagrasib in reducing viability of a panel of KRAS G12C mt cancer cell lines. Accordingly, VS-6766 effectively suppressed MAPK pathway signaling (pMEK, pERK) as a single agent, and the combination of VS-6766 + G12Ci showed improved depth and duration of MAPK pathway inhibition relative to G12Ci alone in KRAS G12C mt NSCLC models in vitro and in vivo. Reverse phase protein array (RPPA) analysis showed stronger inhibition of cell cycle/proliferation markers (e.g. aurora A/B, cyclin B1, pRb, pCDK1, pS6) with the combination than with VS-6766 or G12Ci alone. Additionally, the combination showed stronger activation of pro-apoptotic markers (e.g. cleaved caspase 7) and potential resistance markers (e.g. pFAK) than either agent alone. We are currently testing VS-6766 against a panel of cell lines expressing mutations identified in patients progressing on adagrasib. In KRAS G12C mt NSCLC models in vivo, VS-6766 combination augmented tumor growth inhibition by sotorasib, whereas trametinib combination was much less effective in augmenting sotorasib efficacy. In the H358 KRAS G12C mt NSCLC model, sotorasib monotherapy or sotorasib + trametinib failed to induce substantial tumor regression, whereas sotorasib + VS-6766 induced >25% tumor regression in 10/10 mice. Similarly, in the H2122 KRAS G12C mt NSCLC model, sotorasib + VS-6766 induced >20% tumor regression in 5/10 mice while sotorasib or sotorasib + trametinib were relatively ineffective. These results support the imminent clinical evaluation of VS-6766 in combination with a G12C inhibitor for treatment of KRAS G12C mt NSCLC in both G12Ci naïve patients and patients progressing on G12Ci treatment.
Citation Format: Silvia Coma, Sanjib Chowdhury, Julien Dilly, Monica Musteanu, Mariano Barbacid, Andrew J. Aguirre, Jonathan A. Pachter. Dual RAF/MEK inhibitor VS-6766 enhances antitumor efficacy of KRAS G12C inhibitors through vertical inhibition of RAS, RAF and MEK abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 402.
Abstract
Background: KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C) occur in ~13% of non-small cell lung cancers (NSCLC), ~3% of colorectal cancers (CRC), and less commonly ...in other cancer types. In early phase clinical trials of patients with KRASG12C-mutant cancers, promising antitumor activity has been reported with drugs such as adagrasib (MRTX849) and sotorasib (AMG510) which are direct inhibitors of KRASG12C. These small molecule irreversible inhibitors bind covalently to cysteine 12 within the switch II pocket which is formed, in part, by residues H95, Y96, and Q99. Mechanisms of acquired resistance to these therapies are currently unknown. Methods: Patients with KRASG12C-mutant NSCLC and CRC who were enrolled on adagrasib clinical trials and developed subsequent disease progression were included in this study if they were also consented to institutional review board-approved correlative studies at participating institutions. Tissue biopsies were analyzed histologically; tumor and/or circulating tumor DNA samples underwent next generation sequencing at the time of disease progression which was compared to that from pre-treatment samples when available. Results: A total of 30 patients were included in this study, 23 with NSCLC and 7 with CRC (25 with ctDNA, 7 with tissue sequencing, 2 with both). At the time of acquired resistance to adagrasib, we observed multiple on-target acquired KRAS alterations: mutations of the covalent-binding C12 residue, including C12W, C12F, C12V; a KRAS G13D mutation; high-level amplification of the KRASG12C allele; and mutation of the switch II binding pocket residues R68S, H95D, H95R, and Y96C. Furthermore, we detected several acquired off-target bypass mechanisms of resistance such as EGFR or MET amplification; activating mutations in NRAS (Q61K), BRAF (V600E), MAP2K1 (K57N, I99_K104del, E102_I103del), and RET (M918T); and oncogenic fusions involving RET, BRAF, RAF1, and FGFR. In two NSCLC cases with repeat tissue biopsies, histologic transformation from lung adenocarcinoma to squamous cell carcinoma was observed with no identifiable genomic mechanism of acquired resistance. In several cases, multiple coincident resistance mechanisms were identified in the same patient. Deep scanning mutagenesis studies were performed in parallel and identified the landscape of resistance mutations to adagrasib and sotorasib. While most resistance mutations confer high-level resistance to both therapies, some second-site mutations display differential sensitivity to distinct KRASG12C inhibitors, suggesting potential therapeutic strategies for overcoming drug resistance to specific mutations. Conclusion: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors in patients with cancer. Acquired genomic mutations, amplifications, and rearrangements may be potentially targetable by combining KRASG12C inhibition with available kinase inhibitors or SHP2 inhibitors.
Citation Format: Mark Awad, Shengwu Liu, Kathryn Arbour, Viola Zhu, Melissa Johnson, Rebecca Heist, Tejas Patil, Gregory Riely, Joseph Jacobson, Julien Dilly, Xiaoping Yang, Nicole Persky, David Root, Lynette Sholl, Lee Lim, Kavita Garg, Mark Li, Lars Engstrom, Laura Waters, J. David Lawson, Peter Olson, James Christensen, Piro Lito, Sai-Hong Inatius Ou, Pasi Janne, Andrew Aguirre. Mechanisms of acquired resistance to KRAS G12C inhibition in cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB002.
Abstract KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or ...sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, and Cdk6/Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies.
Acquired Resistance to KRASG12C Inhibition in Cancer Awad, Mark M; Liu, Shengwu; Rybkin, Igor I ...
New England journal of medicine/The New England journal of medicine,
06/2021, Volume:
384, Issue:
25
Journal Article
Peer reviewed
Open access
A study involving 38 patients who initially had a response to adagrasib or who had a long period of stable disease in response to the drug but then had progression yielded diverse mechanisms of ...acquired resistance in 45% of them. Unlike resistance to the tyrosine kinase inhibitors, the cancer cell uses many mechanisms to overcome the inhibition of KRAS.
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