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KRAS is the most frequently mutated oncogene in human tumors and drives tumorigenesis across multiple lineages. Specifically, KRAS is mutated in about 30% of lung cancer and over 90% of pancreatic cancer. Despite recent progress in developing mutant selective KRASG12C inhibitors, patient treatment options for RAS activated cancers remain very limited. Targeting the MAPK pathway by means of MEK and ERK inhibitors has been explored as an alternative strategy for KRAS mutant cancer. However, the clinical benefit is modest due to drug resistance caused by reactivation of the MAPK pathway and potentially other pathways and/or bypass mechanisms. Here we used CRISPR-based screens to identify potential combination therapy targets to enhance trametinib response in KRAS mutant cancers. We screened in five KRAS mutant cell lines of lung and pancreatic lineages and identified both known as well as novel modulators of MEK inhibitor response. Consistent with previous reports, gene knockouts that impair the reactivation of ERK downstream of MEK inhibition scored as top hits in our drop-out screens. Knocking out MAPK1, RAF1, BRAF and PTPN11 sensitized all five cell lines to trametinib treatment supporting these as candidate drug combination targets for MAPK pathway inhibition. Our screens also identified multiple genes within the heparan sulfate pathway (EXT1, EXT2, EXTL3, XYLT2, ALG6, B3GAT3, B4GALT7 and HS2ST1) and the MAPK7 pathway (MAPK7 and MAP2K5) that sensitize multiple but not all cell lines to trametinib. As part of our target discovery platform, we further validated these screening results using various phenotypic assays. Altogether, our results suggest that resistance to MEK inhibitors is driven by reactivation of the MAPK pathway as previously demonstrated and that impairing such reactivation restores the sensitivity of KRAS mutant cancer cells to trametinib. The genetic mechanisms driving the MAPK pathway rebound are likely different in different cancers and understanding such mechanisms will be key for achieving clinical success. Citation Format: Silvia Fenoglio, Aileen M. Cristo, James Tepper, Teng Teng, Samuel R. Meier, Ashley Choi, Hongxiang Zhang, Shan-chuan Zhao, Shangtao Liu, Leanne G. Ahronian, Daniel Aird, Nikitha M. Das, Yi Yu, Robert Tjin Tham Sjin, Jannik N. Andersen, Alan Huang, Fang Li, Xuewen Pan. CRISPR screens identify sensitizers to trametinib in KRAS mutant cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P183.

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRT) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress response via genetically intact MYC-p19(ARF)-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRT.

Following mutations in BRAF and NRAS, the RAC1 c.85C>T single-nucleotide variant (SNV) encoding P29S amino acid change represents the next most frequently observed protein-coding hotspot mutation in melanoma. However, the biologic and clinical significance of the RAC1 P29S somatic mutation in approximately 4% to 9% of patients remains unclear. Here, we demonstrate that melanoma cell lines possessing the RAC1 hotspot variant are resistant to RAF inhibitors (vemurafenib and dabrafenib). Enforced expression of RAC1 P29S in sensitive BRAF-mutant melanoma cell lines confers resistance manifested by increased viability, decreased apoptosis, and enhanced tumor growth in vivo upon treatment with RAF inhibitors. Conversely, RNAi-mediated silencing of endogenous RAC1 P29S in a melanoma cell line with a co-occurring BRAF V600 mutation increased sensitivity to vemurafenib and dabrafenib. Our results suggest RAC1 P29S status may offer a predictive biomarker for RAF inhibitor resistance in melanoma patients, where it should be evaluated clinically. Cancer Res; 74(17); 4845–52. ©2014 AACR.

Malignant neoplasms evolve in response to changes in oncogenic signalling(1). Cancer cell plasticity in response to evolutionary pressures is fundamental to tumour progression and the development of therapeutic resistance(2,3). Here we elucidate the molecular and cellular mechanisms of cancer cell plasticity in a conditional oncogenic Kras mouse model of pancreatic ductal adenocarcinoma (PDAC), a malignancy that displays considerable phenotypic diversity and morphological heterogeneity. In this model, stochastic extinction of oncogenic Kras signalling and emergence of Kras-independent escaper populations (cells that acquire oncogenic properties) are associated with de-differentiation and aggressive biological behaviour. Transcriptomic and functional analyses of Kras-independent escapers reveal the presence of Smarcb1–Myc-network-driven mesenchymal reprogramming and independence from MAPK signalling. A somatic mosaic model of PDAC, which allows time-restricted perturbation of cell fate, shows that depletion of Smarcb1 activates the Myc network, driving an anabolic switch that increases protein metabolism and adaptive activation of endoplasmic-reticulum-stress-induced survival pathways. Elevated protein turnover renders mesenchymal sub-populations highly susceptible to pharmacological and genetic perturbation of the cellular proteostatic machinery and the IRE1-α–MKK4 arm of the endoplasmic-reticulum-stress-response pathway. Specifically, combination regimens that impair the unfolded protein responses block the emergence of aggressive mesenchymal subpopulations in mouse and patient-derived PDAC models. These molecular and biological insights inform a potential therapeutic strategy for targeting aggressive mesenchymal features of PDAC.

SummaryCurrent treatment regimens for pancreatic ductal adenocarcinoma (PDAC) yield poor 5-year survival, emphasizing the critical need to identify druggable targets essential for PDAC maintenance. We developed an unbiased and in vivo target discovery approach to identify molecular vulnerabilities in low-passage and patient-derived PDAC xenografts or genetically engineered mouse model-derived allografts. Focusing on epigenetic regulators, we identified WDR5, a core member of the COMPASS histone H3 Lys4 (H3K4) MLL (1–4) methyltransferase complex, as a top tumor maintenance hit required across multiple human and mouse tumors. Mechanistically, WDR5 functions to sustain proper execution of DNA replication in PDAC cells, as previously suggested by replication stress studies involving MLL1, and c-Myc, also found to interact with WDR5. We indeed demonstrate that interaction with c-Myc is critical for this function. By showing that ATR inhibition mimicked the effects of WDR5 suppression, these data provide rationale to test ATR and WDR5 inhibitors for activity in this disease.