Most lung cancer patients with metastatic cancer eventually relapse with drug-resistant disease following treatment and EGFR mutant lung cancer is no exception. Genome-wide CRISPR screens, to either knock out or overexpress all protein-coding genes in cancer cell lines, revealed the landscape of pathways that cause resistance to the EGFR inhibitors osimertinib or gefitinib in EGFR mutant lung cancer. Among the most recurrent resistance genes were those that regulate the Hippo pathway. Following osimertinib treatment a subpopulation of cancer cells are able to survive and over time develop stable resistance. These 'persister' cells can exploit non-genetic (transcriptional) programs that enable cancer cells to survive drug treatment. Using genetic and pharmacologic tools we identified Hippo signalling as an important non-genetic mechanism of cell survival following osimertinib treatment. Further, we show that combinatorial targeting of the Hippo pathway and EGFR is highly effective in EGFR mutant lung cancer cells and patient-derived organoids, suggesting a new therapeutic strategy for EGFR mutant lung cancer patients.

Figure S1. A subset MPM cell lines respond to FGFR inhibition. A) AZD4547 at a fixed dose of 500nM and B) BGJ-398 at a fixed dose of 300nM. Figure S2. A subset of pleural fluid derived early passage primary cultures (EPL) respond to FGFR inhibition. Figure S3. BAP1 mutation status does not correlate fully with protein expression. Figure S4. BAP1 null cell lines show increased activity of multiple receptor tyrosine kinases. Figure S5. FGF9 activates FGFR3 and modulates growth and phenotype. Figure S6. Gene expression studies comparing H226 BAP1 C91 vs BAP1 wt construct cell line. Figure S7. xenograft Immunohistochemistry for ki67, Caspase 3 under various experimental conditions. Figure S8. Anchor based combination drug screen in MPM using Pi3 kinase inhibitor as anchor drug.

BRAF V600E mutations occur in a subset of colon cancers. These are typically resistant to chemotherapy and are associated with a poor outcome. Combination treatment with BRAF and EGFR inhibitors is superior to standard chemotherapy and has recently received FDA approval, however the early emergence of drug resistance is a significant clinical problem. Clinical studies of resistant patients have identified mutations and amplifications in the MAPK pathway as important resistance drivers. To identify novel non-MAPK dependent resistance mechanisms, genome-wide CRISPR/Cas9 knockout screens were performed to identify genes causing resistance to a combination BRAF/MEK/EGFR inhibitor regimen in the BRAF mutant HT29 and LS411N colon cancer cell lines. A number of strong resistance hits were identified but importantly, only 3 genes (CSK, ARID1A and STK11) were detected as significantly enriched in both cell lines screened. Two of these, CSK and ARID1A, have been shown to play a role in activation of the Hippo signaling pathway. The generation of CSK knockout colon cell lines confirmed resistance to BRAF/MEK/EGFR inhibition both in vitro and in vivo using xenograft models. Furthermore, since CSK is a negative regulator of SRC, re-sensitisation of resistant BRAF mutant CSK knockout cells was achieved by adding Src inhibitors (Dasatinib and Saracatinib) to the combination therapy. Nuclear localisation of the transcription factors YAP1 and WWTR1 and binding to TEAD family members are required for Hippo pathway activation and we confirmed significantly increased nuclear YAP1/WWTR1 in CSK knockout cells. Furthermore, YAP1/WWTR1 nuclear localisation in these cells was reversed by treatment with Src inhibitors. Novel pharmacological TEAD inhibitors have recently been developed and will also be used to confirm that resistance can be overcome by specifically targeting the Hippo pathway. Expression profiling of CSK knockout cells revealed significant enrichment of pathways associated with Hippo signaling. Genes involved in regulating Hippo pathway activation were also identified as CRISPR screen resistance hits in both lung and head and neck cancer cell lines, suggesting that this may be an important mechanism of resistance among other tumour types and not limited to the colon. Here we show that activation of the Hippo pathway is a potential MAPK-independent resistance mechanism in BRAF mutant colon cancer, readily reversible by rational pharmacological targeting. Given the development of specific Hippo pathway inhibitors and plans for their use in clinical trials, activation of Hippo signaling should be considered in resistant BRAF mutant colon cancer where alterations in the MAPK pathway are not detected. Citation Format: Jonathan S. Brammeld, Hannah Thorpe, Mar Arias Garcia, Stacey Price, Jamie Young, Matthias Pfeifer, Barbara Lupo, Kosuke Yusa, Livio Trusolino, Mathew Garnett, Andrea Bertotti, Chris Bakal, Ultan McDermott. Genome-wide CRISPR screens reveal Hippo pathway activation as a resistance mechanism in BRAF mutant colon 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 1081.

Abstract Drug resistance is ultimately the cause of death for most cancer patients – even initially strong responses to treatment are usually followed by the emergence of resistance over time. This suggests the existence of residual or persistent cancer cells, creating a reservoir that ultimately gives rise to stable resistance. These drug tolerant persisters (‘the deadly survivors’) have been described for over a decade in numerous studies; they are often present as a minor fraction of the total tumour population and may exploit non-genetic (transcriptional) programs to allow the cells to survive drug treatment. 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. We carried out parallel genome-wide CRISPR gain and loss of function screens in EGFR mutant lung cancer cell lines treated with EGFR inhibitors, to identify the genes and pathways that may be important in enabling the survival of persister cells. We observed recurrent resistant genes in previously identified resistance pathways including PI3K (PTEN, TSC1, TSC2), MAPK (KRAS, NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, KEAP1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockout demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, key activators of the Hippo pathway. A closer review of the CRISPR screen data confirmed that many resistance hits are members of this pathway - upstream regulators (NF2, AMOTL2), core signalling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signalling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and increased expression of canonical Hippo transcriptional targets. We therefore reasoned that the Hippo pathway might be involved in maintaining the survival of drug tolerant persister cells in this setting. Acute treatment of EGFR mutant cell lines with osimertinib was associated with increased nuclear localisation of YAP1 and WWTR1 and increased expression of canonical Hippo transcriptional targets. Furthermore, the combination of osimertinib and a TEAD inhibitor (K-975) almost completely abolished the survival of drug tolerant persister cells following treatment, indicating that this pathway is an important survival mechanism following drug treatment. Consequently, we propose Hippo signalling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Ercia Banks, Nin Guan, Shanade Dunn, Maria Lisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer [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 P066.

Multiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

Background: Almost 10% of colon cancers harbor the canonical activating V600 BRAF mutation that in melanoma cancers has been shown to dramatically sensitize cells to targeted therapy and prolong survival. In the colon, such tumors are associated with worse survival. Recent clinical trials of combination therapies targeting the EGFR and MAPK pathways in these tumors have demonstrated impressive response rates. However, the clinical benefit has been limited by the rapid emergence of drug resistance. Identifying and targeting the genes complicit in this process and thus re-sensitize resistant cancer cells may be of clinical benefit. The landscape of drug resistance in cancer patients is composed of (a) loss-of-function (LoF), (b) gain-of-function (GoF) and (c) point mutation-mediated perturbations of genes, with often no single mechanism being predominant. Therefore, capturing the full breadth of resistance genes for any drug would require multiple genetic screens to be executed in parallel. Methods: We performed 3 genome-wide genetic screens in parallel in BRAF mutant colon cancer cells treated with a BRAF/MEK/EGFR inhibitor combination, to capture all of the resistance mechanisms described above. To screen for LoF events we used a genome-wide CRISPR/Cas9 sgRNA library. For GoF resistance events, a lentiviral-based insertional mutagenesis vector was used to randomly integrate the SFFV enhancer/promoter sequence throughout the genome. Finally, to saturate the genome of each cell line with point mutations we used N-Ethyl-N-nitrosourea (ENU) to randomly mutagenize every base in the genome. Each genetic screen was analysed separately to identify the relevant genes and pathways that confer drug resistance before aggregating the outputs of all three screens for a more comprehensive view of the drug resistance landscape. Results: The most recurrent and strongest hit from the CRISPR knockout screen was the inhibitor of Src family kinases, CSK. A Gaussian Kernal Convolution analysis for recurrent integrations detected in resistant clones following the insertional mutagenesis screen identified five genes as likely resistance candidates - BRAF, SOS1, MET, FRS2 and KRAS. Finally, Illumina exome sequencing of resistant clones following ENU mutagenesis revealed statistical enrichment for non-synonymous point mutations in three genes in the MAPK pathway, namely MAP2K1, NRAS and KRAS. Of note, loss of CSK was also shown to confer resistance to targeted therapies in other cancer types. Conclusion: Genome-wide genetic screens identified genes in the MAPK pathway likely to cause resistance clinically, and many of which are amenable to therapeutic targeting. Of note, the resistance as a result of loss of CSK instead activates Src signalling. It can be overcome through the addition of a Src inhibitor, and may be relevant across a range of cancer types as a resistance mechanism. Citation Format: Jonathan Brammeld, Marco Ranzani, Elizabeth A. Coker, Stacey Price, Theodoros I. Roumeliotis, Barbara Lupo, Mi Petljak, Steven P. Williams, Francesco Iorio, Francesco Sassi, Nanne Aben, Kosuke Yusa, Livio Trusolino, Lodewyk Wessels, David J. Adams, Mathew Garnett, Bissan Al-Lazikani, Jyoti S. Choudhary, Andrea Bertotti, Ultan McDermott. Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1821.

Cancer hallmarks are evolutionary traits required by a tumour to develop. While extensively characterised, the way these traits are achieved through the accumulation of somatic mutations in key biological pathways is not fully understood. To shed light on this subject, we characterised the landscape of pathway alterations associated with somatic mutations observed in 4,415 patients across ten cancer types, using 374 orthogonal pathway gene-sets mapped onto canonical cancer hallmarks. Towards this end, we developed SLAPenrich: a computational method based on population-level statistics, freely available as an open source R package. Assembling the identified pathway alterations into sets of hallmark signatures allowed us to connect somatic mutations to clinically interpretable cancer mechanisms. Further, we explored the heterogeneity of these signatures, in terms of ratio of altered pathways associated with each individual hallmark, assuming that this is reflective of the extent of selective advantage provided to the cancer type under consideration. Our analysis revealed the predominance of certain hallmarks in specific cancer types, thus suggesting different evolutionary trajectories across cancer lineages. Finally, although many pathway alteration enrichments are guided by somatic mutations in frequently altered high-confidence cancer genes, excluding these driver mutations preserves the hallmark heterogeneity signatures, thus the detected hallmarks’ predominance across cancer types. As a consequence, we propose the hallmark signatures as a ground truth to characterise tails of infrequent genomic alterations and identify potential novel cancer driver genes and networks.

Abstract Purpose: Despite intense research, treatment options for patients with mesothelioma are limited and offer only modest survival advantage. We screened a large panel of compounds in multiple mesothelioma models and correlated sensitivity with a range of molecular features to detect biomarkers of drug response. Experimental design: We utilized a high-throughput chemical inhibitor screen in a panel of 889 cancer cell lines, including both immortalized and primary early-passage mesothelioma lines, alongside comprehensive molecular characterization using Illumina whole-exome sequencing, copy-number analysis and Affymetrix array whole transcriptome profiling. Subsequent validation was done using functional assays such as siRNA silencing and mesothelioma mouse xenograft models. Results: A subgroup of immortalized and primary MPM lines appeared highly sensitive to FGFR inhibition. None of these lines harbored genomic alterations of FGFR family members, but rather BAP1 protein loss was associated with enhanced sensitivity to FGFR inhibition. This was confirmed in an MPM mouse xenograft model and by BAP1 knockdown and overexpression in cell line models. Gene expression analyses revealed an association between BAP1 loss and increased expression of the receptors FGFR1/3 and ligands FGF9/18. BAP1 loss was associated with activation of MAPK signaling. These associations were confirmed in a cohort of MPM patient samples. Conclusions: A subgroup of mesotheliomas cell lines harbor sensitivity to FGFR inhibition. BAP1 protein loss enriches for this subgroup and could serve as a potential biomarker to select patients for FGFR inhibitor treatment. These data identify a clinically relevant MPM subgroup for consideration of FGFR therapeutics in future clinical studies. Clin Cancer Res; 24(1); 84–94. ©2017 AACR.

Drug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases, this is the consequence of specific gene mutations that have the potential to be targeted to resensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here, we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We used an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug-resistant patients.