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Abstract Neuroendocrine (NE) transformation is a mechanism of resistance to targeted therapy in lung and prostate adenocarcinomas leading to poor prognosis. Up to date, even if patients at high risk of transformation can be identified by the occurrence of Tumor Protein P53 (TP53) and Retinoblastoma Transcriptional Corepressor 1 (RB1) mutations in their tumors, no therapeutic strategies are available to prevent or delay histological transformation. Upregulation of the cell cycle kinase Cell Division Cycle 7 (CDC7) occurred in tumors during the initial steps of NE transformation, already after TP53/RB1 co-inactivation, leading to induced sensitivity to the CDC7 inhibitor simurosertib. CDC7 inhibition suppressed NE transdifferentiation and extended response to targeted therapy in in vivo models of NE transformation by inducing the proteasome-mediated degradation of the MYC Proto-Oncogen (MYC), implicated in stemness and histological transformation. Ectopic overexpression of a degradation-resistant MYC isoform reestablished the NE transformation phenotype observed on targeted therapy, even in the presence of simurosertib. CDC7 inhibition also markedly extended response to standard cytotoxics (cisplatin, irinotecan) in lung and prostate small cell carcinoma models. These results nominate CDC7 inhibition as a therapeutic strategy to constrain lineage plasticity, as well as to effectively treat NE tumors de novo or after transformation. As simurosertib clinical efficacy trials are ongoing, this concept could be readily translated for patients at risk of transformation.

Abstract Background Lineage plasticity, the ability to transdifferentiate among distinct phenotypic identities, facilitates therapeutic resistance in cancer. In lung adenocarcinomas (LUADs), this phenomenon includes small cell and squamous cell (LUSC) histologic transformation in the context of acquired resistance to targeted inhibition of driver mutations. LUAD-to-LUSC transdifferentiation, occurring in up to 9% of EGFR-mutant patients relapsed on osimertinib, is associated with notably poor prognosis. We hypothesized that multi-parameter profiling of the components of mixed histology (LUAD/LUSC) tumors could provide insight into factors licensing lineage plasticity between these histologies. Methods We performed genomic, epigenomics, transcriptomics and protein analyses of microdissected LUAD and LUSC components from mixed histology tumors, pre-/post-transformation tumors and reference non-transformed LUAD and LUSC samples. We validated our findings through genetic manipulation of preclinical models in vitro and in vivo and performed patient-derived xenograft (PDX) treatments to validate potential therapeutic targets in a LUAD PDX model acquiring LUSC features after osimertinib treatment. Results Our data suggest that LUSC transdifferentiation is primarily driven by transcriptional reprogramming rather than mutational events. We observed consistent relative upregulation of PI3K/AKT, MYC and PRC2 pathway genes. Concurrent activation of PI3K/AKT and MYC induced squamous features in EGFR-mutant LUAD preclinical models. Pharmacologic inhibition of EZH1/2 in combination with osimertinib prevented relapse with squamous-features in an EGFR-mutant patient-derived xenograft model, and inhibition of EZH1/2 or PI3K/AKT signaling re-sensitized resistant squamous-like tumors to osimertinib. Conclusions Our findings provide the first comprehensive molecular characterization of LUSC transdifferentiation, suggesting putative drivers and potential therapeutic targets to constrain or prevent lineage plasticity.

Abstract Introduction Small Cell Lung Cancer (SCLC) can be classified into transcriptional subtypes with distinct degrees of neuroendocrine (NE) differentiation. Recent evidence supports plasticity among subtypes with a bias toward adoption of low-NE states during disease progression or upon acquired chemotherapy resistance. Here, we identify a role for SMARCA4, the catalytic subunit of the SWI/SNF complex, as a regulator of subtype shift in SCLC. Methods ATACseq and RNAseq experiments were performed in SCLC cells after pharmacological inhibition of SMARCA4. DNA binding of SMARCA4 was characterized by ChIPseq in high-NE SCLC patient derived xenografts (PDXs). Enrichment analyses were applied to transcriptomic data. Combination of FHD-286 and afatinib was tested in vitro and in a set of chemo-resistant SCLC PDXs in vivo. Results SMARCA4 expression positively correlates with that of NE genes in both SCLC cell lines and patient tumors. Pharmacological inhibition of SMARCA4 with FHD-286 induces the loss of NE features and downregulates neuroendocrine and neuronal signaling pathways while activating non-NE factors. SMARCA4 binds to gene loci encoding NE-lineage transcription factors ASCL1 and NEUROD1 and alters chromatin accessibility, enhancing NE programs. Enrichment analysis applied to high-confidence SMARCA4 targets confirmed neuron related pathways as the top GO Biological processes regulated by SMARCA4 in SCLC. In parallel, SMARCA4 also controls REST, a known suppressor of the NE phenotype, by regulating SRRM4-dependent REST transcript splicing. Furthermore, SMARCA4 inhibition drives ERBB pathway activation in SCLC, rendering SCLC tumors sensitive to afatinib. Conclusions This study nominates SMARCA4 as a key regulator of the NE state plasticity and defines a novel therapeutic strategy for SCLC.

Summary: We report a protocol for obtaining high-quality single-cell transcriptomics data from human lung biospecimens acquired from core needle biopsies, fine-needle aspirates, surgical resection, and pleural effusions. The protocol relies upon the brief mechanical and enzymatic disruption of tissue, enrichment of live cells by fluorescence-activated cell sorting (FACS), and droplet-based single-cell RNA sequencing (scRNA-seq). The protocol also details a procedure for analyzing the scRNA-seq data.For complete details on the use and execution of this protocol, please refer to Chan et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: This standardized protocol describes the preparation of PCR amplified and purified samples from human cell lines passaged and collected from CRISPR screening. High-quality samples can be used to perform next-generation sequencing (NGS) to uncover changes in sgRNA abundance from the timepoint at which library-transduced cells are selected to the timepoint when the screen is ended. Here, we describe proper calculation methods for library representation and show how to overcome potential issues often encountered by researchers.For complete information on the use and execution of this protocol, please refer to Wohlhieter et al. (2020).

Summary: Concurrent loss-of-function mutations in STK11 and KEAP1 in lung adenocarcinoma (LUAD) are associated with aggressive tumor growth, resistance to available therapies, and early death. We investigated the effects of coordinate STK11 and KEAP1 loss by comparing co-mutant with single mutant and wild-type isogenic counterparts in multiple LUAD models. STK11/KEAP1 co-mutation results in significantly elevated expression of ferroptosis-protective genes, including SCD and AKR1C1/2/3, and resistance to pharmacologically induced ferroptosis. CRISPR screening further nominates SCD (SCD1) as selectively essential in STK11/KEAP1 co-mutant LUAD. Genetic and pharmacological inhibition of SCD1 confirms the essentiality of this gene and augments the effects of ferroptosis induction by erastin and RSL3. Together these data identify SCD1 as a selective vulnerability and a promising candidate for targeted drug development in STK11/KEAP1 co-mutant LUAD.

Background: Fibroblast growth factor receptor (FGFR)1 and FGFR4 have been associated with tumorigenesis in a variety of tumour types. As a therapeutic approach, their inhibition has been attempted in different types of malignancies, including lung cancer, and was initially focused on FGFR1-amplified tumours, though with limited success. Methods: In vitro and in vivo functional assessments of the oncogenic potential of downregulated/overexpressed genes in isogenic cell lines were performed, as well as inhibitor efficacy tests in vitro and in vivo in patient-derived xenografts (PDXs). mRNA was extracted from FFPE non-small cell lung cancer samples to determine the prognostic potential of the genes under study. Findings: We provide in vitro and in vivo evidence showing that expression of the adhesion molecule N-cadherin is key for the oncogenic role of FGFR1/4 in non-small cell lung cancer. According to this, assessment of the expression of genes in different lung cancer patient cohorts showed that FGFR1 or FGFR4 expression alone showed no prognostic potential, and that only co-expression of FGFR1 and/or FGFR4 with N-cadherin inferred a poorer outcome. Treatment of high-FGFR1 and/or FGFR4-expressing lung cancer cell lines and patient-derived xenografts with selective FGFR inhibitors showed high efficacy, but only in models with high FGFR1/4 and N-cadherin expression. Interpretation: Our data show that the determination of the expression of FGFR1 or FGFR4 alone is not sufficient to predict anti-FGFR therapy efficacy; complementary determination of N-cadherin expression may further optimise patient selection for this therapeutic strategy. Keywords: FGFR1, FGFR4, N-cadherin, Predictive biomarker, FGFR inhibitors

Abstract Background The high incidence and mortality of lung tumours is a major health problem. Therefore, the identification both of biomarkers predicting efficacy for therapies in use and of novel efficacious therapeutic agents is crucial to increase patient survival. MAP17 (PDZK1IP1) is a small membrane-bound protein whose upregulation is reported as a common feature in tumours from diverse histological origins. Furthermore, MAP17 is correlated with tumour progression. Methods We assessed the expression of MAP17 in preclinical models, including cell lines and patient-derived xenografts (PDXs), assessing its correlation with sensitivity to different standard-of-care drugs in lung adenocarcinoma, as well as novel drugs. At the clinical level, we subsequently correlated MAP17 expression in human tumours with patient response to these therapies. Results We show that MAP17 expression is induced during lung tumourigenesis, particularly in lung adenocarcinomas, and provide in vitro and in vivo evidence that MAP17 levels predict sensitivity to therapies currently under clinical use in adenocarcinoma tumours, including cisplatin, carboplatin and EGFR inhibitors. In addition, we show that MAP17 expression predicts proteasome inhibitor efficacy in this context and that bortezomib, an FDA-approved drug, may be a novel therapeutic approach for MAP17-overexpressing lung adenocarcinomas. Conclusions Our results indicate a potential prognostic role for MAP17 in lung tumours, with particular relevance in lung adenocarcinomas, and highlight the predictive pot0065ntial of this membrane-associated protein for platinum-based therapy and EGFR inhibitor efficacy. Furthermore, we propose bortezomib treatment as a novel and efficacious therapy for lung adenocarcinomas exhibiting high MAP17 expression.

Heat shock protein 90 (HSP90) is an important chaperone in lung adenocarcinoma, with relevant protein drivers such as EGFR (epidermal growth factor receptor) and EML4-ALK (echinoderm microtubule-associated protein-like protein4 fused to anaplastic lymphoma kinase) depending on it for their correct function, therefore HSP90 inhibitors show promise as potential treatments for lung adenocarcinoma. To study responses to its inhibition, HSP90 was pharmacologically interrupted by geldanamycin and resorcinol derivatives or with combined inhibition of HSP90 plus HSP70 in lung adenocarcinoma cell lines. Two-dimensional electrophoresis was performed to identify proteomic profiles associated with inhibition which will help to understand the biological basis for the responses. HSP90 inhibition resulted in altered protein profiles that differed according the treatment condition studied. Results revealed 254 differentially expressed proteins after treatments, among which, eukaryotic translation initiation factor3 subunit I (eIF3i) and citrate synthase demonstrated their potential role as response biomarkers. The differentially expressed proteins also enabled signalling pathways involved in responses to be identified; these included apoptosis, serine-glycine biosynthesis and tricarboxylic acid cycle. The proteomic profiles identified here contribute to an improved understanding of HSP90 inhibition and open possibilities for the detection of potential response biomarkers which will be essential to maximize treatment efficacy in lung adenocarcinoma.