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Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis. Non-small cell lung cancer (NSCLC) is often associated with metastasis to the lungs. Here, the authors perform independent screens and identify NuRD as a co-repressor of ZEB1, and demonstrate TBC1D2b as a downstream target of ZEB1/NuRD complex regulating NSCLC metastasis.

Molecular alterations in the PI3K/AKT pathway occur frequently in hormone receptor-positive breast tumors. Patients with ER-positive, HER2-negative metastatic breast cancer are often treated with CDK4/6 inhibitors such as palbociclib in combination with endocrine therapy. Although this is an effective regimen, most patients ultimately progress. The purpose of this study was identifying synthetic lethality partners that can enhance palbociclib’s antitumor efficacy in the presence of PIK3CA / AKT1 mutations. We utilized a barcoded shRNA library to determine critical targets for survival in isogenic MCF7 cells with PIK3CA / AKT1 mutations. We demonstrated that the efficacy of palbociclib is reduced in the presence of PIK3CA / AKT1 mutations. We also identified that the downregulation of discoidin domain receptor 1 ( DDR1 ) is synthetically lethal with palbociclib. DDR1 knockdown and DDR1 pharmacological inhibitor decreased cell growth and inhibited cell cycle progression in all cell lines, while enhanced the sensitivity of PIK3CA/AKT1 mutant cells to palbociclib. Combined treatment of palbociclib and 7rh further induced cell cycle arrest in PIK3CA / AKT1 mutant cell lines. In vivo, 7rh significantly enhanced palbociclib’s antitumor efficacy. Our data indicates that DDR1 inhibition can augment cell cycle suppressive effect of palbociclib and could be effective strategy for targeted therapy of ER-positive, HER2-negative breast cancers with PI3K pathway activation.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development. Statement of significance PDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors. Arginine methylation by PRMTs is dysregulated in cancer. Here, the authors use functional genomics screens and identify PRMT1 as a vulnerability in pancreatic ductal adenocarcinoma, and further show that PRMT1 regulates RNA metabolism and coordinates expression of genes in cell cycle progression, maintaining genomic stability and tumour growth.

Most triple negative breast cancers (TNBCs) are aggressively metastatic with a high degree of intra-tumoral heterogeneity (ITH), but how ITH contributes to metastasis is unclear. Here, clonal dynamics during metastasis were studied in vivo using two patient-derived xenograft (PDX) models established from the treatment-naive primary breast tumors of TNBC patients diagnosed with synchronous metastasis. Genomic sequencing and high-complexity barcode-mediated clonal tracking reveal robust alterations in clonal architecture between primary tumors and corresponding metastases. Polyclonal seeding and maintenance of heterogeneous populations of low-abundance subclones is observed in each metastasis. However, lung, liver, and brain metastases are enriched for an identical population of high-abundance subclones, demonstrating that primary tumor clones harbor properties enabling them to seed and thrive in multiple organ sites. Further, clones that dominate multi-organ metastases share a genomic lineage. Thus, intrinsic properties of rare primary tumor subclones enable the seeding and colonization of metastases in secondary organs in these models. It is unclear how intra-tumoral heterogeneity contributes to metastasis. Here the authors study the clonal dynamics of triple negative breast cancer metastasis using patient derived xenografts and demonstrate that primary tumor clones harbor properties that support seeding and colonization of multiple organs.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.

The implementation of cancer immunotherapeutics for solid tumors including lung cancers has improved clinical outcomes in a small percentage of patients. However, the majority of patients show little to no response or acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and novel combination therapy approaches is imperative to improve responses to immune checkpoint inhibitors. To address this, we performed an in vivo shRNA dropout screen that focused on genes encoding for FDA-approved drug targets (FDAome). We implanted epithelial and mesenchymal Kras/p53 (KP) mutant murine lung cancer cells expressing the FDAome shRNA library into syngeneic mice treated with an anti-PD-1 antibody. Sequencing for the barcoded shRNAs revealed Ntrk1 was significantly depleted from mesenchymal tumors challenged with PD-1 blockade, suggesting it provides a survival advantage to tumor cells when under immune system pressure. Our data confirmed Ntrk1 transcript levels are upregulated in tumors treated with PD-1 inhibitors. Additionally, analysis of tumor-infiltrating T cell populations revealed that Ntrk1 can promote CD8+ T cell exhaustion. Lastly, we found that Ntrk1 regulates Jak/Stat signaling to promote expression of PD-L1 on tumor cells. Together, these data suggest that Ntrk1 activates Jak/Stat signaling to regulate expression of immunosuppressive molecules including PD-L1, promoting exhaustion within the tumor microenvironment.

Colorectal cancer (CRC) is a heterogeneous disease showing significant variability in clinical aggressiveness. Primary and acquired resistance limits the efficacy of available treatments, and identification of effective drug combinations is needed to further improve patients’ outcomes. We previously found that the NEDD8-activating enzyme inhibitor pevonedistat induced tumor stabilization in preclinical models of poorly differentiated, clinically aggressive CRC resistant to available therapies. To identify drugs that can be effectively combined with pevonedistat, we performed a “drop-out” loss-of-function synthetic lethality screening with an shRNA library covering 200 drug-target genes in four different CRC cell lines. Multiple screening hits were found to be involved in the EGFR signaling pathway, suggesting that, rather than inhibition of a specific gene, interference with the EGFR pathway at any level could be effectively leveraged for combination therapies based on pevonedistat. Exploiting both BRAF-mutant and RAS/RAF wild-type CRC models, we validated the therapeutic relevance of our findings by showing that combined blockade of NEDD8 and EGFR pathways led to increased growth arrest and apoptosis both in vitro and in vivo. Pathway modulation analysis showed that compensatory feedback loops induced by single treatments were blunted by the combinations. These results unveil possible therapeutic opportunities in specific CRC clinical settings.

BackgroundUveal melanoma (UM) is a rare tumor characterized by mutually exclusive activating mutations in GNAQ in GNA11, followed by secondary events in BAP1, SF3B1 and EIF1AX. Notably, a large subset of patients presents with copy-loss of chromosome 3 (monosomy 3), which is highly associated with metastatic progression in late-stage disease. Monosomy 3 tumors demonstrate a marked resistance to chemotherapy, targeted therapeutics, and immunotherapy, despite successes observed in cutaneous melanoma.Informing on the biology underlying chromosome 3 copy-loss, and its impact on the tumor microenvironment, is critical towards directing future efforts in targeted therapeutics and immunotherapy. Current limited insight can be attributed to both lack of: (a) preclinical models, and (b) in-depth characterization of paired primary and metastatic tumors in patients.MethodsTo address this, we first induced chromosome 3 copy-loss through CRISPR-based centromere targeting in a well characterized Disomy 3 UM cell line. Disomy 3 (D3) and Monosomy 3 (M3) clones derived from these efforts have enabled us to develop patient derived xenograft (PDX) models to compare D3 and M3 behavior in paired primary and metastatic settings.Additionally, we identified and collected a range of match-paired (primary and metastatic) clinical UM samples across multiple patients.Leveraging these unique samples, we applied spatial transcriptomics to inform on tumor intrinsic and extrinsic features of progressive UM, identifying gene signatures of disease advancement and deconvoluting the evolving tumor microenvironment.ResultsInvestigation of metastatic UM tumor heterogeneity in our models enables us to characterize unique features of phenotypically transformed clones (e.g. depigmentation and growth advantage) (figure 1). We also adapted existing methodology to infer chromosomal copy number events from spatial transcriptomics data to our PDX system, overcoming the lack of same-species microenvironment controls. We complement our preclinical analyses with an investigation of spatial heterogeneity in a patient cohort of paired primary and metastatic tumors (figure 2). Leveraging single-cell deconvolution in this paired dataset, we captured unique immune microenvironments in primary vs. metastatic tumors. Additionally, we integrated our preclinical tumor intrinsic signatures to pair differential gene expression signatures of tumor sub-clones with differential immune cell populations.ConclusionsOur methodology allows for deep characterization of sub-clonal heterogeneity in primary and metastatic settings and informs on the unique microenvironmental heterogeneity underlying invasiveness and outgrowth of M3 tumors. More broadly, comparing these preclinical and patient tumors provides an opportunity to expand on our knowledge of metastatic disease drivers and derive prognostic signatures associated with poor survival and lack of response to immunotherapy.Abstract 1498 Figure 1Spatial Transcriptomics Informs on Liver Outgrowth Phenotypes in Engineered UM Preclinical Models. (A) Spatial slides of two Disomy 3 tumors in the mouse liver and matched clustering following the removal of mouse background demonstrated with differential pigrnentahon and tumor size. (B) Corresponding copy number inference of the D3.5_2 slide demonstrates our ability to identify subclones based on copy number heterogeneity. (C) Spatial slides of two Monosomy 3 tumors in the mouse liver and matched clustering following the removal of mouse background. (D) Corresponding copy number inference of the M3.8_2 slide unable to accurately capture copy number in the absence of microenvironment as a normal control demonstrated by chromosome 3 being inferred as copy neutralAbstract 1498 Figure 2Spatial Transcriptomics on Uveal Melanoma Clinical Samples — Paired Primary Eye and Liver Metastases. (A) Spatial slide with matched primary eye and liver sections from Patient #1. (B) Spatial slide with matched primary eye and liver sections from Patient #2. (C) Predicted cell types observed in matched primary and liver tumors of Patient #1. (D) Predicted cell types in matched primary and liver tumors of Patient #2

Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed 1 , 2 . Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n  = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P  = 0.001; RFS, 64% versus 32% at 2 years, P  = 0.021). The findings were validated in several additional cohorts 2 – 4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy ( n  = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy ( P  = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control ( P  = 0.0006 and P  = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice ( P  = 0.018 and P  = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients ( P  = 0.021 and P  < 0.0001). Together, these results have important implications for therapy. Treatment with neoadjuvant BRAF/MEK-targeted therapy results in higher rates of major pathological response in female compared with male patients with melanoma, and pharmacological inhibition of androgen receptor signalling improved the responses of male and female mice to BRAF/MEK-targeted therapy.