Small cell lung cancer (SCLC) is a highly aggressive cancer with limited treatment options and a generally poor prognosis that has not appreciably changed despite recently approved therapies. Importantly, therapeutic options for SCLC patients have been focused on unselected populations as SCLC was thought to be a relatively homogenous malignancy in the past. Recently, our group and others identified four major distinct subgroups of SCLC (Gay CM et al. Cancer Cell 2021). Three of the four subtypes are defined by the predominant expression of a specific transcription factor, ASCL1 (SCLC-A), NEUROD1 (SCLC-N) and POU2F3 (SCLC-P) while the fourth subtype is defined by an inflamed phenotype (SCLC-I).

While osimertinib has resulted in striking improvements in outcomes for patients with NSCLC harboring classical EGFR mutations (Ex19del, L858R, and/or T790M), patients with atypical EGFR mutations have shown heterogeneous and in some cases inferior responses to EGFR inhibitors. The frequency, structural and clinical implications of atypical EGFR mutations are less understood.

The novel coronavirus SARS-CoV-2 is the cause of the respiratory illness COVID-19—a global pandemic affecting over 4 million individuals worldwide. Viruses efficiently replicate by hijacking host cell machinery to obtain macromolecules and energy by similar mechanisms as cancer cells. Since viral infection is known to alter cellular nutrient requirements, this study explores the metabolites and metabolic pathways associated with SARS-CoV-2 infection. Bulk and single-cell seequencing data from cell lines and tumor samples were retrieved from publically available datasets. Transcriptional data were retrieved from publically available datasets of gefitinib- and erlotinib-resistant EGFR-mutant cell lines and Calu3 and A549 cells mock treated or infected with SARS-CoV-2. Single-cell RNAseq datasets of EGFR-mutant PC-9 mock and osimertinib treated were downloaded from GEO. 225 metabolites were profiled in CCLE cell lines using LC-MS. To identify metabolic features of cells able to be infected by SARS-CoV-2 via the ACE2 receptor, metabolites associated with ACE2 expression were investigated. ACE2 expression positively correlates with glutamine in upper aerodigestive tract cell lines. Consistent with this, ACE2 expression was examined against a list of 253 metabolism-associated genes and GLUL, which encodes an enzyme (glutamine synthetase) responsible for conversion of glutamate to glutamine, was significantly positively correlated in NSCLC, HNSCC, and SCLC cell lines and confirmed in human tumor datasets. Additionally, GLS, which encodes the enzyme (glutaminase) that catalyzes the opposing reaction, is negatively correlated with ACE2 expression. Further, we analyzed RNA sequencing data from NSCLC cell lines infected with SARS-CoV-2 for 24 hours and revealed that upon infection there is a down regulation of GLUL signifying a metabolic-shift away from glutamine as the cells undergo EMT. We show that SARS-CoV-2 targeting of ACE-2 expressing, metabolically-primed epithelial cells is advantageous to exploit the abundance of glutamine to synthesize nucleotides for rapid replication and viral spread.

While EGFR mutant NSCLC patients are initially responsive to EGFR targeted therapies, resistant disease inevitably emerges. In nearly half of resistance cases, tumors lack secondary EGFR mutations such as T790M and are refractory to 2nd- and 3rd-generation EGFR tyrosine kinase inhibitors (TKI). We and others have also observed that EGFR-independent resistant tumor cells may undergo a histologic and functional transformation through epithelial-to-mesenchymal transition (EMT) (Byers et al., 2013; Chung et al., 2011; Uramoto et al., 2010; Zhang et al., 2012), which can occur concurrently with other genomic alterations. The lack of treatment regimens with efficacy against EGFR-independent EGFR TKI resistance remains a major clinical challenge. We investigated transcriptomic and proteomic alterations that occur in NSCLC cells with acquired resistance to EGFR TKIs that occurs independent of EGFR and c-Met and screened >1,300 compounds to identify targetable vulnerabilities. T790M-negative EGFR TKI resistance was associated with evidence of a mesenchymal transition along with increased activation of the YAP/FOXM1 transcriptional program and a broad-spectrum multidrug resistance phenotype. EGFR TKI resistant cells displayed increased expression of spindle assembly checkpoint (SAC) proteins PLK1, Aurora kinases, survivin, and KSP, and expression of these proteins was dependent on the YAP/FOXM1 axis. Consistent with recent reports (Bertran-Alamillo et al., 2019; Shah et al., 2019), EGFR TKI resistant cells were found to be sensitive to aurora kinase inhibitors. We further determined that EGFR TKI resistant cells were likewise highly sensitive to inhibitors of components of the spindle assembly checkpoint (SAC) pathway including PLK1, KSP, and survivin, and treatment with these agents resulted in the accumulation of cells in the G2/M phase of the cell cycle and mitotic catastrophe. Using a patient-derived model of T790M negative EGFR TKI resistance, we observed that treatment with SAC component inhibitors, alisertib, ispinesib, or volasertib significantly inhibited tumor growth compared with vehicle-treated tumors. Analysis of NSCLC clinical data revealed that FOXM1 expression correlated with expression of SAC components including PLK1, Aurora kinases, KSP, and survivin. Moreover, in EGFR mutant NSCLC patients, high FOXM1 expression was associated with a worse clinical outcome compared to EGFR mutant NSCLC patients with low expression of FOXM1. In resistant models, targeting of YAP reduced FOXM1 expression and expression of SAC components. In conclusion, we provide novel insights into the molecular alterations associated with EGFR TKI resistance and demonstrate that upregulation of SAC components in EGFR TKI resistant cells occurs through the activation of the YAP/FOXM1 pathway. These results support the future targeting of these pathways in NSCLC patients with EGFR-independent resistance to EGFR-targeted agents.

Patients with thoracic cancers affected by the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), appear to have poor clinical outcomes. AXL, a TAM (Tyro3, AXL, Mer) family receptor tyrosine kinase, is a known mediator of epithelial to mesenchymal transition (EMT) and therapeutic resistance in non-small cell lung cancer (NSCLC) and other cancers. Additionally, AXL plays a role in efficient Ebola and Zika viral entry and infection and AXL inhibition has demonstrated antiviral activities. Recently, bemcentinib, a highly selective and potent AXL inhibitor with antiviral activity, has been fast-tracked as the first potential treatment for assessment in the United Kingdom's ACcelerating COVID-19 Research & Development (ACCORD) multicenter, randomized phase II trial. We analyzed mRNA expression of AXL and other TAM family members as well as angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, in treatment-naïve (n=1016) and previously treated (n=239) NSCLC tumors and in a panel of NSCLC cell lines (n=70). We also analyzed AXL mRNA levels in NSCLC cell lines (n=3) infected with SARS-CoV-2. In treatment-naïve and previously-treated NSCLC tumors, AXL mRNA expression was higher in mesenchymal tumors, as expected, and inversely correlated with ACE2. Similarly, in NSCLC cell lines, high ACE2 expression was associated with low AXL mRNA and protein expression. Notably, expression of ACE2 was downregulated while that of AXL and ZEB1, an EMT transcription factor, were upregulated in NSCLC cells infected with SARS-CoV-2 as compared to mock infected cells, suggesting a shift to a more mesenchymal phenotype. Treatment with bemcentinib for 24h downregulated ZEB1 expression in mesenchymal cell lines, reversing EMT. These data, in the context of ACE2's role in preventing acute respiratory distress syndrome, suggest a shift from ACE2-expressing epithelial cells to a more mesenchymal phenotype characterized by low ACE2 and high AXL expression, upon infection of NSCLC cells with SARS-CoV-2. In addition to bemcentinib's antiviral activity, it can also reverse EMT, further supporting AXL and EMT as novel therapeutic targets for COVID-19 treatment.

SARS-CoV-2 infection is the cause of the respiratory illness COVID-19, which presents most frequently with respiratory symptoms. SARS-CoV-2 cell entry requires interactions with ACE2 and TMPRSS2 on the surface of the host cell. Cancer patients and, specifically, those with thoracic malignancies seem to experience poorer clinical outcomes. We utilized bulk and single-cell transcriptional data from a combination of normal and malignant tissues and cells from aerodigestive and respiratory tracts to explore mechanisms governing the expression of ACE2 and TMPRSS2. Additionally, we determined the effect of EMT induction, ZEB1 modulation, and SARS-CoV-2 infection on ACE2 expression. Our bulk data suggests that aerodigestive and lung cancer models express a broad range of ACE2 and TMRPSS2, particularly in epithelial cells, and would serve as good models for studying SARS-CoV-2 infection. We assessed the relationship between ACE2 and epithelial differentiation in numerous datasets, and found consistent positive correlations with transcriptional and microRNA signifiers of epithelial differentiation. The miR-200 family – zinc finger E-box-binding homeobox 1 (ZEB1) pathway, which is an established regulator of EMT, also directly regulates ACE2 expression, likely via putative ZEB1 repressor sites located in the ACE2 promoter. Furthermore, SARS-CoV-2 infection reduces ACE2 expression and shifts cells to a more mesenchymal phenotype with loss of EPCAM and upregulation of ZEB1 and other EMT-associated genes. ACE2-positive cells are almost exclusively epithelial and unexpectedly rare, considering the devastating impact of this infection. Following viral entry, SARS-CoV-2 infection induces molecular changes within the cells that are reminiscent of EMT, including increased ZEB1. ZEB1, in turn, appears to directly repress the expression of ACE2. This SARS-CoV-2-induced ACE2 deficiency, compounded by the downregulation of genes, including claudins, which play a critical role in restricting epithelial and endothelial permeability, exposes respiratory cells to increased risk of edema and acute respiratory distress syndrome (ARDS).

Accounting for 15% of all lung cancer diagnoses, small cell lung cancer (SCLC) is an aggressive malignancy with dismal clinical outcomes, due in part to failure to define clinical biomarkers predictive of unique, targetable vulnerabilities. Recent data has begun to delineate molecular subsets of SCLC by uncovering inter-tumoral heterogeneity in features such as DNA damage response, EMT, and neuroendocrine (NE) status. However, it remains unclear whether the subsets defined by these features are predictive of response to cancer therapies and could be employed as patient selection criteria. Using RNAseq data from 81 resected SCLC tumor samples and 62 SCLC cell lines, we applied non-negative matrix factorization (NMF) to optimize delineation of transcriptionally defined clusters. Reverse phase protein array (RPPA) and drug response data for cell lines were analyzed post-clustering to compare features between clusters. Clustering analyses were validated in vivo using CTC-derived patient xenograft (CDX) models, while single-cell RNAseq (scRNAseq) from these same models was used to assess intratumoral heterogeneity among clusters. Results: NMF identifies four biologically distinct clusters among SCLC tumor samples and cell lines, each defined almost solely by differential expression of the transcription factors ASCL1 (SCLC-A, 36%), NEUROD1 (SCLC-N, 31%), and POU2F3 (SCLC-P, 16%), including a cluster defined by the absence of all three (SCLC-Inflamed/Mesenchymal, or SCLC-IM, 17%). SCLC-A are neuroendocrine, epithelial tumors with susceptibility to drug classes including BCL-2 inhibitors. SCLC-N are neuroendocrine, cMYC-high tumors with susceptibilities including Aurora kinase inhibitors that are neither epithelial nor mesenchymal. SCLC-P are non-neuroendocrine, epithelial tumors vulnerable to PARP inhibitors and nucleoside analogs. Lastly, SCLC-IM consists of mesenchymal, non-neuroendocrine tumors with high-expression of immune checkpoints, STING-related genes, and inflammatory markers that may represent those SCLC which are sensitive to immune checkpoint blockade. scRNAseq reveals intratumoral heterogeneity among cluster assignment within tumors that fluctuates coincident with the onset of therapeutic resistance. SCLC tumors can be assigned to one of four molecular subtypes on the basis of differential expression of three transcription factors. These subtype assignments reflect profound distinctions in underlying biology and susceptibility to a range of candidate drug classes. While subtype assignment on a single-cell basis within a tumor is largely homogeneous, rare cells from distinct subtypes (or representing multiple subtypes), as well as shifting assignments following treatment indicate the possibility of subtype-switching, or subtype-selection, as mechanisms of therapeutic resistance.

Recent success using immune checkpoint blockade (ICB) in the metastatic setting has raised the need to understand the immune microenvironment (IME) in early-stage disease. Moreover, pre-clinical evidence suggests that cytotoxic agents can modulate this IME. A recent study conducted by our group showed that non-small cell lung cancer (NSCLC) patients who received neoadjuvant chemotherapy followed by surgery (NCT), as compared to patients who received upfront surgery (US), had higher densities of CD3+ lymphocytes and CD68+ tumor-associated macrophages (TAMs). CD3+CD4+ lymphocytes and TAMs also correlated with better clinical outcomes. In this study, we explored the relationships between NCT and the IME by harvesting tumor samples of multiple surgical NSCLC cohorts. The PROSPECT microarray database was queried in NCT (n=45) and US (n=200) patients to investigate differentially expressed genes related to immunogenic cell death (ICD), susceptibility to CD8+ T cell and NK cell cytotoxicity, priming of antigen presenting cells, immunosuppressive enzymes and intra-tumoral cytokines. Available data from the ImmunogenomiC prOfiling of NSCLC (ICON) and other surgical NSCLC cohorts was evaluated to determine: 1) differential immune profiling using FACS (NCT=17; US=39) and multiplex IHC imaging (NCT=10; US=72); 2) plasma circulating cytokines (NCT=18; US=73); 3) tumor mutational burden (TMB) (NCT=40; US=61). Participants who received NCT or US were excluded according to these criteria: 1) concurrent treatment in addition to NCT; 2) sarcomatoid and small cell histologies; 3) clinical or pathological TNM Stage 4 disease; 4) synchronous malignancies other than lung. PROSPECT NCT patients expressed increased damage-associated molecular pattern (DAMP) genes (HSPA2, HSPA4, HSPE1, and S100A2; p<0.05) and T cell-related chemotaxis and antigen presentation genes (CXCR7, CD1A; p<0.05). Concordantly, the ICON cohort FACS results showed that NCT patients display increases in: 1) infiltration of CD8+ T cells (p=0.004); 2) proliferating Ki67+CD8+ T cells (p=0.02); 3) tissue resident memory CD8+CD103+ (p=0.02) and CD4+CD103+ non-Treg cells (p=0.01). Trends from the ICON multiplex IHC also highlighted increases in CD8+ T cells (p=0.09), CD20+ cells (p=0.08), as well as PD-L1+ malignant cells (p=0.08) and PD-L1+ TAMs (p=0.08) in NCT patients, the latter finding being supported by increased circulating MCP-1 (p=0.03). TMB was similar between NCT and US groups (p=0.912). Our data provides the first evidence of ICD (i.e., increased DAMP gene expression) following NCT in human early-stage NSCLC. Furthermore, our data highlights the association of NCT with a favorable IME (i.e., increased T cell infiltration), supporting the rationale of NCT and ICB combinations in localized NSCLC.

Although immune checkpoint inhibitors of the PD-1/PD-L1 axis provide significant clinical benefit for patients with lung cancer, effective use of these agents is encumbered by a high rate of primary or acquired resistance. Strategies for optimal therapeutic application of immunotherapy require a thorough understanding of resistance mechanisms. To date, there have been only a few studies reporting potential mechanisms of resistance to PD-1/PD-L1 blockade. In multiple immunocompetent syngeneic and spontaneous animal models of K-ras/p53 mutant lung cancer, we explored the resistance mechanisms to PD-1/PD-L1 blockade using both pharmacologic and genetic approaches (therapeutic antibody treatment and CRISPR/Cas9-mediated editing). The molecular and immune profiles of the tumor microenvironment were evaluated. Additionally, to determine the applicability to patients with lung cancer, we analyzed 259 tumor specimens with IHC staining and mRNA expression, and further confirmed the analyses in publically-available TCGA datasets. In multiple models of antibody blockade and genetic knockout of PD-L1, we identified the up-regulation of CD38 on tumor cells as a marker of treatment resistance. Furthermore, by manipulating CD38 on a panel of lung cancer cell lines we demonstrated in vitro and in vivo that CD38 expression inhibits CD8+ T cell proliferation, anti-tumor cytokine secretion, and tumor cell killing capability. The T cell suppressive effect is dependent upon the ectoenzyme activity of CD38 that regulates the extracellular levels of adenosine. To test whether CD38 blockade might be therapeutically efficacious to prevent anti-PD-L1/PD-1 resistance, we applied combination therapy with anti-CD38 and anti-PD-L1 and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Additionally, in a set of 259 resected lung cancer specimens, ∼15% exhibited positive staining for CD38 on tumor cells, and the expression correlated with cytolytic T cell score and an immune/inflammatory signature across multiple large datasets. CD38 was found to be a novel mechanism for tumor escape from immune checkpoint PD-1/PD-L1 inhibitor therapy. Targeting this resistance pathway may broaden the benefit of PD-L1/PD-1 axis blockade for lung cancer treatment.

Metastatic lung cancer is one of the most lethal forms of cancer and molecular pathways driving metastasis are still not clearly elucidated. Metastatic cancer cells undergo an epithelial–mesenchymal transition (EMT) where they lose their epithelial properties and acquire a migratory and invasive phenotype. Here we identify that the expression of microRNAs from the miR-200 family and the miR-183~96~182 cluster are significantly co-repressed in non-small cell lung cancer cell lines and primary tumors from multiple TCGA dataset with high EMT scores. Ectopic expression of the miR-183~96~182 cluster inhibited cancer cell migration and invasion, whereas its expression was tightly modulated by miR-200. We identified Foxf2 as a common, novel and direct target of both these microRNA families. Foxf2 expression tightly correlates with the transcription factor Zeb1 and is elevated in mesenchymal-like metastatic lung cancer cells. Foxf2 expression induced robust EMT, migration, invasion and metastasis in lung cancer cells, whereas Foxf2 inhibition significantly repressed these phenotypes. We also demonstrated that Foxf2 transcriptionally represses E-cadherin and miR-200, independent of Zeb1, to form a double-negative feedback loop. We, therefore, identified a novel mechanism whereby the miR-200 family and the miR-183~96~182 cluster inhibit lung cancer invasion and metastasis by targeting Foxf2.

BACKGROUNDMalignant pleural mesothelioma (MPM) is a lethal neoplasm exhibiting resistance to most treatment regimens and requires effective therapeutic options. Though an effective strategy in many cancer, targeted therapy is relatively unexplored in MPM because the therapeutically important oncogenic pathways and networks in MPM are largely unknown.MATERIALS AND METHODSWe carried out gene expression microarray profiling of 53 surgically resected MPMs tumors along with paired normal tissue. We also carried out whole transcriptomic sequence (RNA-seq) analysis on eight tumor specimens. Taqman-based quantitative Reverse-transcription polymerase chain reaction (qRT-PCR), western analysis and immunohistochemistry (IHC) analysis of mitotic arrest deficient-like 1 (MAD2L1) was carried out on tissue specimens. Cell viability assays of MPM cell lines were carried out to assess sensitivity to specific small molecule inhibitors.RESULTSBioinformatics analysis of the microarray data followed by pathway analysis revealed that the mitotic spindle assembly checkpoint (MSAC) pathway was most significantly altered in MPM tumors with upregulation of 18 component genes, including MAD2L1 gene. We validated the microarray data for MAD2L1 expression using quantitative qRT-PCR and western blot analysis on tissue lysates. Additionally, we analyzed expression of the MAD2L1 protein by IHC using an independent tissue microarray set of 80 MPM tissue samples. Robust clustering of gene expression data revealed three novel subgroups of tumors, with unique expression profiles, and showed differential expression of MSAC pathway genes. Network analysis of the microarray data showed the cytoskeleton/spindle microtubules network was the second-most significantly affected network. We also demonstrate that a nontaxane small molecule inhibitor, epothilone B, targeting the microtubules have great efficacy in decreasing viability of 14 MPM cell lines.CONCLUSIONSOverall, our findings show that MPM tumors have significant deregulation of the MSAC pathway and the microtubule network, it can be classified into three novel molecular subgroups of potential therapeutic importance and epothilone B is a promising therapeutic agent for MPM.

BACKGROUND Human epidermal growth factor receptor 2 (HER2) Ile655Val polymorphism may affect the efficacy of trastuzumab treatment of breast cancer. PATIENTS AND METHODS HER2 Ile655Val polymorphism was determined in 4167 patients with primary breast cancer using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. We investigated the associations between the HER2 Ile655Val polymorphism and clinical outcomes in women with HER2-negative breast cancer and with HER2-positive breast cancer who received trastuzumab or who did not. RESULTS At a median follow-up of 44 months, HER2 Ile655Val polymorphism was not significantly associated with survival either in the entire study population of 4167 patients or in 2976 HER2-negative breast cancer patients. Among 816 HER2-positive patients who received adjuvant chemotherapy and/or endocrine therapy without trastuzumab treatment, patients with the Val/Ile or the Val/Val genotype had a significantly worse disease-free survival (DFS) and distant DFS (DDFS) than those with the Ile/Ile genotype (DFS, adjusted hazard ratio [HR] 1.5; 95% confidence interval [CI] 1.0-2.3; P = 0.037; DDFS, adjusted HR 1.9; 95% CI 1.2-2.9 P = 0.005). In contrast, among 212 HER2-positive patients who received chemotherapy in combination with trastuzumab treatment, patients with the Val/Ile or the Val/Val genotype had a significantly better DFS and DDFS than those with the Ile/Ile genotype (5-year DFS, 100% versus 83%; P = 0.008; 5-year DDFS, 100% versus 89%; P = 0.031). CONCLUSIONS HER2 Ile655Val polymorphism affects the function of HER2 gene only restricted in HER2-positive breast cancers. HER2-positive breast cancer patients with the Val variant have an aggressive phenotype, but are sensitive to trastuzumab treatment.

BACKGROUND:BRCA1 function is inactivated through BRCA1 promoter methylation in a substantial number of triple-negative breast cancers. We investigated the impact of BRCA1-methylation status on the efficacy of adjuvant chemotherapy in patients with triple-negative breast cancer or with non-triple-negative breast cancer.METHODS:BRCA1 promoter methylation was assessed in 1163 unselected breast cancer patients. Methylation was evaluated using a methylation-specific PCR (MSP) assay.RESULTS:In the subgroup of 167 triple-negative breast cancer patients who received adjuvant chemotherapy, patients with BRCA1-methylated tumors had a superior 10-year disease-free survival (DFS)(78% versus 55%, P = 0.009) and 10-year disease-specific survival (DSS) (85% versus 69%, P = 0.024) than those with BRCA1-unmethylated tumors, and BRCA1 methylation was an independent favorable predictor of DFS and DSS in a multivariate analysis in this subgroup [DFS: hazard ratio (HR) = 0.45; 95% confidence interval (CI) 0.24-0.84; P = 0.019; DSS: HR = 0.43; 95% CI = 0.19-0.95; P = 0.044]. In contrast, in 675 non-triple-negative breast cancer patients who received adjuvant chemotherapy, BRCA1 methylation was an unfavorable predictor of DFS and DSS in univariate analysis (DFS: HR = 1.56; 95% CI 1.16-2.12; P = 0.003; DSS: HR = 1.53; 95% CI = 1.05-2.21; P = 0.026).CONCLUSIONS:Triple-negative breast cancer patients with BRCA1-methylated tumors are sensitive to adjuvant chemotherapy and have a favorable survival compared with patients with BRCA1-unmethylated triple-negative tumors.