Faculty Bibliography

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

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.

INTRODUCTION/BACKGROUND:Recent studies have identified subtypes of small cell lung carcinoma (SCLC) defined by RNA expression of ASCL1, NEUROD1, POU2F3 and YAP1 transcriptional regulators. There are only limited data on distribution of these markers at the protein level and associated pathologic characteristics in clinical SCLC samples. METHODS:Expression of ASCL1, NEUROD1, POU2F3 and YAP1 was analyzed by immunohistochemistry in 174 SCLC patient samples. Subtypes defined by these markers were correlated with histologic characteristics, expression of neuroendocrine markers (synaptophysin, chromogranin A, CD56, INSM1) and other SCLC markers including neuroendocrine phenotype-associated marker DLL3. RESULTS:. CONCLUSIONS:This is the first comprehensive immunohistochemical and histopathologic analysis of novel SCLC subtypes in patient samples. We confirm that ASCL1/NEUROD1-double-negative tumors represent a distinct neuroendocrine-low subtype of SCLC which is either uniquely associated with POU2F3 or lacks a known dominant regulator. Expression profiles of these markers appear more heterogeneous in native samples than in experimental models, particularly in regard to high prevalence of ASCL1/NEUROD1 co-expression. These findings may have prognostic and therapeutic implications and warrant further clinical investigation.

The WNT pathway is a fundamental regulator of intestinal homeostasis and hyperactivation of WNT signaling is the major oncogenic driver in colorectal cancer (CRC). To date, there are no described mechanisms that bypass WNT dependence in intestinal tumors. Here, we show that while WNT suppression blocks tumor growth in most organoid and in vivo CRC models, the accumulation of CRC-associated genetic alterations enables drug resistance and WNT-independent growth. In intestinal epithelial cells harboring mutations in KRAS or BRAF, together with disruption of p53 and SMAD4, transient TGFB exposure drives YAP/TAZ-dependent transcriptional reprogramming and lineage reversion. Acquisition of embryonic intestinal identity is accompanied by a permanent loss of adult intestinal lineages, and long-term WNT-independent growth. This work identifies genetic and microenvironmental factors that drive WNT inhibitor resistance, defines a new mechanism for WNT-independent CRC growth and reveals how integration of associated genetic alterations and extracellular signals can overcome lineage-dependent oncogenic programs.

Despite advancements in treatment options, the overall cure and survival rates for non-small cell lung cancers (NSCLC) remain low. While small-molecule inhibitors of epigenetic regulators have recently emerged as promising cancer therapeutics, their application in patients with NSCLC is limited. To exploit epigenetic regulators as novel therapeutic targets in NSCLC, we performed pooled epigenome-wide CRISPR knockout screens in vitro and in vivo and identified the histone chaperone nucleophosmin 1 (NPM1) as a potential therapeutic target. Genetic ablation of Npm1 significantly attenuated tumor progression in vitro and in vivo. Furthermore, KRAS-mutant cancer cells were more addicted to NPM1 expression. Genetic ablation of Npm1 rewired the balance of metabolism in cancer cells from predominant aerobic glycolysis to oxidative phosphorylation and reduced the population of tumor-propagating cells. Overall, our results support NPM1 as a therapeutic vulnerability in NSCLC.

Small cell lung cancer (SCLC) represents ∼15% of all lung cancer diagnoses in the US and has a particularly poor prognosis. We hypothesized that kinases regulating SCLC survival pathways represent therapeutically targetable vulnerabilities whose inhibition may improve SCLC outcome. A shRNA library targeting all human kinases was introduced into seven chemonaive patient derived xenografts (PDX), and cells cultured in vitro and in vivo. On harvest, lost, or depleted, shRNAs were considered as regulating cell survival pathways, and deemed essential kinases. Unsupervised hierarchical cluster analysis of recovered shRNAs separated the PDX into two clusters suggesting kinase based heterogeneity among the SCLC PDX. Twenty-three kinases were identified as essential in two or more PDX, with mTOR a candidate essential kinase in four. mTOR phosphorylation (p-mTOR) status correlated with PDX sensitivity to mTOR kinase inhibition, and mTOR inhibition sensitized PDX to cisplatin/etoposide. In PDX where mTOR was defined as essential, mTOR inhibition caused a 43% decrease in tumor volume at 21 days (P<0.01). Combining mTOR inhibition with cisplatin/etoposide decreased PDX tumor volume 96% compared to cisplatin/etoposide alone at 70 days (P<0.002). Chemoresistance did not develop with the combination of mTOR inhibition and cisplatin/etoposide in mTOR essential PDX over 105 days. The prevalence of p-mTOR in a tissue microarray of chemonaive SCLC was 27% identifying a significant SCLC subtype that might benefit by the addition of mTOR inhibition to standard chemotherapy. These studies show that kinases can define SCLC subgroups, can identify therapeutic vulnerabilities, and can potentially be used to optimize therapeutic approaches.

Tumor evolution from a single cell into a malignant, heterogeneous tissue remains poorly understood. Here, we profile single-cell transcriptomes of genetically engineered mouse lung tumors at seven stages, from pre-neoplastic hyperplasia to adenocarcinoma. The diversity of transcriptional states increases over time and is reproducible across tumors and mice. Cancer cells progressively adopt alternate lineage identities, computationally predicted to be mediated through a common transitional, high-plasticity cell state (HPCS). Accordingly, HPCS cells prospectively isolated from mouse tumors and human patient-derived xenografts display high capacity for differentiation and proliferation. The HPCS program is associated with poor survival across human cancers and demonstrates chemoresistance in mice. Our study reveals a central principle underpinning intra-tumoral heterogeneity and motivates therapeutic targeting of the HPCS.

Amplification and oncogenic mutations of ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADCs) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2/HER2-amplified or mutant lung cancers. We show that co-treatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.

Small cell lung cancer patient outcomes have not yet been significantly impacted by the revolution in precision oncology, primarily due to a paucity of genetic alterations in actionable driver oncogenes. Nevertheless, systemic therapies that include immunotherapy are beginning to show promise in the clinic. While these results are encouraging, many patients do not respond to or rapidly recur after current regimens, necessitating alternative or complementary therapeutic strategies. In this review, we discuss ongoing investigations into the pathobiology of this recalcitrant cancer and the therapeutic vulnerabilities that are exposed by the disease state. Included within this discussion is a snapshot of the current biomarker and clinical trial landscapes for small cell lung cancer. Finally, we identify key knowledge gaps that should be addressed in order to advance the field in pursuit of reduced small cell lung cancer mortality. This review largely summarizes work presented at the Third Biennial IASLC Small Cell Lung Cancer Meeting.