Faculty Bibliography

Background: A minority of small cell lung carcinomas (SCLC) has minimal or absent expression of neuroendocrine (NE) markers, which can present a diagnostic challenge. Recent studies have suggested that POU2F3-a marker of chemosensory tuft cell lineage-is expressed specifically in NE-low SCLC. Here, we aimed to examine expression of POU2F3 in SCLC with extremely low or negative NE markers and to determine its specificity relative to non-NE lung carcinomas.
Design(s): POU2F3 expression was examined immunohistochemically in 152 SCLC and 116 non-small cell lung carcinomas (NSCLC; 53 adenocarcinomas, 63 squamous cell carcinomas). SCLC comprised 144 unselected cases and 8 additional pre-selected NE-minimal or negative SCLC. All SCLC were tested for 4 conventional NE markers (CNM; synaptophysin, chromogranin, CD56, and INSM1), and tumors with combined NE score (average H-score of 4 CNM) <50 were defined as NE-low and those with score <10 (staining isolated cells only) as NE-minimal. TTF-1 expression was also evaluated.
Result(s): POU2F3 was expressed in 8% of unselected SCLC (11/140), but was completely negative in all 116 NSCLC. In the whole cohort, compared to POU2F3-negative cases (n=134), POU2F3-positive SCLC (n=18) had fewer positive CNM (mean 1.8 vs 3.7, p<0.0001), lower combined NE score (mean 60 vs 183, p<0.0001) and lower rate of TTF-1 expression (6% vs 81%, p<0.0001), respectively. A total of 15 SCLC were NE-low (n=10), NEminimal (n=4) or NE-entirely negative (n=1). POU2F3 was positive in 10/15 (67%) of these cases, including all 5 NE-minimal/negative SCLC. POU2F3 expression in these cases was typically strong and diffuse (mean H-score 147; range 60-235).
Conclusion(s): POU2F3 expression is highly specific for SCLC relative to NSCLC, and is significantly enriched in NE-low SCLC, particularly in cases with minimal or negative NE marker expression. We suggest that POU2F3 represents a novel helpful diagnostic marker of SCLC

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).

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.

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.

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.

Patient-derived xenografts are high fidelity in vivo tumor models that accurately reflect many key aspects of human cancer. In contrast to either cancer cell lines or genetically engineered mouse models, the utility of PDXs has been limited by the inability to perform targeted genome editing of these tumors. To address this limitation, we have developed methods for CRISPR-Cas9 editing of PDXs using a tightly regulated, inducible Cas9 vector that does not require in vitro culture for selection of transduced cells. We demonstrate the utility of this platform in PDXs (1) to analyze genetic dependencies by targeted gene disruption and (2) to analyze mechanisms of acquired drug resistance by site-specific gene editing using templated homology-directed repair. This flexible system has broad application to other explant models and substantially augments the utility of PDXs as genetically programmable models of human cancer.

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.