Faculty Bibliography

PURPOSE/OBJECTIVE:A small percentage of patients with small cell lung cancer (SCLC) experience durable responses to immune checkpoint blockade (ICB). Defining determinants of immune response may nominate strategies to broaden the efficacy of immunotherapy in patients with SCLC. Prior studies have been limited by small numbers and/or concomitant chemotherapy administration. METHODS:CheckMate 032, a multicenter, open-label, phase 1/2 trial evaluating nivolumab alone or with ipilimumab in patients with previously treated advanced or metastatic solid tumors was the largest study of ICB alone in patients with SCLC. We performed comprehensive RNA sequencing of 286 pretreatment SCLC tumor samples from patients enrolled on this study. We evaluated outcome based on defined SCLC subtypes (SCLC-A, -N, -P, and -Y), and explored expression signatures associated with durable benefit, defined as progression-free survival ≥6 months. Potential biomarkers were further explored by immunohistochemistry. RESULTS:None of the subtypes were associated with progression-free or overall survival. YAP1 gene expression across the dataset was associated with an inflammation signature (R=0.25, p=0.00014), and SCLC-Y associated with expression of antigen presentation machinery (APM) (p<0.00001). The APM signature (p=0.000032) and presence of ≥ 1% infiltrating CD8+ T cells by immunohistochemistry (HR 0.51; 95% confidence interval 0.27 - 0.95) both correlated with overall survival in patients treated with nivolumab. Pathway enrichment analysis demonstrated association between durable benefit from immunotherapy and antigen processing and presentation. CONCLUSIONS:Tumor antigen processing and presentation is a key correlate of ICB efficacy in patients with SCLC. As antigen presentation machinery is frequently epigenetically suppressed in SCLC, this study defines a targetable mechanism by which we might improve clinical benefit of ICB for patients with SCLC.

UNLABELLED:. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)-key downstream effectors of LKB1- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCE/CONCLUSIONS:tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

Although lung disease is the primary clinical outcome in COVID-19 patients, how SARS-CoV-2 induces lung pathology remains elusive. Here we describe a high-throughput platform to generate self-organizing and commensurate human lung buds derived from hESCs cultured on micropatterned substrates. Lung buds resemble human fetal lungs and display proximodistal patterning of alveolar and airway tissue directed by KGF. These lung buds are susceptible to infection by SARS-CoV-2 and endemic coronaviruses and can be used to track cell type-specific cytopathic effects in hundreds of lung buds in parallel. Transcriptomic comparisons of infected lung buds and postmortem tissue of COVID-19 patients identified an induction of BMP signaling pathway. BMP activity renders lung cells more susceptible to SARS-CoV-2 infection and its pharmacological inhibition impairs infection by this virus. These data highlight the rapid and scalable access to disease-relevant tissue using lung buds that recapitulate key features of human lung morphogenesis and viral infection biology.

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

Background: Hepatocyte transplantation is being pursued as an alternative to liver transplantation. This strategy requires hepatocyte proliferation following transplantation, but the extent to which transplanted hepatocytes can proliferate is unknown. Primary human hepatocytes (PHH) from some donors can efficiently humanize liver chimeric mice yet the relative contributions of engraftment and proliferation have not been quantified. We here aimed to define how many PHH engraft after transplantation in chimeric mice and test their proliferation limit.
Method(s): PHH were serially transplanted into immunodeficient Fah-/- mice with liver injury. PHH were transduced with lentiviruses to deliver fluorophores or barcodes prior to transplantation to quantify engraftment and proliferation.
Result(s): On a population level PHH expanded approximately 200-fold per round of transplantation resulting in ~10⁸ cells per liver. Fluorophore labeling showed PHH islands to be of clonal origin. Barcode labeling indicated that approximately 104 cells engrafted per mouse liver. After three rounds of efficient repopulation humanization deteriorated >10-fold per round.
Conclusion(s): PHH can efficiently expand approximately 1012 -fold (104 per transplantation for 3 serial transplantations) in liver chimeric mice, after which their engraftment and/or proliferation potential diminishes. This limit of ~40 cell divisions in mouse livers may guide therapeutic PHH transplantation protocols. (Figure Presented)

BACKGROUND:Diffuse pleural mesothelioma (DPM) is an aggressive malignancy that, despite recent treatment advances, has unacceptably poor outcomes. Therapeutic research in DPM is inhibited by a paucity of preclinical models that faithfully recapitulate the human disease. METHODS:We established 22 patient-derived xenografts (PDX) from 22 patients with DPM and performed multi-omic analyses to deconvolute the mutational landscapes, global expression profiles, and molecular subtypes of these PDX models and compared features to those of the matched primary patient tumors. Targeted next-generation sequencing (NGS; MSK-IMPACT), immunohistochemistry, and histologic subtyping were performed on all available samples. RNA sequencing was performed on all available PDX samples. Clinical outcomes and treatment history were annotated for all patients. Platinum-doublet progression-free survival (PFS) was determined from the start of chemotherapy until radiographic/clinical progression and grouped into < or ≥ 6 months. RESULTS:PDX models were established from both treatment naïve and previously treated samples and were noted to closely resemble the histology, genomic landscape, and proteomic profiles of the parent tumor. After establishing the validity of the models, transcriptomic analyses demonstrated overexpression in WNT/β-catenin, hedgehog, and TGF-β signaling and a consistent suppression of immune-related signaling in PDXs derived from patients with worse clinical outcomes. CONCLUSIONS:These data demonstrate that DPM PDX models closely resemble the genotype and phenotype of parental tumors, and identify pathways altered in DPM for future exploration in preclinical studies.

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).

Small cell lung cancer (SCLC) is characterized by morphologic, epigenetic and transcriptomic heterogeneity. Subtypes based upon predominant transcription factor expression have been defined that, in mouse models and cell lines, exhibit potential differential therapeutic vulnerabilities, with epigenetically distinct SCLC subtypes also described. The clinical relevance of these subtypes is unclear, due in part to challenges in obtaining tumor biopsies for reliable profiling. Here we describe a robust workflow for genome-wide DNA methylation profiling applied to both patient-derived models and to patients' circulating cell-free DNA (cfDNA). Tumor-specific methylation patterns were readily detected in cfDNA samples from patients with SCLC and were correlated with survival outcomes. cfDNA methylation also discriminated between the transcription factor SCLC subtypes, a precedent for a liquid biopsy cfDNA-methylation approach to molecularly subtype SCLC. Our data reveal the potential clinical utility of cfDNA methylation profiling as a universally applicable liquid biopsy approach for the sensitive detection, monitoring and molecular subtyping of patients with SCLC.

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apoB48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cell expression systems are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The cell culture system was developed by ablating the endogenous MTTP gene in human hepatoma Huh-7 cells using sgRNA and CRISPR/Cas9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA, or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins, and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.