Faculty Bibliography

In the Supplementary Information originally published with this article, a lane was missing in the β-actin blot in Supplementary Fig. 2. The lane has been added. The error has been corrected in the Supplementary Information associated with this article.

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS-mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS-mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Tregs) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1 inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras+/LSL-G12D; Trp53L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared to either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma.

PURPOSE/OBJECTIVE:mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH. CONCLUSIONS:mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy.

Despite extensive efforts, oncogenic KRAS remains resistant to targeted therapy. Combined downstream RAL-TBK1 and MEK inhibition induces only transient lung tumor shrinkage in KRAS-driven genetically engineered mouse models (GEMMs). Using the sensitive KRAS;LKB1 (KL) mutant background, we identify YAP1 upregulation and a therapy-induced secretome as mediators of acquired resistance. This program is reversible, associated with H3K27 promoter acetylation, and suppressed by BET inhibition, resensitizing resistant KL cells to TBK1/MEK inhibition. Constitutive YAP1 signaling promotes intrinsic resistance in KRAS;TP53 (KP) mutant lung cancer. Intermittent treatment with the BET inhibitor JQ1 thus overcomes resistance to combined pathway inhibition in KL and KP GEMMs. Using potent and selective TBK1 and BET inhibitors we further develop an effective therapeutic strategy with potential translatability to the clinic.

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing anti-tumor immune responses. Through an unbiased approach, we have established that IL-6 mediates crosstalk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL-6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant 3D organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL-6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL-6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head-and-neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL-6 is a major contributor to the dynamic crosstalk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL-6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.

Tumor mutational burden correlates with response to immune checkpoint blockade in multiple solid tumors, although in microsatellite-stable tumors this association is of uncertain clinical utility. Here we uniformly analyzed whole-exome sequencing (WES) of 249 tumors and matched normal tissue from patients with clinically annotated outcomes to immune checkpoint therapy, including radiographic response, across multiple cancer types to examine additional tumor genomic features that contribute to selective response. Our analyses identified genomic correlates of response beyond mutational burden, including somatic events in individual driver genes, certain global mutational signatures, and specific HLA-restricted neoantigens. However, these features were often interrelated, highlighting the complexity of identifying genetic driver events that generate an immunoresponsive tumor environment. This study lays a path forward in analyzing large clinical cohorts in an integrated and multifaceted manner to enhance the ability to discover clinically meaningful predictive features of response to immune checkpoint blockade.

N⁶-methyladenosine (m⁶A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m⁶A homeostasis is linked to cancer^1-5. m⁶A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon⁵. METTL3 can promote translation but the mechanism and relevance of this process remain unknown¹. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also m⁶A-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.

CDK4 is emerging as a target in KRAS-mutant non-small cell lung cancer (NSCLC). We demonstrate that KRAS-mutant NSCLC cell lines are initially sensitive to the CDK4/6 inhibitor palbociclib, but readily acquire resistance associated with increased expression of CDK6, D-type cyclins and cyclin E. Resistant cells also demonstrated increased ERK1/2 activity and sensitivity to MEK and ERK inhibitors. Moreover, MEK inhibition reduced the expression and activity of cell cycle proteins mediating palbociclib resistance. In resistant cells, ERK activated mTOR, driven in part by upstream FGFR1 signaling resulting from the extracellular secretion of FGF ligands. A genetically-engineered mouse model of KRAS-mutant NSCLC initially sensitive to palbociclib similarly developed acquired resistance with increased expression of cell cycle mediators, ERK1/2 and FGFR1. In this model, resistance was delayed with combined palbociclib and MEK inhibitor treatment. These findings implicate an FGFR1-MAP kinase-mTOR pathway resulting in increased expression of D-cyclins and CDK6 that confers palbociclib resistance and indicate that CDK4/6 inhibition acts to promote MAP kinase dependence.

Human melanomas frequently harbor amplifications of EZH2. However, the contribution of EZH2 to melanoma formation has remained elusive. Taking advantage of murine melanoma models, we show that EZH2 drives tumorigenesis from benign Braf^V600E- or Nras^Q61K-expressing melanocytes by silencing of genes relevant for the integrity of the primary cilium, a signaling organelle projecting from the surface of vertebrate cells. Consequently, gain of EZH2 promotes loss of primary cilia in benign melanocytic lesions. In contrast, blockade of EZH2 activity evokes ciliogenesis and cilia-dependent growth inhibition in malignant melanoma. Finally, we demonstrate that loss of cilia enhances pro-tumorigenic WNT/β-catenin signaling, and is itself sufficient to drive metastatic melanoma in benign cells. Thus, primary cilia deconstruction is a key process in EZH2-driven melanomagenesis.

Small cell lung cancer (SCLC) has the highest malignancy among all lung cancers, exhibiting aggressive growth and early metastasis to distant sites. For 30 years, treatment options for SCLC have been limited to chemotherapy, warranting the need for more effective treatments. Frequent inactivation of TP53 and RB1 as well as histone dysmodifications in SCLC suggest that transcriptional and epigenetic regulations play a major role in SCLC disease evolution. Here we performed a synthetic lethal screen using the BET inhibitor JQ1 and an shRNA library targeting 550 epigenetic genes in treatment-refractory SCLC xenograft models and identified HDAC6 as a synthetic lethal target in combination with JQ1. Combined treatment of human and mouse SCLC cell line-derived xenograft tumors with the HDAC6 inhibitor ricolinostat (ACY-1215) and JQ1 demonstrated significant inhibition of tumor growth; this effect was abolished upon depletion of NK cells, suggesting that these innate immune lymphoid cells play a role in SCLC tumor treatment response. Collectively, these findings suggest a potential new treatment for recurrent SCLC.