Faculty Bibliography

Autophagy has been shown to be elevated in pancreatic adenocarcinoma (PDAC) and its role in promoting established tumor growth has made it a promising therapeutic target. However, due to limitations of prior mouse models as well as the lack of potent and selective autophagy inhibitors, the ability to fully assess the mechanistic basis of how autophagy supports pancreatic cancer has been limited. To test the feasibility of treating PDAC using autophagy inhibition and further our understanding of the mechanisms of pro-tumor effects of autophagy, we developed a novel mouse model that allowed the acute and reversible inhibition of autophagy. We observed that autophagy inhibition causes significant tumor regression in an autochthonous mouse model of PDAC. A detailed analysis of these effects indicated that the tumor regression was likely multifactorial, involving both tumor cell intrinsic as well as host effects. Thus, our study supports autophagy inhibition in PDAC may have future utility in the treatment of pancreatic cancer and illustrates the importance of assessing complex biological processes in relevant autochthonous models.

PURPOSE/OBJECTIVE:HER2 (or ERBB2) aberrations, including both amplification and mutations, have been classified as oncogenic drivers that contribute to 2-6 percent of lung adenocarcinomas. HER2 amplification is also an important mechanism for acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). However, due to limited preclinical studies and clinical trials, currently there is still no available standard of care for lung cancer patients with HER2 aberrations. To fulfill the clinical need for targeting HER2 in non-small cell lung cancer (NSCLC) patients, we performed a comprehensive pre-clinical study to evaluate the efficacy of a third-generation TKI, osimertinib (AZD9291).  Experimental Design:Three genetically modified mouse models (GEMMs) mimicking individual HER2 alterations in NSCLC were generated and osimertinib was tested for its efficacy against these HER2 aberrations in vivo. RESULTS:Osimertinib treatment showed robust efficacy in HER2wt overexpression and EGFR del19/HER2 models but not in HER2 exon 20 insertion tumors. Interestingly, we further identified that combined treatment with osimertinib and the BET inhibitor JQ1 significantly increased the response rate in HER2-mutant NSCLC while JQ1 single treatment did not show efficacy. CONCLUSIONS:Overall, our data indicated robust anti-tumor efficacy of osimertinib against multiple HER2 aberrations in lung cancer, either as a single agent or in combination with JQ1. Our study provides a strong rationale for future clinical trials using osimertinib either alone or in combination with epigenetic drugs to target aberrant HER2 in NSCLC patients.

An increasingly recognized component of resistance to tyrosine kinase inhibitors (TKI) involves persistence of a drug-tolerant subpopulation of cancer cells which survive despite effective eradication of the majority of the cell population. Multiple groups have demonstrated that these drug-tolerant persister cells undergo transcriptional adaptation via an epigenetic state change that promotes cell survival. Because this mode of TKI drug tolerance appears to involve transcriptional addiction to specific genes and pathways, we hypothesized that systematic functional screening of EGFR TKI/transcriptional inhibitor combination therapy would yield important mechanistic insights and alternative drug escape pathways. We therefore performed a genome-wide CRISPR/Cas9 enhancer/suppressor screen in EGFR-dependent lung cancer PC9 cells treated with erlotinib + THZ1 (CDK7/12 inhibitor) combination therapy,a combination previously shown to suppress drug tolerant cells in this setting. As expected, suppression of multiple genes associated with transcriptional complexes (EP300, CREBBP and MED1) enhanced erlotinib/THZ1 synergy. Unexpectedly, we uncovered nearly every component of the recently described ufmylation pathway in the synergy suppressor group. Loss of ufmylation did not affect canonical downstream EGFR signaling. Instead, absence of this pathway triggered a protective unfolded protein response (UPR) associated with STING upregulation, promoting pro-tumorigenic inflammatory signaling but also unique dependence on Bcl-xL. These data reveal that dysregulation of ufmylation and ER stress comprise a previously unrecognized TKI drug tolerance pathway that engages survival signaling, with potentially important therapeutic implications.

Acquired drug resistance is a major factor limiting the effectiveness of targeted cancer therapies. Targeting tumors with kinase inhibitors induces complex adaptive programs that promote the persistence of a fraction of the original cell population, facilitating the eventual outgrowth of inhibitor-resistant tumor clones. We show that the addition of a newly identified CDK7/12 inhibitor, THZ1, to targeted therapy enhances cell killing and impedes the emergence of drug-resistant cell populations in diverse cellular and in vivo cancer models. We propose that targeted therapy induces a state of transcriptional dependency in a subpopulation of cells poised to become drug tolerant, which THZ1 can exploit by blocking dynamic transcriptional responses, remodeling of enhancers and key signalling outputs required for tumor cell survival in the setting of targeted therapy. These findings suggest that the addition of THZ1 to targeted therapies is a promising broad-based strategy to hinder the emergence of drug-resistant cancer cell populations.

The bromodomain and extraterminal (BET) family of proteins comprises four members-BRD2, BRD3, BRD4 and the testis-specific isoform BRDT-that largely function as transcriptional coactivators and play critical roles in various cellular processes, including the cell cycle, apoptosis, migration and invasion. BET proteins enhance the oncogenic functions of major cancer drivers by elevating the expression of these drivers, such as c-Myc in leukemia, or by promoting the transcriptional activities of oncogenic factors, such as AR and ERG in prostate cancer. Pathologically, BET proteins are frequently overexpressed and are clinically linked to various types of human cancer; they are therefore being pursued as attractive therapeutic targets for selective inhibition in patients with cancer. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed and have shown promising outcomes in early clinical trials. Although resistance to BET inhibitors has been documented in preclinical models, the molecular mechanisms underlying acquired resistance are largely unknown. Here we report that cullin-3^SPOP earmarks BET proteins, including BRD2, BRD3 and BRD4, for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the degradation of BET proteins, leading to their elevated abundance in SPOP-mutant prostate cancer. As a result, prostate cancer cell lines and organoids derived from individuals harboring SPOP mutations are more resistant to BET-inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor-suppressor role of SPOP in prostate cancer in which it acts as a negative regulator of BET protein stability and also provide a molecular mechanism for resistance to BET inhibitors in individuals with prostate cancer bearing SPOP mutations.

Understanding how murine models can elucidate the mechanisms underlying antitumor immune responses and advance immune-based drug development is essential to advancing the field of cancer immunotherapy. The Society for Immunotherapy of Cancer (SITC) convened a workshop titled, "Challenges, Insights, and Future Directions for Mouse and Humanized Models in Cancer Immunology and Immunotherapy" as part of the SITC 31st Annual Meeting and Associated Programs on November 10, 2016 in National Harbor, MD. The workshop focused on key issues in optimizing models for cancer immunotherapy research, with discussions on the strengths and weaknesses of current models, approaches to improve the predictive value of mouse models, and advances in cancer modeling that are anticipated in the near future. This full-day program provided an introduction to the most common immunocompetent and humanized models used in cancer immunology and immunotherapy research, and addressed the use of models to evaluate immune-targeting therapies. Here, we summarize the workshop presentations and subsequent panel discussion.

Background: The genomic landscape of primary resistance to PD-1 blockade in lung adenocarcinoma (LUAD) is largely unknown. We previously reported that co-mutations in STK11/LKB1 (KL) or TP53 (KP) define subgroups of KRAS-mutant LUAD with distinct therapeutic vulnerabilities and immune profiles. Here, we present updated data on the clinical efficacy of PD-1/PD-L1 inhibitors in co-mutation defined KRAS mutant and wild-type LUAD patients and examine the relationship between genetic alterations in individual genes, tumor cell PD-L1 expression and tumor mutational burden (TMB) using cohorts form the SU2C/ACS Lung Cancer Dream Team and Foundation Medicine (FM). Method: The cohorts included 924 LUAD with NGS (FM cohort) and 188 patients with KRAS non-squamous NSCLC (SU2C cohort) who received at least one cycle of PD-1/PD-L1 inhibitor therapy and had available molecular profiling. Tumor cell PD-L1 expression was tested using E1L3N IHC (SU2C) and the VENTANA PD-L1 (SP142) assay (FM). TMB was defined as previously described and was classified as high (TMB-H), intermediate (TMB-I) or low (TMB-L). Result: 188 immunotherapy-treated (83.5% nivolumab, 11.7% pembrolizumab, 4.8% anti-PD1/PD-L1 plus anti-CTLA-4) pts with KRASmutant NSCLC were included in the efficacy analysis. The ORR differed significantly between the KL (8.8%), KP (35.9%) and K-only subgroups (27.3%) (P=0.0011, Fisher's exact test). KL LUAC exhibited significantly shorter PFS (mPFS 1.8m vs 2.7m, HR=0.53, 95% CI 0.34- 0.84, P<0.001, log-rank test) and OS (mOS 6.8m vs 15.6m, HR 0.53, 95% CI 0.34 to 0.84, P=0.0072, log rank test) compared to KRASmutant NSCLC with wild-type STK11. Loss-of function (LOF) genetic alterations in STK11 were the only significantly enriched event in PDL1 negative, TMB-I/H compared to PD-L1 high positive (TPS>=50%), TMB-I/H tumors in the overall FMI cohort (Bonferroni adjusted P=2.38x10^-4, Fisher's exact test) and among KRAS-mutant tumors (adjusted P=0.05, Fisher's exact test). Notably, PD-1 blockade demonstrated activity among 10 PD-L1-negative KP tumors, with 3 PRs and 4SDs recorded. In syngeneic isogenic murine models PD-1 blockade significantly inhibited the growth of Kras mutant tumors with wild-type LKB1 (K), but not those with LKB1 loss (KL), providing evidence that LKB1 loss can play a causative role in promoting PD-1 inhibitor resistance. Conclusion: Loss of function genomic alterations in STK11 represents a dominant driver of de novo resistance to PD-1/PDL1 blockade in KRAS-mutant NSCLC. In addition to tumor PD-L1 status and tumor mutational burden precision immunotherapy approaches should take into consideration the STK11 status of individual tumors

Background: Proinflammatory cytokine Interleukin (IL)-17A (IL-17A) is the prototypical member of the IL-17 family of pro-inflammatory cytokines. It is produced by Th17 cells, CD8 T cells, gdT cells, and Natural Killer (NK) cells in the tumor microenvironment. The inflammatory milieu can contribute to lung cancer growth by further production of tumor promoting cytokines, reduction in cytotoxic T cells, and development of myeloid derived suppressor cells. IL-17A and its receptors are expressed across different tumor types; however, their exact role in tumor development, progression, and response to therapeutic regimens is unclear. IL-17A is overexpressed in a subset of patients with lung cancer. We hypothesized that IL-17A promotes a protumorigenic inflammatory phenotype, and inhibits anti-tumor immune responses. Method: IL-17A is expressed at high levels in a subset of lung cancers. Interestingly, we observed that IL-17A could not be detected in Bronchoalveolar lavage fluids (BALFs) from immunocompetent mouse lung cancer models. To characterize the role of IL-17A in Kras mutant lung tumors, we developed a mouse model of chronic inflammation that more closely resembles human KRAS mutant lung cancer through expressing IL-17A constitutively in the lung epithelium and then introducing this allele into lox-stop-lox Kras G12D mutant mice. We performed immune phenotyping of mouse lungs, survival analysis, and treatment studies with antibodies either blocking PD-1 or IL6, or depleting neutrophils. To support preclinical findings, we analyzed human gene expression datasets and immune profiled patient lung tumors. Result: Tumors in IL-17:Kras G12Dmice grew more rapidly, resulting in a significantly shorter survival as compared to Kras G12D. IL-6, G-CSF, MFG-E8, and CXCL1 were increased in the lungs of IL17:Kras mice. Time course analysis revealed that tumor-associated neutrophils were significantly elevated, and lymphocyte recruitment was significantly reduced in IL17:Kras G12D mice as compared to Kras G12D. In therapeutic studies PD-1 blockade was not effective in treating IL-17:Kras G12D tumors. In contrast, blocking IL-6 or depleting neutrophils with an anti-Ly-6G antibody in the IL17:Kras G12D tumors resulted in a clinical response associated with T cell activation. In tumors from lung cancer patients with KRAS mutation we found a correlation among higher levels of IL-17A and the colony stimulating factor (CSF3), and a significant correlation among high neutrophil and lower T cell numbers. Conclusion: Here we show that an increase in a single cytokine, IL-17A, without additional mutations, can promote lung cancer growth by promoting inflammation, which contributes to resistance to PD-1 blockade and sensitizes tumors to cytokine/ neutrophil depletion

Background: There is growing appreciation for the role of tumordraining lymph nodes (TDLN) in the dynamic of immuno-editing orchestrated by non-small cell lung cancers (NSCLC). By comparing Tcell subsets and gene expression in TDLN and non-draining lymph nodes (NDLN), we aim to determine whether there is tumor-regional variation in immunophenotype. Method: Patients undergoing endobronchial ultrasound-guided transbronchial needle aspiration for the diagnosis/staging of NSCLC were recruited. Aspirates were obtained from TDLN (N1/N2 nodes with increased fluorodeoxyglucose-F-18 (FDG) avidity and/or enlarged >1cm) and NDLN (non-enlarged/non- FDG-avid N2/N3 nodes) along with peripheral blood. Samples were stained with fluorophore-conjugated antibodies (CD4-FITC, CD8-V450, CD25-PECy7, CD127-APCR700, CD45RO-PECF594) and analyzed by flow cytometry. CD4+CD25- and CD8+ effector T-cells (Teff) were sorted. Gene expression profiling was performed on sorted Teff using the NanostringTM platform to measure differential expression between TDLN and NDLNs. Result: We compared T-cell subpopulations in TDLN and paired NDLN from 16 subjects. There were significantly fewer CD4+ T-cells in TDLN vs NDLN (10.1% vs 28.9%, p=0.0039), with more Tregs (12.1% vs 7.3%, p=0.1563) suggesting a pattern of tumorregional immunosuppression in the TDLN. This was more consistent when tumor histology was adenocarcinoma compared to squamous cell cancer with respect to both depletion of Teff and higher proportion of Tregs (Fig 1). A more immunosuppressive TDLN phenotype was also observed with high tumor PD-L1 expression (>50%), with 36% fewer CD4+ T-cells in TDLN relative to paired NDLN when PD-L1 expression was high relative to just 3.2% fewer CD4+ T-cells with low PD-L1 expression. Gene expression in Teff has preliminarily demonstrated upregulation of genes mediating T-cell exhaustion (CTLA-4, PD-1, TGFb) and downregulation of co-stimulatory/recruitment factors (CD28, ICOS, ICAM2) in TDLN suggesting impaired activation of tumorregional Teff. Conclusion: Our findings suggest that TDLNs in patients with NSCLC display a tolerogenic phenotype, with more marked immunosuppression in the setting of adenocarcinoma and high tumor PD-L1 expression. (Figure Presented)