Faculty Bibliography

Background: Approximately 10% of EGFR mutant NSCLCs have an insertion/mutation in exon 20 of EGFR resulting in primary resistance to currently available tyrosine kinase inhibitors (TKIs). We previously reported that the structural features of poziotinib could potentially enable it to circumvent the steric hindrance induced by exon 20 mutations. Here we further characterize the preclinical activity of poziotinib and report on initial clinical activity of poziotinib in patients with EGFR exon 20 mutations from an ongoing phase II study. Method: We evaluated poziotinib activity in vitro using human NSCLC cell lines and the BAF3 model as well as several patient-derived xenograft (PDX) models and genetically engineered mouse models (GEMMs) of exon 20 insertion. We launched a phase 2 investigator-initiated trial of poziotinib in patients with metastatic NSCLC with EGFR exon 20 insertions (NCT03066206). Result: In vitro poziotinib was approximately 100x more potent than osimertinib and 40x more potent than afatinib against a common panel of EGFR exon 20 insertions. Furthermore, it had w65-fold greater potency against common exon 20 insertions compared with EGFR T790M mutations; 3rd generation inhibitors osimertinib, EGF816, and rociletinib were all significantly less potent for exon 20 mutations/ insertions compared with T790M. in vivo poziotinib led to >85% reduction in tumor burden in GEM models of EGFR exon 20 insertion (D770insNPG) NSCLC and the PDX model LU0387 (H773insNPH). To date, 8 platinum-refractory patients with EGFR exon 20 insertion mutation metastatic NSCLC have been enrolled in the clinical trial and treated with poziotinib at a dose of 16 mg PO daily. Two patients have reached the first interval-imaging time point (at 8 weeks of therapy per protocol). Both patients exhibited dramatic partial response, with one patient reporting improvement in dyspnea and cough at one week of therapy. In this early stage of the study, one case of grade 3 paronchycia was observed. One additional platinum- and erlotinib-refractory patient with EGFR exon 20 insertion was treated with poziotinib on compassionate basis. The patient achieved partial response after three weeks of treatment. Conclusion: Poziotinib has selective activity against EGFR exon 20 mutations and potent activity in cell lines, PDX, and GEM models. Three platinum-refractory patients with EGFR exon 20 mutations have been treated thus far and are evaluable for response; all three had partial responses at the time of the initial scan. Updated data from the ongoing phase 2 clinical trial of poziotinib will be presented at the meeting

Notch1 transactivates Notch3 to drive terminal differentiation in stratified squamous epithelia. Notch1 and other Notch receptor paralogs cooperate to act as a tumor suppressor in squamous cell carcinomas (SCCs). However, Notch1 can be stochastically activated to promote carcinogenesis in murine models of SCC. Activated form of Notch1 promotes xenograft tumor growth when expressed ectopically. Here, we demonstrate that Notch1 activation and epithelial-mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-beta present in the tumor microenvironment. We find that TGFbeta activates the transcription factor ZEB1 to repress Notch3, thereby limiting terminal differentiation. Concurrently, TGFbeta drives Notch1-mediated EMT to generate tumor initiating cells characterized by high CD44 expression. Moreover, Notch1 is activated in a small subset of SCC cells at the invasive tumor front and predicts for poor prognosis of esophageal SCC, shedding light upon the tumor promoting oncogenic aspect of Notch1 in SCC.

Purpose:KRAS-activating mutations are the most common oncogenic driver in non-small cell lung cancer (NSCLC), but efforts to directly target mutant KRAS have proved a formidable challenge. Therefore, multitargeted therapy may offer a plausible strategy to effectively treat KRAS-driven NSCLCs. Here, we evaluate the efficacy and mechanistic rationale for combining mTOR and WEE1 inhibition as a potential therapy for lung cancers harboring KRAS mutations.Experimental Design: We investigated the synergistic effect of combining mTOR and WEE1 inhibitors on cell viability, apoptosis, and DNA damage repair response using a panel of human KRAS-mutant and wild type NSCLC cell lines and patient-derived xenograft cell lines. Murine autochthonous and human transplant models were used to test the therapeutic efficacy and pharmacodynamic effects of dual treatment.Results: We demonstrate that combined inhibition of mTOR and WEE1 induced potent synergistic cytotoxic effects selectively in KRAS-mutant NSCLC cell lines, delayed human tumor xenograft growth and caused tumor regression in a murine lung adenocarcinoma model. Mechanistically, we show that inhibition of mTOR potentiates WEE1 inhibition by abrogating compensatory activation of DNA repair, exacerbating DNA damage in KRAS-mutant NSCLC, and that this effect is due in part to reduction in cyclin D1.Conclusions: These findings demonstrate that compromised DNA repair underlies the observed potent synergy of WEE1 and mTOR inhibition and support clinical evaluation of this dual therapy for patients with KRAS-mutant lung cancers. Clin Cancer Res; 23(22); 6993-7005. (c)2017 AACR.

Cells lacking the tumor suppressor gene LKB1/STK11 alter their metabolism to match the demands of accelerated growth, leaving them highly vulnerable to stress. However, targeted therapy for LKB1-deficient cancers has yet to be reported. In both Kras/p53/Lkb1 cell lines and a genetically engineered mouse model of Kras/p53/Lkb1-induced lung cancer, much higher rates of DNA damage occur, resulting in increased dependence on Chk1 checkpoint function. Here we demonstrate that short-term treatment with the Chk1 inhibitor AZD7762 reduces metabolism in pembrolizumab tumors, synergizing with the DNA-damaging drug gemcitabine to reduce tumor size in these models. Our results offer preclinical proof of concept for use of a Chk1 inhibitor to safely enhance the efficacy of gemcitabine, particularly in aggressive KRAS-driven LKB1-deficient lung adenocarcinomas. Cancer Res; 77(18); 5068-76. (c)2017 AACR.

Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.

HYPOTHESIS: Proinflammatory cytokine Interleukin (IL)-17A (IL-17A) is overexpressed in a subset of patients with lung cancer. We hypothesized that IL-17A promotes a pro-tumorigenic inflammatory phenotype, and inhibits anti-tumor immune responses. EXPERIMENTAL DESIGN: We generated bi-transgenic mice expressing a conditional IL-17A allele along with conditional KrasG12D and 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. RESULTS: Tumors in IL-17:KrasG12D mice grew more rapidly, resulting in a significantly shorter survival as compared to KrasG12D. 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 (TANs) were significantly elevated, and lymphocyte recruitment was significantly reduced in IL17:KrasG12D mice as compared to KrasG12D. In therapeutic studies PD-1 blockade was not effective in treating IL-17:KrasG12D tumors. In contrast, blocking IL-6 or depleting neutrophils with an anti-Ly-6G antibody in the IL17:KrasG12D 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. CONCLUSIONS: 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.

Effective therapies for non-small cell lung cancer (NSCLC) remain challenging despite an increasingly comprehensive understanding of somatically altered oncogenic pathways. It is now clear that therapeutic agents with potential to impact the tumor immune microenvironment potentiate immune-orchestrated therapeutic benefit. Herein we evaluated the immunoregulatory properties of histone deacetylase (HDAC) and bromodomain inhibitors, two classes of drugs that modulate the epigenome, with a focus on key cell subsets that are engaged in an immune response. By evaluating human peripheral blood and NSCLC tumors, we show that the selective HDAC6 inhibitor ricolinostat promotes phenotypic changes that support enhanced T cell activation and improved function of antigen presenting cells. The bromodomain inhibitor JQ1 attenuated CD4+Foxp3+ T regulatory cell suppressive function and synergized with ricolinostat to facilitate immune-mediated tumor growth arrest, leading to prolonged survival of mice with lung adenocarcinomas. Collectively, our findings highlight the immunomodulatory effects of two epigenetic modifiers that, together, promote T cell-mediated anti-tumor immunity and demonstrate their therapeutic potential for treatment of NSCLC.

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial-mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically.

Response to immune checkpoint blockade in mesenchymal tumors is poorly characterized, but immunogenomic dissection of these cancers could inform immunotherapy mediators. We identified a treatment-naive patient who has metastatic uterine leiomyosarcoma and has experienced complete tumor remission for >2 years on anti-PD-1 (pembrolizumab) monotherapy. We analyzed the primary tumor, the sole treatment-resistant metastasis, and germline tissue to explore mechanisms of immunotherapy sensitivity and resistance. Both tumors stained diffusely for PD-L2 and showed sparse PD-L1 staining. PD-1+ cell infiltration significantly decreased in the resistant tumor (p = 0.039). Genomically, the treatment-resistant tumor uniquely harbored biallelic PTEN loss and had reduced expression of two neoantigens that demonstrated strong immunoreactivity with patient T cells in vitro, suggesting long-lasting immunological memory. In this near-complete response to PD-1 blockade in a mesenchymal tumor, we identified PTEN mutations and reduced expression of genes encoding neoantigens as potential mediators of resistance to immune checkpoint therapy.

OBJECTIVES: Immune checkpoint inhibitors have demonstrated clinical benefit in recurrent, metastatic (R/M) squamous cell carcinoma of the head and neck (SSCHN), but lacking are biomarkers that predict response. We sought to define an inflamed tumor immunophenotype in this R/M SCCHN population and correlate immune metrics with clinical parameters and survival. METHODS: Tumor samples were prospectively acquired from 34 patients to perform multiparametric flow cytometry and multidimensional clustering analysis integrated with next-generation sequencing data, clinical parameters and outcomes. RESULTS: We identified an inflamed subgroup of tumors with prominent CD8+ T cell infiltrates and high PD-1/TIM3 co-expression independent of clinical variables, with improved survival compared with a non-inflamed subgroup (median overall survival 84.0 vs. 13.0months, p=0.004). The non-inflamed subgroup demonstrated low CD8+ T cells, low PD-1/TIM3 co-expression, and higher Tregs. Overall non-synonymous mutational burden did not correlate with response to PD-1 blockade in a subset of patients. CONCLUSION: R/M SCCHN patients with an inflamed tumor immunophenotype demonstrate improved survival. Further prospective studies are needed to validate these findings and explore the use of immunophenotype to guide patient selection for immunotherapeutic approaches.