Alissa Poteete

Mechanisms and clinical activity of an EGFR and HER2 exon 20–selective kinase inhibitor in non–small cell lung cancer

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.Poziotinib is a candidate inhibitor for a subset of EGFR or HER2 mutant non–small cell lung cancers that lack effective therapy.

Stress hormones promote EGFR inhibitor resistance in NSCLC: Implications for combinations with β-blockers

Chronic stress hormones promote EGFR inhibitor resistance in EGFR mutant non–small cell lung cancer. Destressing cancer with β-blockers Common wisdom holds that stress is not good for cancer patients, but it can be difficult to avoid considering that both the diagnosis of cancer and the associated treatments are quite stressful for the mind and body. Nilsson et al. investigated this phenomenon in non–small cell lung cancer, providing insights into the underlying mechanism and a potential intervention. The authors found that stress hormones activate β2-adrenergic receptors on cancer cells, activating a signaling cascade that promotes tumor resistance to EGFR inhibitors, a key therapy for this disease. Conversely, β-blockers—a common class of drugs used in humans—blocked this mechanism of resistance and may become a useful adjunct to lung cancer therapy regimens. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance mediated by T790M-independent mechanisms remains a major challenge in the treatment of non–small cell lung cancer (NSCLC). We identified a targetable mechanism of EGFR inhibitor resistance whereby stress hormones activate β2-adrenergic receptors (β2-ARs) on NSCLC cells, which cooperatively signal with mutant EGFR, resulting in the inactivation of the tumor suppressor, liver kinase B1 (LKB1), and subsequently induce interleukin-6 (IL-6) expression. We show that stress and β2-AR activation promote tumor growth and EGFR inhibitor resistance, which can be abrogated with β-blockers or IL-6 inhibition. IL-6 was associated with a worse outcome in EGFR TKI–treated NSCLC patients, and β-blocker use was associated with lower IL-6 concentrations and improved benefit from EGFR inhibitors. These findings provide evidence that chronic stress hormones promote EGFR TKI resistance via β2-AR signaling by an LKB1/CREB (cyclic adenosine 3′,5′-monophosphate response element–binding protein)/IL-6–dependent mechanism and suggest that combinations of β-blockers with EGFR TKIs merit further investigation as a strategy to abrogate resistance.

Abstract 5648: Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models

Introduction: Immunotherapy with single agent anti-PD-1 antibody confers modest overall survival benefits in patients with recurrent/metastatic HNSCCs. Combined immune checkpoint targeting has shown promising results for melanomas and other solid tumors. Thus, evaluation of combination immunotherapy in animal models specific to HNSCCs could inform prioritization and design of human clinical trials for this disease. We conducted this pre-clinical study to test the hypothesis that combining anti-PD-1 with either anti-CTLA-4 or co-stimulatory immune checkpoint agonist antibodies would be more effective than anti-PD-1 monotherapy in a mouse model of oral carcinoma. We also aimed at identifying the combination regimen with highest anti-tumor activity. Methods: C57BL/6 mice were subcutaneously grafted with 2x106 mouse oral cancer 1 (MOC1) cells derived from a carcinogen-induced tumor molecularly similar to human HNSCC. After 25 days, mice were randomized into one of 12 treatment groups (IgG, or antibodies targeting PD-1, CTLA-4, CD40, OX-40, GITR, 4-1BB, PD-1+CTLA-4, PD1+CD40, PD-1+OX-40, PD-1+GITR, or PD-1+4-1BB), N=8-9 mice/group. Antibodies were administered every 4 days for 3 doses. Tumors were measured twice per week. The primary endpoint was overall survival. Mice were sacrificed when tumor diameter was >2 cm or tumor ulceration was >5 mm. Survival curves were computed using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test. Results: In analogy to human clinical trials, anti-PD-1 therapy led to a modest improvement in survival compared to IgG [HR=0.41; 95%CI 0.06-0.53; P=0.015], indicating that this model recapitulates, in part, the effects of immunotherapy in HNSCC patients; none of the animals survived long term. Monotherapy with anti-CTLA-4, anti-CD40, anti-OX-40, or anti-4-1BB also improved survival compared to IgG (P=0.0001, 0.003, 0.02, and 0.003, respectively), but were not more effective than anti-PD-1 alone (P=0.09, 0.12, 0.72, and 0.77, respectively). Combination therapy did not prolong survival compared to anti-PD-1 alone, with the exception of anti-PD-1+ anti-CTLA4 [HR=0.36; 95% CI 0.07-0.6; P=0.016], and anti-PD-1+ anti-CD40 [HR=0.125, 95% CI 0.02-0.24; P=0.0003]. Notably, 50% of mice receiving anti-PD-1+ anti-CD40 antibodies had complete tumor eradication and are alive and disease-free 120 days after tumor grafting. Conclusions: Single agent immunotherapy targeting PD-1, CTLA-4, CD40, OX-40 and 4-1BB modestly improved survival in a mouse model of oral cancer. Combination with anti-PD-1+ anti-CTLA4 and anti-PD-1+ anti-CD40 antibodies outperformed anti-PD-1 monotherapy. Complete responses were exclusively seen with PD-1+CD40 dual targeting. This regimen should be prioritized for evaluation in HNSCC clinical trials. Citation Format: Jose Augusto Monteiro de Oliveira Novaes, Alissa R. Poteete, Marlese A. Pisegna, Uma Giri, Fahao Zhang, Patrick Hwu, John V. Heymach, William N. William. Combined immune checkpoint targeting with anti-PD-1 plus anti-CD40 antibodies as the most effective approach to eradicate head and neck squamous cell carcinomas (HNSCCs) in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5648. doi:10.1158/1538-7445.AM2017-5648

Abstract 3272: LKB1 loss is associated with resistance to VEGF inhibitors in non-small cell lung cancer (NSCLC)

Background LKB1 is a protein kinase that is mutated and down-regulated in 20-30% of NSCLC. LKB1 mutations co-occur with KRAS alterations in 7%-10% of NSCLC, resulting in an aggressive phenotype with short survival, and frequent metastases. Because LKB1 activates AMPK, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in the cells. However LKB1 also plays an important role in regulating tumor progression, metastasis and angiogenesis, likely as a compensatory strategy to overcome energetic depletion of tumor microenvironment. Bevacizumab, the anti-VEGF antibody improves PFS in NSCLC patients combined with chemotherapy but the benefit is modest and transient and often patients develop resistance. Our laboratory has identified alterations in cell metabolism and in vasculature of LKB1 deficient tumors when compared to LKB1 wild type. These findings may indicate that loss of LKB1 could alter the tumor vasculature in NSCLC. Methods mRNA expression of angiogenesis related genes were analyzed in wild type and LKB1 deficient NSCLC tumors (TCGA). In vitro validation was performed by qPCR and western blot. CD31 IHC was performed to analyze microvasculature density (MVD) in Krasmut and Krasmut LKB1f/f mice tumors. HUVEC tube formation and migration assays were performed with conditioned medium of LKB1 expressing and deficient NSCLC isogeneic cell lines. Xenograft NSCLC models were established via s.c. injection of H460 (LKB1 deficient) and H460 LKB1 (LKB1 expressing) cells in nude mice. Treatment consisting of human and/or mouse bevacizumab and nintedanib were administrated. Tumor volumes were measured and vasculature analysis was performed. Results In vitro HUVEC cells exhibited an increase migration and differences in endothelial network formation when incubated with conditioned medium from LKB1 deficient cells compared to LKB1 expressing cells medium (p Conclusions LKB1 deficiency may promote resistance to anti-angiogenic therapy by regulating compensatory angiogenic pathways along metabolic adaptations to energetic stress in NSCLC. Citation Format: Irene Guijarro, Alissa Poteete, Chao Yang, Emily Roarty, Monique Nilsson, Huiying Sun, Pan Tong, Edward Chang, Jaime Rodriguez-Canales, Barbara Mino, Edwin Parra, Ignacio Wistuba, Jing Wang, Timothy Heffernan, John V. Heymach. LKB1 loss is associated with resistance to VEGF inhibitors in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3272.