KRAS-driven Lung Cancer Research Articles

Modulation of Bax and mTOR for Cancer Therapeutics.

A rationale exists for pharmacologic manipulation of the serine (S)184 phosphorylation site of the proapoptotic Bcl2 family member Bax as an anticancer strategy. Here, we report the refinement of the Bax agonist SMBA1 to generate CYD-2-11, which has characteristics of a suitable clinical lead compound. CYD-2-11 targeted the structural pocket proximal to S184 in the C-terminal region of Bax, directly activating its proapoptotic activity by inducing a conformational change enabling formation of Bax homooligomers in mitochondrial membranes. In murine models of small-cell and non-small cell lung cancers, including patient-derived xenograft and the genetically engineered mutant KRAS-driven lung cancer models, CYD-2-11 suppressed malignant growth without evident significant toxicity to normal tissues. In lung cancer patients treated with mTOR inhibitor RAD001, we observed enhanced S184 Bax phosphorylation in lung cancer cells and tissues that inactivates the propaoptotic function of Bax, contributing to rapalog resistance. Combined treatment of CYD-2-11 and RAD001 in murine lung cancer models displayed strong synergistic activity and overcame rapalog resistance in vitro and in vivo Taken together, our findings provide preclinical evidence for a pharmacologic combination of Bax activation and mTOR inhibition as a rational strategy to improve lung cancer treatment. Cancer Res; 77(11); 3001-12. ©2017 AACR.

Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours.

KRAS is one of the most frequently mutated oncogenes in human non-small cell lung cancers (NSCLCs). RAS proteins trigger multiple effector signalling pathways including the highly conserved RAF-MAPK pathway. CRAF, a direct RAS effector protein, is required for KRAS-mediated tumourigenesis. Thus, the molecular mechanisms driving the activation of CRAF are intensively studied. Prohibitin 1 (PHB1) is an evolutionarily conserved adaptor protein and interaction of CRAF with PHB1 at the plasma membrane is essential for CRAF activation. Here, we demonstrate that PHB1 is highly expressed in NSCLC patients and correlates with poor survival. Targeting of PHB1 with two chemical ligands (rocaglamide and fluorizoline) inhibits epidermal growth factor (EGF)/RAS-induced CRAF activation. Consistently, treatment with rocaglamide inhibited proliferation, migration and anchorage-independent growth of KRAS-mutated lung carcinoma cell lines. Surprisingly, rocaglamide treatment inhibited Ras-GTP loading in KRAS-mutated cells as well as in EGF-stimulated cells. Rocaglamide treatment further prevented the oncogenic growth of KRAS-driven lung cancer allografts and xenografts in mouse models. Our results suggest rocaglamide as a RAS inhibitor and that targeting plasma membrane-associated PHB1 with chemical ligands would be a viable therapeutic strategy to combat KRAS-mediated NSCLCs.

All for one and FOSL1 for all: FOSL1 at the crossroads of lung and pancreatic cancer driven by mutant KRAS

ABSTRACTKRAS proto-oncogene, GTPase (KRAS) remains refractory to current therapies. We devised an integrative cross-tumor approach to expose common core elements up-regulated in mutant KRAS cancers that could provide new treatment opportunities. This approach identified FOSL1 (Fos-like antigen 1) as a clinically and functionally relevant gene in mutant KRAS-driven lung and pancreatic cancers, and unveiled downstream transcriptional targets amenable to pharmacological inhibition

Abstract B27: Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer

Abstract Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p<0.004). In a separate study, similar tumor regressions were noted in the FGFR-driven GEMM at 1 week of erdafitinib with or without anti-PD-1 treatment, while no such response to these treatments was noted in a KRAS-driven lung cancer GEMM. Immune profiling of tumor specimens revealed high baseline expression of programmed death ligand-1 (PD-L1) expression by IHC and flow cytometry. Following combination treatment, subsequent immunohistochemistry (IHC) analyses showed a significant decrease in Ki67 and PD-L1 positive tumor cells, accompanied by an increase in cluster of differentiation 3 (CD3) positive tumor-infiltrating cells in combination group as compared to control. T cell function is not inhibited by erdafitinib, as measured in vitro by mixed lymphocyte reaction and cytomegalovirus recall assays. Additional changes observed in lung tumors across treatment groups in immune cell infiltration, functionality, and T-cell clonality will be discussed. These data suggest that combination treatment of erdafitinib and PD-1 blockade drives improved survival in FGFR2-driven model of lung cancer by simultaneous inhibition of FGFR pathway in tumor cells and enhancement of anti-tumor immunity. Thus, data here provide rationale for the combined clinical testing of erdafitinib and PD-1 blockade in patients with FGFR-altered lung cancers. Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.

Abstract IA05: Autophagy prevents fatal nucleotide pool depletion in Ras-driven cancer cells

Abstract Autophagy degrades and recycles proteins and organelles essential for quality control and survival in starvation. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes tumor growth and malignancy. Autophagy-deficient tumor-derived cell lines (TDCLs) are respiration impaired, starvation sensitive, and glutamine-dependent. To survive starvation, autophagy may eliminate defective mitochondria or supply mitochondrial substrates. Here, we sequenced the mitochondrial genome from Kras-driven lung tumors and found higher mutant allele frequencies without autophagy-related-7 (Atg7). But the low pathogenic allele frequency was insufficient to account for metabolic defects. In starvation, Atg7 deficiency attenuated substrate supply for mitochondrial oxygen consumption and increased nucleotide degradation. The resulting nucleotide pool depletion and energy crisis in starvation was rescued by nucleoside supplementation. Thus the role for autophagy in Kras-driven tumor cells is to provide substrates to mitochondria to maintain nucleotide pools and energy homeostasis, which promotes survival to nutrient limitation. Citation Format: Eileen White. Autophagy prevents fatal nucleotide pool depletion in Ras-driven cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA05.

P3.02c-070 Combination Immunotherapy with MEK Inhibitor for Treatment of Kras-Mutant Lung Cancer in Animal Model: Topic: IT Biomarkers

Lung cancer remains a major cause of cancer mortality. Malignant lesions are normally endogenously corrected by the immune surveillance system. However, tumors evade this immunity by inducing immunosuppressive microenvironments during cancer progression. Recent studies demonstrate that multiple cancer types, including melanoma, lung, kidney, bladder, and stomach, respond to immune checkpoint inhibitors, such as PD-L1 and PD-1 with 11-30% response rates and durable responses. However, a substantial number of patients still fail to respond to immunotherapy and the refractory mechanisms are largely unknown. In this study, we focus on KRas-driven lung cancers, as there are no clinically effective targeted drugs available for treating this type of lung cancer. We examined tumor infiltrated immune cells using FACS, CyTOF2, and Immunostaining of lung sections during the progression of lung tumors in KRas mutation and p53 knockout-driven lung cancer mouse models; KRasG12D/+;p53-/- (KP). Using this mouse model, we determined the anti-cancer efficacy of combined inhibition of MEK and immune checkpoint molecules. We demonstrate that there is a gradual increase in the number of myeloid derived suppressor cells (MDSC) and that the combination of either anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor shows anticancer efficacy in these animal models. These combinations, in comparison to either single agent alone, effectively blocks the growth of subcutaneously injected syngeneic mouse lung cancer cells in immune competent transgenic KP mice, significantly increasing the survival rates: 37.5% (for anti-PD-1 antibody and MEK inhibitor), 62.5% (for anti-PD-L1 antibody and MEK inhibitor) vs. 0% single agents or control at the end of treatment. We find that the tumors in the control treated group harbor a substantial number of immune cells, including PD-L1 expressing MDSC. The combination treatment with either an anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor dramatically modulates the composition and the activity of tumor infiltrated immune cells. Tumors in the combination treatment group show a significant decrease in PD-L1 expressing MDSC in comparison with control tumors. Additionally, combination treatment blocks PD-L1 activity of the infiltrated PD-L1 expressing MDSC in malignant tumors and thus lead to improved survival. These results point to a potential therapeutic opportunity for currently untargetable KRas-driven lung cancers.

Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells.

Autophagy degrades and is thought to recycle proteins, other macromolecules, and organelles. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes malignancy. Autophagy-deficient tumor-derived cell lines are respiration-impaired and starvation-sensitive. However, to what extent their sensitivity to starvation arises from defective mitochondria or an impaired supply of metabolic substrates remains unclear. Here, we sequenced the mitochondrial genomes of wild-type or autophagy-deficient (Atg7(-/-)) Kras-driven lung tumors. Although Atg7 deletion resulted in increased mitochondrial mutations, there were too few nonsynonymous mutations to cause generalized mitochondrial dysfunction. In contrast, pulse-chase studies with isotope-labeled nutrients revealed impaired mitochondrial substrate supply during starvation of the autophagy-deficient cells. This was associated with increased reactive oxygen species (ROS), lower energy charge, and a dramatic drop in total nucleotide pools. While starvation survival of the autophagy-deficient cells was not rescued by the general antioxidant N-acetyl-cysteine, it was fully rescued by glutamine or glutamate (both amino acids that feed the TCA cycle and nucleotide synthesis) or nucleosides. Thus, maintenance of nucleotide pools is a critical challenge for starving Kras-driven tumor cells. By providing bioenergetic and biosynthetic substrates, autophagy supports nucleotide pools and thereby starvation survival.

Abstract 297: Epigenetic activation of IGF1R signaling promotes resistance to momelotinib and MEK inhibition in KRAS-driven lung cancer

Abstract KRAS is the most frequently mutated oncogene in non-small cell lung cancer (NSCLC), yet remains resistant to targeted therapy. Previously, we reported that RAL-TBK1 activation downstream of KRAS promotes an autocrine cytokine circuit that fuels tumorigenesis. Interruption of this signaling by treatment with momelotinib, an inhibitor of TBK1/IKKϵ and JAK kinases, together with MEK inhibition, was effective in the aggressive Kras-p53 (KP) mouse lung cancer model. These findings have prompted a human clinical trial of momelotinib and trametinib in advanced refractory KRAS mutant NSCLC. Because this combination leads to acquired resistance in mice, we sought to understand additional pathways that could compensate for TBK1/JAK and MEK inhibition. Receptor tyrosine kinase (RTK) profiling of A549 cells treated with momelotinib revealed delayed activation of IGF1 receptor (IGF1R) signaling. Indeed, IGF1 supplementation partially rescued momelotinib-induced growth arrest of A549 cells, while combination of momelotinib with the dual IGF1R/insulin receptor (IR) inhibitor linsitinib cooperated to impair cell viability, which was more pronounced in KRAS/STK11 (encoding LKB1) mutant cell lines compared with LKB1 wild type cell lines. Triple momelotinib/trametinib/linsitinib therapy also improved tumor shrinkage in Kras-Lkb1 (KL) mice compared with KP mice. However, chronic treatment with this triple combination required pulse therapy due to toxicity, and was inferior to momelotinib/trametinib dosed daily, which itself promoted deep responses in KL mice lasting 6 weeks. In parallel, we analyzed mediators of acquired resistance to chronic momelotinib and MEK inhibitor exposure in A549 cells. Interestingly, IGF1 expression was dramatically upregulated in resistant cells, in contrast to IGF2, and enhanced autocrine activation of IGF1R signaling. Importantly, both IGF1 upregulation and inhibitor resistance were completely reversible after drug withdrawal for several passages, yet reappeared quickly upon drug readdition, suggesting epigenetic reprogramming. Consistent with this hypothesis, whereas global H3K27 histone acetylation was decreased in resistant cells, this epigenetic mark was strongly enriched at the IGF1 promoter and linked tightly with both IGF1 levels and drug resistance. Treatment of resistant A549 cells with the BRD4 inhibitor JQ1 completely suppressed IGF1 expression and downstream IGF1R activation, and impaired cell viability. Combination JQ1 treatment also prevented momelotinib/MEK inhibitor induction of IGF1 in A549 cells, synergized to reduce outgrowth of resistant cells, and was well tolerated as daily triple combination therapy in mice. Together, these findings suggest the potential of BET inhibition to synergize with and prevent the development of acquired resistance to momelotinib and MEK inhibitor treatment in KRAS mutant NSCLC. Citation Format: Shunsuke Kitajima, Hajime Asahina, Jillian D. Cavanaugh, Ting Chen, Ashley A. Merlino, Thai C. Tran, Kwonk-Kin Wong, David A. Barbie. Epigenetic activation of IGF1R signaling promotes resistance to momelotinib and MEK inhibition in KRAS-driven lung cancer. [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 297.

Abstract 4184: Pre-clinical study using KRAS mutant mouse models for lung cancer immunotherapy together with MEK inhibition

Abstract Activating KRAS mutations are identified as the most common driver oncogene for lung adenocarcinomas, accounts for 20∼30% of lung cancer patients. The development of therapeutic strategy for KRAS mutant lung cancer patients remains to be an unmet need, due to poor prognosis and therapeutic resistance to conventional therapy. The purpose of our study is through utilizing different types of genetically engineered mouse models (GEMMs), to understand the molecular heterogeneity of KRAS mutant lung cancer, to define the immune microenvironment, and to determine the feasibility and strategy of using MEK inhibition in combination with immune checkpoint inhibitors for the treatment of KRAS mutant lung cancer. We generated two different types of GEMMs with KRAS mutations that develop tumor in the lung: Kras with G12D mutation or KRAS with G12C mutation. Continuous treatment of either KrasG12D or KRASG12C mice with MEK inhibitor Selumetinib (AZD6244, ARRY-142886) shows short-term response followed by drug resistance to single regimen treatment. The immuno-profiling shows that Selumetinib can increase cytotoxic T cells percentage and decrease PD-L1 expression on both myeloid cells and lymphocytes. Prolonged treatment of Selumetinib on KRAS mutant mice will lead to down-regulation of the expression of immune-checkpoint inhibitory factors Ctla-4 and Pd-1 on both CD4+ and CD8+ T cells. When KRAS mutant mice are treated with Selumetinib intermittently, they show improved response comparing with continuous treatment. When combining MEK inhibition with immune checkpoint blockade by using Selumetinib together with Pd-1 antibody treatment, Tim3 and Ctla-4 expression on T cells is increased potentially leading to T cell exhaustion and immune suppression in lung cancer. These pre-clinical results provide molecular insight for the immune response to MEK inhibition in KRAS driven lung cancer. Furthermore, these data support our hypothesis that MEK inhibition combination with immune checkpoint blockade treatment will have better outcome for lung cancer patients that have KRAS mutation through activating T cell response. Citation Format: Jiehui Deng, Shuai Li, Grit Herter-Sprie, Paul D. Smith, Gordon J. Freeman, Jeffrey A. Engelman, Peter Hammerman, Kwok-Kin Wong. Pre-clinical study using KRAS mutant mouse models for lung cancer immunotherapy together with MEK inhibition. [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 4184.

Abstract 2020: Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer death in the United States and in the world. Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1, in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce either oncogene induced senescence or apoptosis. However, the key functions of TWIST1 that are required for its transcriptional activity in cancer are unknown. In the current study, we engineered domain-specific mutations in TWIST1 to determine the impact of altered DNA binding, dimerization and post-translational modifications on its transcriptional activity by luciferase reporter assays. Testing the promoter activity at three TWIST1-regulated loci involved in tumorigenesis, YBX1, SNAI2, and FLIP, we found that DNA binding and nuclear localization were uniformly required for TWIST1 transcriptional function. However, phosphorylation, TWIST1 box function, and ability to form homo- versus hetero- dimers impacted TWIST1 activity in a locus-specific manner. Previous studies have demonstrated that TWIST1 dimerizes with the E2A proteins, E12 and E47. We have shown that silencing of E2A phenocopies loss of TWIST1 in NSCLC and that formation of the TWIST1-E2A heterodimer results in a reciprocal stabilization of the binding partner. Therefore, we sought to determine how differential dimerization by TWIST1 might modulate tumorigenic gene expression. We utilized tethered TWIST1 dimers with either TWIST1 or the E2A proteins to form TWIST1-TWIST1 (homodimer), TWIST1-E12 or -E47 (heterodimers), and tested their transcriptional activities at several TWIST1-regulated promoters. We found that TWIST1-E2A heterodimers could enhance TWIST1 transcriptional activity compared to that of the TWIST-TWIST homodimer. In addition, we determined that the TWIST-E2A heterodimer is degraded by harmine, a harmala alkaloid that we have shown degrades TWIST1, whereas the TWIST-TWIST homodimer is resistant to harmine degradation. This suggests that the TWIST1-E2A heterodimer is the key target of harmine. Furthermore, we have found that overexpression of the TWIST1, E2A proteins, or the TWIST1-E2A heterodimer is able to partially rescue harmine induced cytotoxicity in KRAS mutant NSCLC. Taken together, these data suggest that E2A is essential for TWIST1 mediated tumorigenesis and that targeting of the TWIST1-E2A axis may be an effective therapeutic strategy against oncogene-driven NSCLC. Citation Format: Susheel K. Khetarpal. Structure-function analysis of twist1-e2a transcriptional activity in kras-driven non-small cell lung cancer. [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 2020.

Abstract 2334: Anti-cancer efficacy of the combination with immunomodulatory antibodies and MEK inhibitor for Kras mutation and p53 knockout driven lung cancer

Abstract Lung cancer remains a major cause of cancer mortality. Malignant lesions are normally endogenously corrected by the immune surveillance system. However, tumors evade this immunity by inducing immunosuppressive microenvironments during cancer progression. It has been thought that there are two critical components of tumors making them capable of maintaining the immunosuppressive microenvironment. One is the inhibition of cytotoxic T lymphocytes (CTL) activity against the tumor through immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1), which are negative regulators of CTL, and the other is by the induction of tumor infiltrating immune suppressive cells, which can deactivate the CTL. Recent studies demonstrate that multiple cancer types, including melanoma, lung, kidney, bladder, and stomach, respond to immune checkpoint inhibitors, such as PD-L1 and PD-1 with 11-30% response rates and durable responses. However, a substantial number of patients still fail to respond to immunotherapy and the refractory mechanisms are largely unknown. In this study, we focus on KRas-driven lung cancers, as there are no clinically effective targeted drugs available for treating this type of lung cancer. We demonstrate that during the progression of tumors in KRas mutation and p53 knockout-driven lung cancer mouse models; KRasG12D/+;p53-/- (KP), there is a gradual increase in the number of myeloid derived suppressor cells (MDSC) and that the combination of either anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor, which targets downstream of KRas signaling, shows anticancer efficacy in these animal models. These combinations, in comparison to either single agent alone, effectively blocks the growth of subcutaneously (s.c.) injected syngeneic mouse lung cancer cells in immune competent transgenic KP mice, significantly increasing the survival rates: 37.5% (for anti-PD-1 antibody and MEK inhibitor), 62.5% (for anti-PD-L1 antibody and MEK inhibitor) vs. 0% single agents or control at the end of treatment. We find that the tumors in the control treated group harbor a substantial number of immune cells, including PD-L1 expressing MDSC. The combination treatment with either an anti-PD-1 or anti-PD-L1 antibody along with a MEK inhibitor dramatically modulates the composition and the activity of tumor infiltrated immune cells. Tumors in the combined treated group show a significant decrease in PD-L1 expressing MDSC in comparison with control tumors. Additionally, a combined treatment would block the PD-L1 activity of the infiltrated PD-L1 expressing MDSC in malignant tumors and thus lead to improved survival. These results point to a potential therapeutic opportunity for currently untargetable KRas-driven lung cancers. Citation Format: Jong Woo Lee, Yu Zhang, Hee Sun Park, Sin Aye Park, JaSeok Peter Koo, Roy S. Herbst. Anti-cancer efficacy of the combination with immunomodulatory antibodies and MEK inhibitor for Kras mutation and p53 knockout driven lung cancer. [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 2334.

Abstract 1253: Inhibition of FN14 to suppress KRAS-driven lung cancer migration and survival

Abstract Lung cancer remains the leading cause of cancer related mortality throughout the world, and is estimated to kill more than one million people this year. Mutations in KRAS occur in about 1/3 of all lung adenocarcinomas, the most common histologic subtype of lung cancer. Mutations in KRAS are correlated with poor patient prognosis, advanced disease, therapeutic resistance and metastatic outcomes. There is an unmet clinical need to target KRAS-driven NSCLC towards abating the high mortality rate of this disease. We hypothesize that the tumor necrosis factor receptor, FN14 is induced and sustained by mutant KRAS signaling, and represents a therapeutic target in KRAS-driven NSCLC. Employing The Cancer Genome Atlas (TCGA) data, we showed that within a subgroup of adenocarcinoma patients harboring mutant KRAS, elevated FN14 expression conferred significantly worse survival outcomes compared to those with lower FN14 expression. Alone, elevated expression of FN14 conferred a poor prognosis in adenocarcinoma patients compared to lower expression of FN14. In lung cancer cell lines driven by mutant KRAS, elevated FN14 protein expression was observed. The ectopic induction of mutant KRAS in normal rat lung epithelial cells was sufficient to induce FN14 protein expression, an induction dependent on ERK signaling. In an animal model of mutant KRAS-driven NSCLC, FN14 protein was highly expressed and correlated with metastasis in vivo. TWEAK, the only known ligand for FN14, induced migration in NSCLC cell lines harboring mutant KRAS. Conversely, depletion of FN14 expression through siRNA suppressed KRAS-driven cell migration in vitro. We further demonstrated using a clonogenic assay that depletion of FN14 by siRNA was sufficient to reduce KRAS-driven NSCLC cell survival and significantly enhanced the cell killing effects of standard-of-care treatments such as radiation or cisplatin. These data suggest that NSCLC tumors harboring mutant KRAS may be vulnerable to inhibition of FN14 signaling. The recent development of FN14-targeted therapeutics provides an avenue towards clinical utility. As no therapeutics exist to target KRAS directly, a therapeutic strategy targeting FN14 might impact survival in KRAS-driven tumors and reduce the high mortality rate in this subgroup. Citation Format: Vashti M. Carson, Seungchan Kim, Landon J. Inge, Timothy G. Whitsett. Inhibition of FN14 to suppress KRAS-driven lung cancer migration and survival. [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 1253.

Abstract LB-168: MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer

Abstract Lung cancer is the leading cause of cancer deaths and yet the genes that drive this malignancy are not entirely known. MicroRNA (miRNA) are important regulators of gene expression and aberrant miRNA expression has been linked to lung oncogenesis; however, little known about their contribution in tumor initiation. We’ve determined that miR-31 is overexpressed in human lung adenocarcinoma and this overexpression independently correlates with decreased patient survival. miR-31 promotes lung cell proliferation and adenocarcinoma growth in vivo. We also developed a transgenic mouse model that allows for lung-specific expression of miR-31 to test the oncogenic potential of miR-31 in the lung. Using this model, we observed that miR-31 induction results in lung hyperplasia, followed by adenoma formation and later adenocarcinoma development. We identified 6 negative regulators of RAS/MAPK signaling (RASA1, SPRED1, SPRED2, SPRY1, SPRY3, SPRY4) as direct targets of miR-31. Analysis of human lung adenocarcinoma revealed that high miR-31 expression correlated to decreased target mRNA expression. miR-31 overexpression in lung cells results in increased RAS signaling and pharmacologic inhibition of MEK ablates miR-31 induced lung cell proliferation. Induced expression of miR-31 in mice cooperated with mutant KRAS to accelerate lung tumorigenesis. Our study demonstrates for the first time that a miRNA can drive an epithelial cancer. Specifically we identified miR-31 as a driver of lung tumorigenesis that promotes mutant KRAS-mediated oncogenesis and demonstrate that miR-31 directly targets and reduces expression of negative regulators of RAS/MAPK signaling. Citation Format: Mick D. Edmonds, Kelli L. Boyd, Tamara K. Moyo, Ramkrishna Mitra, Robert J. Duszynski, Maria P. Arrate, Xi Chen, Zhongming Zhao, Timothy S. Blackwell, Christine M. Eischen. MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer. [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 LB-168.

Abstract 1255: Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein

Abstract KRAS is frequently mutated in lung cancers. Innovative strategies are needed to combat KRAS mutant lung cancers because these tumors are often resistant to therapy. This study reports that loss of the deubiquitinase USP18 leads to destabilization of the KRAS oncogenic protein in panels of murine and human lung cancer cells. In marked contrast, engineered gain of USP18 expression using retroviral vectors introduced into the same panel of murine and human lung cancer cells stabilized KRAS protein. Loss of USP18 expression in KRAS mutant-expressing lung cancer cells inhibited their growth while gain of USP18 expression opposed this effect. We sought to identify mechanisms engaged in this KRAS protein destabilization. Intriguingly, immunoprecipitation assays established that KRAS conjugates with the ubiquitin-like protein ISG15. This leads to KRAS protein destabilization. Using the protein synthesis inhibitor cycloheximide, USP18 knockdown was shown to destabilize mutant KRAS more prominently than wild-type KRAS protein. To independently confirm that KRAS directly complexes with ISG15, site-directed mutagenesis was performed to render the C-terminal domain of KRAS lysine-less. This led to stabilization of KRAS, despite knockdown of USP18. These studies were confirmed and extended in the in vivo setting of KRAS-driven lung cancers in KRASLA2/+ mice within the FVB strain background. We crossed these mice with USP18 null (USP18-/-) mice to obtain KRASLA2/+;USP18-/- compound mice. Strikingly, these compound mice had statistically significantly (P < 0.05) reduced lung cancers as compared to parental KRASLA2/+ mice. To establish the biological importance of this finding, USP18 immunohistochemical expression was compared in these engineered mice versus a second engineered mouse lung cancer model that was not driven by KRAS, but by aberrant cyclin E expression. USP18 immunohistochemical expression was substantially higher in the KRAS-driven than cyclin E-dependent lung cancers. To explore the translational relevance of this work, lung cancer tissue arrays were examined in 551 lung cancers where a clinical database was available. USP18 immunohistochemical expression was significantly (P < 0.001) higher in KRAS mutant adenocarcinomas as compared to wild-type cases. There was also a significant increase in USP18 expression in adenocarcinoma versus squamous cell carcinoma cases. Taken together, these studies broaden the role of USP18 as an antineoplastic target to combat lung cancers that harbor KRAS mutations. Citation Format: Lisa Maria Mustachio, Yun Lu, Laura J. Tafe, Angeline S. Andrew, Vincent Memoli, Jaime Rodriguez-Canales, Pamela A. Villalobos, Ignacio Wistuba, Jun Yu, Jack J. Lee, Fadzai Chinyengetere, David J. Sekula, Xi Liu, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of the ubiquitin protease USP18 represses KRAS mutant lung cancer tumorigenicity in mice by destabilizing KRAS protein. [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 1255.

An integrative cross-tumors approach identifies FOSL1 as an oncogene dependency in KRAS-driven lung cancer

The Ontario Clinical Oncology Group (OCOG) – ALMERA trial (NCT02186847) is a Canadian multicenter randomized phase II trial funded by the Canadian Institute for Health Research, which is designed to examine whether metformin has chemo-radio-sensitization activity as well as activity as a single agent in advanced localized NSCLC. A total of 94 patients will be randomized in this screening trial to standard CRT with or without treatment with 2000mg of metformin, which is delivered during CRT (2 cycles of cisplatin based chemotherapy, concurrent with chest RT of 63 Gy in 30 fractions) but continues also as a single agent for a total of 12 months. ALMERA is designed to detect a 20% improvement in lung cancer progression free survival at 12 months. This trial is now open in several centers across Canada. The primary outcome is 12 month Progression Free Survival (PFS) and secondary outcomes include 1 and 2 y overall survival (OAS), time to loco-regional progression, distant progression free survival, adverse events and collection of biospecimens for biomarker studies. These will involve investigation of circulating serum and blood cell biomarkers of metformin activity and tumor biomarkers such as tumor histology, TP53, LKB1 and K-Rasmutation status. When completed ALMERAwill provide some of the first prospective evidence on the potential of metformin to improve the outcomes of standard cytotoxic therapy in locally advanced NSCLC. Biospecimen collect will allow us to investigate serum and tumors biomarkers ofmetformin response. Comparison of the results of ALMERA with other studies, like NRG-LU001, may be able to provide evidence on the value of metformin as maintenance therapy and assist in the design of future phase III trials with metformin in the setting of locally advanced NSCLC.

Polycomb Repressive Complex 2 Is a Barrier to KRAS-Driven Inflammation and Epithelial-Mesenchymal Transition in Non-Small-Cell Lung Cancer

Polycomb repressive complexes (PRC) are frequently implicated in human cancer, acting either as oncogenes or tumor suppressors. Here, we show that PRC2 is a critical regulator of KRAS-driven non-small cell lung cancer progression. Modulation of PRC2 by either Ezh2 overexpression or Eed deletion enhances KRAS-driven adenomagenesis and inflammation, respectively. Eed-loss-driven inflammation leads to massive macrophage recruitment and marked decline in tissue function. Additional Trp53 inactivation activates a cell-autonomous epithelial-to-mesenchymal transition program leading to an invasive mucinous adenocarcinoma. A switch between methylated/acetylated chromatin underlies the tumor phenotypic evolution, prominently involving genes controlled by Hippo/Wnt signaling. Our observations in the mouse models were conserved in human cells. Importantly, PRC2 inactivation results in context-dependent phenotypic alterations, with implications for its therapeutic application.

Abstract B22: Acyl-CoA synthetase long-chain family member 3 dependent lipid homeostasis is required for mutant KRAS driven lung cancer

Abstract Lung cancer is the leading cause of cancer related deaths in the USA and worldwide. Lung tumorigenesis is a multistep process that involves several genetic aberrations. Activating mutations of the proto-oncogene KRAS (mutant KRAS) occur in ~30% of the cases of human non-small cell lung cancer (NSCLC), which is associated with aggressive, therapy-resistant disease. Despite the recent discovery of low affinity inhibitors, mutant KRAS is a challenging therapeutic target and there is a dearth of therapeutic options for these tumors. Mutant KRAS not only promotes tumorigenesis but also the survival of established lung cancer, both in mouse models and in certain human NSCLC lines. Therefore, in the absence of clinically-relevant effective inhibitors of mutant KRAS, there has been an intense clinical interest in the development of inhibitors of its downstream effectors. Importantly, mutant KRAS cancer cells undergo oncogene-directed metabolic reprogramming in order to meet the energetic and biosynthetic challenges of cell survival, growth and proliferation. Activation of certain pathways of fatty acid synthesis has been observed in many cancer types including lung cancer. Till date, fatty acid synthase (FASN) has been the candidate for drug development. Unfortunately, the inhibitors against FASN have poor pharmacokinetics and target related toxicity concerns. There is an urgent need for discovery of additional targets that inhibit lipid metabolism specifically in cancer cells that could be exploited for therapeutic gain. The goal of our study was to identify the cellular networks that mediate the maintenance of mutant KRAS lung cancer which further could be used as high priority therapeutic targets. To this end, we functionally analyzed the transcriptome of transgenic mouse lung tumors manipulated in vivo to undergo mutant Kras extinction, providing isogenic comparisons between mutant KRAS extinguished versus non-extinguished tumors for the discovery of new therapeutic targets. We determined that mutant KRAS controls tumor metabolism by regulating lipid homeostasis. We found that Acyl-CoA synthetase long-chain family member 3 (ACSL3), which converts fatty acids into fatty Acyl-CoA esters, the substrate for lipid synthesis and β-oxidation, is required for the survival of mutant KRAS lung cancer cells. These effects were not due to generalized toxicity, since we did not observe them in immortalized human bronchoalveolar cells and several NSCLC cells expressing wild type KRAS and in Acsl3 null mouse embryonic fibroblasts. We confirmed that ACSL3 is a mutant KRAS responsive gene expressed in the respiratory epithelium, in lung cancer cells and in primary human cancers. With mechanistic experiments we determined that mutant KRAS stimulates, in an ACSL3-dependent manner, the uptake and retention of fatty acids by lung cancer cells as well as their β-oxidation. As predicted by these experiments, ACSL3 is essential for the ability of mutant KRAS human lung cancer cells to form colonies in soft agar or to establish xenografts in immunocompromised mice. In addition, our preliminary results shows reduced tumor size and tumor burden in KrasG12D;Acsl3-/- mice. The detailed characterization of these mice is currently in progress. Our data demonstrate that mutant KRAS reprograms lipid homeostasis in lung cancer, establishing a cancer specific metabolic vulnerability. Thus, ACSL3 could be a viable therapeutic target for NSCLC driven by mutant KRAS. Citation Format: Mahesh S. Padanad, Georgia Konstantinidou, Chendong Yang, Margherita Melegari, Niranjan Venkateswaran, Kimberly Batten, Kenneth E. Huffman, Jerry W. Shay, John D. Minna, Ralph J. DeBerardinis, Pier P. Scaglioni. Acyl-CoA synthetase long-chain family member 3 dependent lipid homeostasis is required for mutant KRAS driven lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B22.

Abstract B124: Drugging the undruggable: development of small molecule activators of protein phosphatase 2A for cancer treatment

Abstract KRAS is the most common recurrent oncogenomic mutations driving the growth of NSCLC. Patients with KRAS mutations respond poorly to current therapies. Thus, novel therapies, are critically needed, to improve the lives of patients suffering from KRAS driven lung cancers. While oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Through reverse engineering of tricyclic neuroleptic drugs, we developed a first-in-class series of small molecule activators of PP2A activators (SMAPs) molecules, as represented that have favorable pharmaceutic properties directly bind and activate the serine/threonine phosphatase 2A (PP2A). A critical role for PP2A as a tumor suppressor has previously been established, and PP2A inactivation is common feature in human lung cancers. Furthermore, protein phosphatase 2A (PP2A) accounts for the majority of cellular serine/threonine phosphatase activity, and its dominant and best-defined targets are oncogenic protein kinases including ERK and AKT. In this study, we sought to determine both the association of PP2A inactivation in lung cancer with specific molecular genotypes and the biological and functional consequences of PP2A reactivation in lung cancer. To understand the effects of SMAPs on cell viability and survival, we used MTT and colony formation assays in lung cancer cell lines. Apoptosis was evaluated through annexin V staining and cell cycle profile analysis. Additionally, global phosphoproteomic profiling was performed. Effects of SMAPs in vivo were assessed using A549, HH41 and H358 xenograft and Kras LA2 transgenic mouse models. Treatment of lung cancer cell lines with TRC resulted in decreased cell viability, decreased colony formation, and an increase in apoptosis. Global phosphoproteomic analysis of SMAP treated cell lines revealed ERK signaling as a commonly perturbed pathway which was confirmed by western blotting. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Furthermore, combination based therapy with kinase inhibitors and our novel phosphatase activators resulting in marked synergy and tumor regressions in vivo. Importantly, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity both alone or in combination. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. While research and clinical effort has largely focused on development of inhibitors of oncogenic kinases, the identification of small molecule activators of tumor suppressor proteins has remained elusive. Activation of such proteins could offer the opportunity to identify novel synergistic strategies for the treatment of a number of cancer types. Nevertheless, translation of a PP2A activation strategy into clinical medicine has required pharmaceutically tractable agents for development. Our studies represent a first step into that new territory and highlight the potential for the development of small molecule activators of other protein phosphatases and tumor suppressor proteins Citation Format: Jaya Sangodkar, Daniel McQuaid, Janna Kisselar, David Brautigan, Mark Chance, Michael Ohlmeyer, David Kastrinsky, Yiannis Ioannou, Goutham Narla. Drugging the undruggable: development of small molecule activators of protein phosphatase 2A for cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B124.

Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis.