Preclinical Models Of Non-small Cell Lung Cancer Research Articles

Jagged2 targeting in lung cancer activates anti-tumor immunity via Notch-induced functional reprogramming of tumor-associated macrophages

Signaling through Notch receptors intrinsically regulates tumor cell development and growth. Here, we studied the role of the Notch ligand Jagged2 on immune evasion in non-small cell lung cancer (NSCLC). Higher expression of JAG2 in NSCLC negatively correlated with survival. In NSCLC pre-clinical models, deletion of Jag2, but not Jag1, in cancer cells attenuated tumor growth and activated protective anti-tumor Tcell responses. Jag2-/- lung tumors exhibited higher frequencies of macrophages that expressed immunostimulatory mediators and triggered Tcell-dependent anti-tumor immunity. Mechanistically, Jag2 ablation promoted Nr4a-mediated induction of Notch ligands DLL1/4 on cancer cells. DLL1/4-initiated Notch1/2 signaling in macrophages induced the expression of transcription factor IRF4 and macrophage immunostimulatory functionality. IRF4 expression was required for the anti-tumor effects of Jag2 deletion in lung tumors. Antibody targeting of Jagged2 inhibited tumor growth and activated IRF4-driven macrophage-mediated anti-tumor immunity. Thus, Jagged2 orchestrates immunosuppressive systems in NSCLC that can be overcome to incite macrophage-mediated anti-tumor immunity.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

Abstract C104: Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC

Abstract Aberrant expression of the transcription factor MYC is found in most cancers including non-small cell lung cancer (NSCLC) and has critical roles in a wide range of oncogenic processes including immune evasion and EGFR inhibitor resistance. Despite MYC’s therapeutic attractiveness, potent inhibitors of MYC activity have been elusive, due to its intrinsically disordered protein structure and tightly autoregulated expression. We have developed NSCLC-specific programmable epigenomic mRNA therapy, termed a MYC Omega epigenomic controller (NSCLC MYC-OEC) that is designed to target the insulated genomic domain of MYC. We have previously shown that NSCLC MYC-OEC effectively downregulates MYC expression pre-transcriptionally and have demonstrated that NSCLC MYC-OEC inhibits NSCLC patient-derived organoid viability in vitro and abrogates xenograft tumor growth in vivo. To identify combination treatments with NSCLC MYC-OEC, we retrospectively evaluated baseline MYC expression and real-world progression-free survival (PFS) in a cohort of 283 NSCLC patients treated with pembrolizumab, an anti-PD1 monoclonal antibody. This analysis revealed that NSCLC patients with MYC-overexpressing tumors exhibited shorter PFS when treated with pembrolizumab. Moreover, analysis of PD1 or PD-L1 transcript levels in tumors from 13,230 lung adenocarcinoma patients revealed a significant positive correlation between PD1 or PD-L1 and MYC mRNA levels, consistent with a role for MYC in regulation of the immune response. In vivo evaluation of NSCLC MYC-OEC activity and immune checkpoint immunotherapy (ICI) treatment of preclinical Lewis lung carcinoma syngeneic tumor models demonstrated that the combination of NSCLC MYC-OEC with anti-PD-L1 antibody enhanced inhibition of tumor growth and prolonged survival over administration when compared to either NSCLC MYC-OEC or anti-PD-L1 antibody monotherapy. Retrospective analysis of a cohort of 301 NSCLC patients also showed that patients with MYC-overexpressing tumors exhibited significantly shorter PFS when treated with osimertinib, a third generation EGFR inhibitor. To further understand this, we investigated the effects of NSCLC MYC-OEC and osimertinib treatment alone or in combination in EGFR mutant cells, H1975 and PC9. While NSCLC MYC-OEC and osimertinib monotherapy treatments resulted in downregulation of MYC protein expression in both cell lines, combination treatment led to a significantly greater decrease in MYC expression. Furthermore, the combination synergistically reduced H1975 and PC9 cell viability in vitro and led to a significantly greater inhibition of H1975 xenograft growth relative to each single agent in vivo. Together, these data provide preclinical proof-of-concept in NSCLC for development of OEC-mediated pre-transcriptional epigenomic inhibition of MYC expression in combination with immune checkpoint and EGFR inhibitors. Furthermore, these findings suggest that NSCLC MYC-OEC combination with ICI or EGFR-targeted therapy may improve patient outcomes. Citation Format: Defne Yarar, Eugine Lee, Padraich Flahardy, Cameron Vergato, Graeme Hodgson, Thomas McCauley. Targeted epigenomic inhibition of MYC enhances responses to immune checkpoint and EGFR inhibitors in preclinical models of NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C104.

Abstract B124: MYTX-011 is a highly internalized ADC with anti-tumor activity across a spectrum of NSCLC preclinical models with various levels of cMET expression

Abstract cMET overexpression occurs in non-small cell lung cancer (NSCLC) and antibody-drug conjugates targeting cMET (cMET-ADCs) have been developed to treat cMET+ NSCLC tumors irrespective of dependency on cMET signaling. cMET-ADCs have shown promising clinical activity, but largely in a subset of NSCLC patients whose tumors express the highest cMET levels. MYTX-011, an investigational, pH-sensitive ADC incorporating the clinically validated vcMMAE linker-payload, was specifically designed to potentially benefit a broader population of patients including those whose tumors express lower cMET levels. We’ve previously demonstrated that pH-sensitive cMET binding of MYTX-011 drives higher internalization in cMET+ tumor cells, and greater cytotoxicity against a panel of cMET+ cancer cell lines in vitro. MYTX-011 also shows superior efficacy in NSCLC xenograft models with high or moderate cMET expression levels when compared to the matched non-pH-sensitive Parent ADC or a Benchmark ADC generated by hinge conjugation of a clinical stage anti-cMET ADC antibody to vcMMAE. Here we demonstrate that the MYTX-011 monoclonal antibody, which is engineered to selectively and rapidly lose affinity at the acidic pH of endolysosomes, rapidly internalizes and accumulates in cells expressing moderate cMET levels compared to the matched parent antibody. MYTX-011 in vitro cytotoxicity correlated with cMET expression levels and conferred sensitivity to cells that were otherwise insensitive to ADCs generated with the parent or Benchmark antibodies. Importantly, cells that were only sensitive to MYTX-011, but not matched controls, expressed low and moderate levels of cMET. Furthermore, MYTX-011 was active against the low cMET-expressing NCI-H2122 xenograft model in vivo whereas the Benchmark ADC was inactive. In the moderate cMET-expressing NCI-H1975 xenograft model, MYTX-011 was more active than both the Benchmark ADC and a second benchmark ADC generated by hinge conjugation of a clinical stage biparatopic anti-cMET ADC antibody to a maytansinoid linker/toxin. MYTX-011 activity was further evaluated against a panel of NSCLC patient derived xenograft (PDX) models with relevant histology, cMET expression, and genetic profiles. MYTX-011 was highly active in cMET+ squamous and adenocarcinoma PDX, EGFR WT and mutant PDX, in a PDX model with MET ex14 skipping mutation, and in PDX with heterogenous cMET expression. Together, these findings highlight the potential of MYTX-011 as a therapeutic candidate for treating a broader range of cMET+ NSCLC malignancies than other cMET-ADCs. Citation Format: William C Comb, Deepak Kanojia, Federico Colombo, Vijay Ramesh, Nimish Gera, Thomas Chittenden, Brian Fiske. MYTX-011 is a highly internalized ADC with anti-tumor activity across a spectrum of NSCLC preclinical models with various levels of cMET expression [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B124.

Small-molecule MX-C2/3 suppresses non-small cell lung cancer progression via p53 activation

p53 inactivation is a common feature in non-small cell lung cancer (NSCLC) resulting in NSCLC malignant transformation. Targeting serine 392 phosphorylation to restore p53 anticancer activity has proven to be an effective therapeutic strategy against NSCLC. A synthetic p53 activator, NA-17, has been developed that shows promise in preclinical models of NSCLC. However, NA-17 exhibits limited therapeutic efficacy in oncogene-driven tumors as well as relatively high toxicity to normal cells. It is possible that high efficiency and low toxicity p53 activators can be obtained by optimizing the leading molecule. Here, we performed high-throughput screening of compounds optimized based on NA-17 to identify new p53 activators. Two promising candidates named MX-C2 and MX-C3 were identified, both exhibited considerable therapeutic efficacy in oncogene-driven tumor models. Similar to NA-17, MX-C2/3 induced p53 activation via phosphorylating serine-392 without DNA damage. Both compounds showed broad antitumor activity in NSCLC cells and limited toxicity in normal cell lines. Moreover, MX-C2/3 suppressed tumor progression by arresting the cell cycle at G2/M phase, exhibiting a different mechanism of cell cycle arrest than NA-17. In addition, MX-C2/3 promoted the enrichment of p-p53 (s392) in mitochondria, leading to the conformational activation of Bak for cell apoptosis, which is consistent with NA-17. Finally, we demonstrated that MX-C2 significantly inhibited tumor growth without obvious systemic toxicity in oncogene-driven HCC-827 xenograft models. Collectively, we report two p53 activators with high-efficiency and low-toxicity that target p53 serine 392 phosphorylation for anticancer translational investigation.

Abstract 6346: Identification of ERK inhibitor-based combinations targeting LKB1-mutated NSCLC

Abstract Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. It is a very heterogeneous disease where some frequent mutations remain untargetable. Among them there are those affecting STK11/LKB1, the third most commonly mutated gene in NSCLC adenocarcinomas. Patients harboring LKB1-mutated tumors often have a poor prognosis due to the aggressiveness of this type of cancer and the lack of specific and efficacious therapies. Previous studies in our laboratory demonstrated a peculiar in vitro and in vivo activity of ERK inhibitors (ERKi) on LKB1-mutated NSCLC preclinical models. However, due to the huge heterogeneity of this type of tumor, it is likely that ERKi alone will have limited clinical application. In this scenario, the aim of this project is to find ERKi synthetic lethal partners in order to enlarge NSCLC patient population amenable to this therapy. Methods: We performed high-throughput screenings of a FDA-approved drug library on two different cell line systems (LU99 and H358) each composed by the LKB1 wild-type (wt) parental cell line and the CRISPR-CAS9-derived LKB1 deleted clone. In both the screenings, we treated the cell lines with the FDA-approved library alone or in combination with a subtoxic concentration of the ERKi. Results: According to our aim, for each screening, we analyzed the results selecting as hits those combinations preferentially active on the LKB1-deleted clones compared to their LKB1-wt cell lines. We verified that the FDA-approved compounds were subtoxic when used as single treatment in both the parental cell lines and their clones. We highlighted 28 and 42 hit combinations for LU99 and H358 isogenic systems, respectively. Some hits were common between the two screenings and the most represented FDA-approved drug’s classes of the hit combinations were PI3K/Akt/mTOR inhibitors, tyrosine kinase receptor inhibitors, MAPK inhibitors and compounds involved in anti-inflammatory pathways neuronal signaling, metabolism and DNA damage. Among the hits, we have cross-validated 4 ERKi-based combinations (a tyrosin kinase inhibitor, a MAPK inhibitor, a serine-threonine selective protein inhibitor and a farnesyl transferase inhibitor) in four different isogenic systems and we are now performing further validation on a panel of NSCLC cell lines with different LKB1-status as well as on NSCLC organoids. Conclusions: From the FDA-approved drug library screenings, different ERKi-based combinations showed promising results worthy to be deeply studied. Further analyses are ongoing to verify their efficacy on more complex models as NSCLC 3D organoids and to clarify the mechanism of action at the basis of the hit combinations. Positive results could give the chance to develop effective and specific therapies for patient with LKB1-mutated NSCLCs. Citation Format: Marika Colombo, Matteo Demetrio Tripodi, Gabriel Caso, Elisa Perin, Mirko Marabese, Massimo Broggini, Elisa Caiola. Identification of ERK inhibitor-based combinations targeting LKB1-mutated NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6346.

Abstract P084: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models

Abstract Background: Activating mutations in EGFR have been reported in ~30% of patients with non-small cell lung cancer (NSCLC). Three generations of small molecule EGFR kinase inhibitors have been approved by the FDA to treat these patients, however, multiple mechanisms of resistance cause cancer progression. In addition to drug-resistant mutations that arise and re-activate EGFR, other signaling pathways can be activated to cause resistance. Although EGFR inhibitors such as osimertinib and afatinib (also a pan-ErbB inhibitor) have demonstrated clear clinical benefits, patients inevitably develop resistance. Herein we demonstrate mutant EGFR NSCLCs activate autophagy upon treatment with EGFR inhibitors as a drug resistance mechanism. Therefore, a combination of an EGFR inhibitor with an autophagy inhibitor has the potential to deepen and prolong responses and improve patient outcomes. Materials and Methods: Human cancer cells with EGFR mutations were cultured using recommended complete medium. Inhibition of ULK1/2 was measured through standard biochemical assays and cellular readouts including NanoBRET and ELISA-based ATG13 phosphorylation assays. Autophagosome formation was measured using the CytoID assay (Enzo Life Sciences). For in vivo studies, NCI-H1975 cells that harbor an EGFR T790M resistance mutation were inoculated into BALB/c nude mice. Statistical analyses for the in vivo studies were performed using the Student’s t-test. Results: Erlotinib, gefitinib, osimertinib, and afatinib activated autophagy 3–4-fold over basal levels in the HCC827 cell line (EGFR exon 19 deletion) as measured by increases in phosphorylated ATG13, a cellular substrate of the autophagy-initiating kinases ULK1/2. DCC-3116, an investigational potent and selective dual inhibitor of ULK1 (IC50 6 nM) and ULK2 (9 nM) in cellular assays, inhibited both EGFR-induced and basal phosphorylation of ATG13 with IC50 values of 61–66 nM. Treatment of the NCI-H1975 EGFR mutated (L858R/T790M) NSCLC cell line with osimertinib or afatinib induced autophagy 3-fold over basal levels. DCC-3116 potently inhibited osimertinib and afatinib induced phosphorylation of ATG13 with IC50 values of 91 nM and 71 nM, respectively, and inhibited the increase in autophagosomes induced by these agents. Importantly, these in vitro effects translated to in vivo efficacy. The combination of DCC-3116 with osimertinib or afatinib resulted in significantly greater tumor responses than single agent treatments in the NCI-H1975 NSCLC xenograft model (combination vs. osimertinib p = 0.0005; combination vs. afatinib p = 0.0001; osimertinib combination vs vehicle p < 0.0001; afatinib combination vs. vehicle p < 0.0001). These data provide a strong rationale to study the combination of the ULK inhibitor DCC-3116 with EGFR inhibitors such as osimertinib and afatinib in cancer patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with a documented RAS or RAF mutation (NCT04892017). Citation Format: Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Yu Zhan, Bryan D. Smith, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with EGFR inhibitors osimertinib and afatinib in NSCLC preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P084.

Canagliflozin Mediates Effective Blockade of the mTORC1 - HIF-1a Pathway, Inhibits Growth and Enhances Non-Small Cell Lung Cancer (NSCLC) Response to Radiotherapy (RT)

Alterations in NSCLC cell metabolism contribute to rapid tumor growth, resistance to RT and poor prognosis. The mammalian Target of Rapamycin Complex 1 (mTORC1) - Hypoxia-Inducible Factor-1α (HIF-1α) pathway mediates expression of the Warburg glycolytic phenotype in NSCLC and induces RT resistance. Earlier, we showed that the new diabetes agent Canagliflozin (CANA), developed to control hyperglycemia through inhibition of the renal Na+-glucose co-transporter 2 (SGLT2), also inhibits mitochondrial complex I respiration and blocks mTORC1 signaling. In this study, we analyzed CANA’s transcriptional and post-translational effects and its regulation on RT response in preclinical models of NSCLC. Cell proliferation and clonogenic survival assays were used to assess the efficacy of CANA (0–30 μM) and RT (0–8 Gy) in adenocarcinoma (A549, H1299, H1975) and squamous cell carcinoma (SK-MES-1) NSCLC cells. Protein immunoblotting, RT-qPCR and RNAseq of A549 NSCLC cells were performed to study regulation of pathway activation and gene expression in response to CANA and RT. In-vivo effects of the two agents were examined with ectopic A549 xenografts grown in immunodeficient nude mice treated with CANA (100 mg/kg/day in chow diet) and/or RT (5 Gy). RNAseq showed that clinically achievable CANA doses (10 μM) mediate profound regulation of NSCLC cell transcriptional activity with over 100 upregulated and 80 downregulated genes. CANA significantly downregulates HIF-1α pathway-related genes that mediate HIF-1α stability and nuclear transfer, glycolysis and lipogenesis, tyrosine kinase receptor signaling and inhibition of apoptosis. CANA treatment led to activation of AMP-activated protein kinase (AMPK) and blockade of the mammalian target of rapamycin complex 1 (mTORC1) pathway. It abolished phosphorylation of the mitosis marker, histone H3. This was consistent with long-term induction of the G1/S cyclin-dependent kinase inhibitor p27kip1. Clinically relevant doses of CANA suppressed proliferation and clonogenic survival in all NSCLC cell lines but this was enhanced in the K-Ras mutant/LKB1null A549 cells. Combination index analysis indicated additivity and sensitization interaction between the CANA and RT anti-proliferative effects. In the A549 xenograft model, CANA enhanced the cytotoxic effects of RT in an additive manner. CANA mediates effective suppression of the mTORC1-HIF-1α pathway, regulates gene expression and mediates potent anti-tumor activity in NSCLC models in-vitro and in-vivo. Given that CANA is a widely-used, well-tolerated and economical agent, this work indicates strong potential for this agent to be investigated rapidly in NSCLC clinical trials in combination with RT.

Palbociclib treatment alters nucleotide biosynthesis and glutamine dependency in A549 cells

BackgroundAberrant activity of cell cycle proteins is one of the key somatic events in non-small cell lung cancer (NSCLC) pathogenesis. In most NSCLC cases, the retinoblastoma protein tumor suppressor (RB) becomes inactivated via constitutive phosphorylation by cyclin dependent kinase (CDK) 4/6, leading to uncontrolled cell proliferation. Palbociclib, a small molecule inhibitor of CDK4/6, has shown anti-tumor activity in vitro and in vivo, with recent studies demonstrating a functional role for palbociclib in reprogramming cellular metabolism. While palbociclib has shown efficacy in preclinical models of NSCLC, the metabolic consequences of CDK4/6 inhibition in this context are largely unknown.MethodsIn our study, we used a combination of stable isotope resolved metabolomics using [U-13C]-glucose and multiple in vitro metabolic assays, to interrogate the metabolic perturbations induced by palbociclib in A549 lung adenocarcinoma cells. Specifically, we assessed changes in glycolytic activity, the pentose phosphate pathway (PPP), and glutamine utilization. We performed these studies following palbociclib treatment with simultaneous silencing of RB1 to define the pRB-dependent changes in metabolism.ResultsOur studies revealed palbociclib does not affect glycolytic activity in A549 cells but decreases glucose metabolism through the PPP. This is in part via reducing activity of glucose 6-phosphate dehydrogenase, the rate limiting enzyme in the PPP. Additionally, palbociclib enhances glutaminolysis to maintain mitochondrial respiration and sensitizes A549 cells to the glutaminase inhibitor, CB-839. Notably, the effects of palbociclib on both the PPP and glutamine utilization occur in an RB-dependent manner.ConclusionsTogether, our data define the metabolic impact of palbociclib treatment in A549 cells and may support the targeting CDK4/6 inhibition in combination with glutaminase inhibitors in NSCLC patients with RB-proficient tumors.

Blood Outgrowth Endothelial Cells as a Cellular Carrier for Oncolytic Vesicular Stomatitis Virus Expressing Interferon-β in Preclinical Models of Non-Small Cell Lung Cancer

Oncolytic viruses have demonstrated efficacy in numerous tumor models including non-small cell lung cancer (NSCLC). One limitation of viral therapy for metastatic lung cancer is that systemic administration can be hindered by complement and antiviral immunity. Thus, we investigated whether ex vivo-infected blood outgrowth endothelial cells (BOECs) could be used to deliver VSV-IFNβ in preclinical models of NSCLC. BOECs were obtained from human donors or C57/Bl6 mice. VSV was engineered to produce GFP or IFNβ. Human and murine BOECs could be infected by VSV-GFP and VSV-IFNβ. Infected BOECs resulted in killing of NSCLC cells in vitro and shielded VSV-IFNβ from antibody neutralization. Mouse BOECs localized to lungs of mice bearing syngeneic LM2 lung tumors, and infected murine BOECs reduced tumor burden in this model. In an immune-deficient A549 xenograft model, mice treated with VSV-IFNβ-infected human BOECs exhibited superior antitumor activity and survival of mice (n = 10, P < .05 compared to VSV-IFNβ alone). We conclude that BOECs can be used as a carrier for delivery of oncolytic VSV-IFNβ. This may be an effective strategy for clinical translation of oncolytic virotherapy for patients with metastatic NSCLC.

B31 Development of Multicell-Type Organoid Cultures for Preclinical Studies of Immunotherapeutics for Lung Cancer

Macrophages are key regulators of the immune landscape within the tumor microenvironment (TME). The plasticity of macrophage phenotypes in the TME has previously been correlated with prognosis within non-small cell lung cancer (NSCLC). Depending on their phenotype, macrophages in the TME can secrete protumor cytokine and chemokines, ultimately suppressing the function of other immune cells in the TME. The purpose of our study was to explore the ability of individual NSCLC preclinical models to alter macrophage phenotype in organoid cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. We hypothesize that immune suppression occurs through tumor-secreted signaling molecules, and if blocked, macrophage suppression can be alleviated, resulting in a better antitumor immune response. We developed an in vitro organoid coculture system (NSCLC tumor cells, human cancer-associated fibroblasts, CAFs, and mouse macrophages) to interrogate cancer cell features causing heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured (with 4-7 replicates for each NSCLC): mRNA expression in mouse macrophages with a panel of qPCR probes for important macrophage-related genes (Arg, NOS2, IL1beta, IL-6, CHIL-3, SOCS3), and in selected cases whole-genome RNAseq; and protein expression using cytokine arrays measuring expression of 40 inflammatory cytokines. Positive controls were stimulation with LPS and IL-4. Using our platform, we characterized 70 NSCLC patient-derived lines by their ability to alter mouse macrophage phenotype. We found: 1. the macrophage phenotypes induced by any one NSCLC were highly reproducible; 2. three major clusters of cancer polarized macrophage phenotypes: high Arg (immune suppressive), high IL-1beta (inflammatory) or high SOCS3 (cGAS-STING pathway) expression; and 3. the major oncogenotypes (KRAS, TP53, STK11, EGFR, BRAF) have no correlation to the induced macrophage phenotype. We selected 7 NSCLC “exemplar” lines representing each of these 3 clusters for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters we found: 1. suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling (M2 immune suppressive phenotype); and 2. increased expression of human IL6, IL8, and MCP1 proteins, which have been implicated in suppressing innate immune tumor sensing. Analyses of differences between the 3 clusters is ongoing. Patient-derived NSCLC preclinical models have reproducible effects on altering macrophage phenotypes in organoid cultures. Three major classes of NSCLC initiated macrophage alteration, which are not linked to oncogenotype. Cytokines secreted by the NSCLCs appear responsible for these macrophage changes, and this system provides an experimental mechanism to systematically test each as potential therapeutic targets.

Abstract LB-083: Combination of an ERK1/2 inhibitor (LY3214996) with pan-RAF inhibitor enhances anti-tumor activity in KRAS mutant colorectal cancer (CRC) and non-small cell lung cancer (NSCLC)

Abstract Cancer patients with KRAS mutations have poor prognosis and represent an unmet medical need. Mutant KRAS driven MAPK activation is present in ~ 40% and ~ 30% of CRC and NSCLC patients, respectively. ERK1/2, a key downstream effector of KRAS mutations, is involved in the critical signaling network to drives cell proliferation, survival, metastasis and drug resistance. LY3214996, an ERK1/2 inhibitor which is in Phase I clinical trial (NCT02857270), has potent anti-tumor activities in KRAS mutant tumor cells in vitro and in vivo. However, some KRAS mutant cancer cells were less sensitive to LY3214996 single agent, suggesting that combination therapy is needed to maximize the benefit from ERK1/2 inhibition. The combined inhibition of RAS/RAF/MEK/ERK pathway components (such as the inhibition of both BRAF and MEK) has shown the promising anti-cancer activity in melanoma. Based on interaction between activated RAS with RAF which is responsible for cancer progression and resistance in KRAS mutant cancers, we hypothesized that inhibiting both ERK and RAF may augment efficacy of the single agents and delay the resistance. Therefore in this study we have investigated the combination effect of LY3214996 with a pan-RAF inhibitor LY3009120 in KRAS mutant CRC and NSCLC in vitro and in vivo. The combination of LY3214996 and LY3009120 showed synergistic or additive inhibition of cell proliferation in all KRAS mutant CRC (n=8) and NSCLC (n=8) cell lines tested. The LY3214996 and LY3009120 combination significantly decreased the phosphorylation levels of ERK, Rb, S6 and H3, and induced more complete cell death in NCI-H2122 (G-12C), A549 (G-12S), NCI-H441 (G-12V), HCT-116 (G-13D) and in LY3009120-resistant HCT-116 cells compared with either single agent. Subsequent combination efficacy was evaluated in the HCT-116 xenograft model. LY3214996 alone, LY3009120 alone and the combination of LY3214996 and LY3009120 resulted in 52%, 68% and 94% tumor growth inhibition, respectively; and the combination significantly augmented the efficacy (p &amp;lt;0.001). Furthermore the combination efficacy of LY3214996 with a different pan-RAF inhibitor (LSN3074753, a surrogate and an analogue of LY3009120) was also assessed in the H441 NSCLC orthotopic model, and the combination increased the inhibition of both primary tumor growth and spontaneous metastasis (including lymph node metastasis and chest wall metastasis) when compared with the respective single agent activity. Overall, the combined inhibition of ERK1/2 and RAF was tolerated and enhanced antitumor efficacy in KRAS mutant CRC and NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with a pan-RAF inhibitor as a promising strategy for the treatment of KRAS mutant CRC and NSCLC. Citation Format: Wenjuan Wu, Shripad Bhagwat, Lisa Kindler, William McMillen, Sajan Joseph, Sean Buchanan, Christoph Reinhard, Ramon V. Tiu, Sheng-Bin Peng. Combination of an ERK1/2 inhibitor (LY3214996) with pan-RAF inhibitor enhances anti-tumor activity in KRAS mutant colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-083.

Third generation EGFR inhibitor osimertinib combined with pemetrexed or cisplatin exerts long-lasting anti-tumor effect in EGFR-mutated pre-clinical models of NSCLC

BackgroundThe third generation Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor (TKI) osimertinib has been initially approved for T790M positive Non-Small Cell Lung Cancer (NSCLC) and more recently for first-line treatment of EGFR-mutant T790M negative NSCLC patients. Similarly to previous generation TKIs, despite the high response rate, disease progression eventually occurs and current clinical research is focused on novel strategies to delay the emergence of osimertinib resistance. In this study we investigated the combination of osimertinib with pemetrexed or cisplatin in EGFR-mutated NSCLC cell lines and xenografts.MethodsTumor growth was evaluated in a PC9T790M xenograft model and tissue composition was morphometrically determined. PC9, PC9T790M and HCC827 cell lines were employed to test the efficacy of osimertinib and chemotherapy combination in vitro. Cell viability and cell death were evaluated by MTT assay and fluorescence microscopy. Protein expression and gene status were analysed by Western blotting, fluorescence in situ hybridization analysis, next-generation sequencing and digital droplet PCR.ResultsIn xenograft models, osimertinib significantly inhibited tumor growth, however, as expected, in 50% of mice drug-resistance developed. A combination of osimertinib with pemetrexed or cisplatin prevented or at least delayed the onset of resistance. Interestingly, such combinations increased the fraction of fibrotic tissue and exerted a long-lasting activity after stopping therapy. In vitro studies demonstrated the stronger efficacy of the combination over the single treatments in inhibiting cell proliferation and inducing cell death in PC9T790M cells as well as in T790M negative PC9 and HCC827 cell lines, suggesting the potential role of this strategy also as first-line treatment. Finally, we demonstrated that osimertinib resistant clones, either derived from resistant tumors or generated in vitro, were less sensitive to pemetrexed prompting to use a chemotherapy regimen non-containing pemetrexed in patients after progression to osimertinib treatment.ConclusionsOur results identify a combination between osimertinib and pemetrexed or cisplatin potentially useful in the treatment of EGFR-mutated NSCLC patients, which might delay the appearance of osimertinib resistance with long-lasting effects.

Initial reporting of NRG-LU001 (NCT02186847), randomized phase II trial of concurrent chemoradiotherapy (CRT) +/- metformin in locally advanced Non-Small Cell Lung Cancer (NSCLC).

8502 Background: Metformin, a diabetes agent that inhibits mitochondria complex I, enhances radiotherapy and chemotherapy responses in pre-clinical models of NSCLC. NRG-LU001 examined whether metformin can improve outcomes of curative CRT in locally advanced (LA)-NSCLC. Methods: The primary endpoint of this trial was 1-year progression free survival (PFS). Unresected, non-diabetic, stage IIIA/B NSCLC patients were randomized (1:1) to either carboplatin-paclitaxel chemotherapy concurrent with chest RT (60Gy), followed by consolidation carboplatin-paclitaxel chemotherapy (Control Arm) or the same and oral metformin (2000mg daily) during cytotoxic therapy (Experimental Arm). PFS and overall survival (OS) were estimated with the Kaplan-Meier method; time to local-regional progression (TTLRP), time to distant metastasis (TTDM) were estimated using the cumulative incidence method. Adverse events (AEs) were graded with CTCAE v.4.0. Results: Between Aug.2014 and Dec.2016, 170 patients were accrued. Analysis was planned at 102 PFS events (Feb. 2019). There was no significant difference in rates or grade of toxicity between the two arms. 1- and 2-year PFS was 60.4% (95% CI: 48.5, 70.4) and 40.1% (95% CI: 29.0, 51.0) in Control vs 51.3% (95% CI: 39.8, 61.7) and 34.5% (95% CI: 24.2, 45.1) in the Metformin arm (multivariable Cox proportional HR=1.20 (95% CI: 0.81, 1.78), p=0.36). OS at 2 years was 65.4% (95% CI: 53.5, 75.0) for Control vs 64.9% (95% CI: 53.1, 74.5) for the Metformin arm (HR=1.03 (95% CI: 0.64, 1.68)), while deaths due to disease were 90% vs 71%, respectively. No significant differences were found for TTLRP or TTDM. Conclusions: NRG-LU001 center reported outcomes show that oral daily metformin was well-tolerated in combination with CRT treatment for LA-NSCLC. However, metformin did not improve PFS and OS and did not alter the rates of local-regional failure or distant metastasis. Acknowledgements: TT and HS are Co-Principal Investigators. This project was supported by National Cancer Institute (NCI) grants: U10CA180868 (NRG Oncology Operations), U10CA180822 (NRG SDMC), UG1CA189867 (NCORP), U24CA180803 (IROC). Clinical trial information: NCT02186847.

MERTK Mediates Intrinsic and Adaptive Resistance to AXL-targeting Agents.

The TAM (TYRO3, AXL, MERTK) family receptor tyrosine kinases (RTK) play an important role in promoting growth, survival, and metastatic spread of several tumor types. AXL and MERTK are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. AXL is the most well-characterized TAM receptor and mediates resistance to both conventional and targeted cancer therapies. AXL is highly expressed in aggressive tumor types, and patients with cancer are currently being enrolled in clinical trials testing AXL inhibitors. In this study, we analyzed the effects of AXL inhibition using a small-molecule AXL inhibitor, a monoclonal antibody (mAb), and siRNA in HNSCC, TNBC, and NSCLC preclinical models. Anti-AXL-targeting strategies had limited efficacy across these different models that, our data suggest, could be attributed to upregulation of MERTK. MERTK expression was increased in cell lines and patient-derived xenografts treated with AXL inhibitors and inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibition. Dual targeting of AXL and MERTK led to a more potent blockade of downstream signaling, synergistic inhibition of tumor cell expansion in culture, and reduced tumor growth in vivo Furthermore, ectopic overexpression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These observations suggest that therapeutic strategies cotargeting both AXL and MERTK could be highly beneficial in a variety of tumor types where both receptors are expressed, leading to improved survival for patients with lethal malignancies. Mol Cancer Ther; 17(11); 2297-308. ©2018 AACR.

Abstract A140: MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents

Abstract The TAM family of receptor tyrosine kinases (RTKs) has been discovered to play a predominant role in promoting the growth, survival, and metastatic spread of several tumor types. AXL and MERTK are two TAM family RTKs that are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. The AXL receptor is the most well-characterized TAM receptor and has been found to mediate resistance to both conventional and targeted cancer therapies. Since AXL is overexpressed in aggressive tumor types, cancer patients are currently being enrolled in clinical trials testing AXL inhibitors. In the current study, we analyzed the efficacy of AXL inhibitors—both small molecule and monoclonal antibody therapy—in HNSCC, TNBC, and NSCLC preclinical models. We observed limited efficacy of anti-AXL targeting strategies across these different models, which was attributed to the upregulation of MERTK. MERTK was robustly overexpressed in cell lines and patient-derived xenografts treated with AXL inhibitors. Inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibitors, leading to a more potent blockade of downstream signaling, decreased expansion of tumor cells in culture, and reduced tumor growth in vivo. Furthermore, ectopic overexpression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These results suggest that cotargeting both AXL and MERTK may be highly beneficial in a variety of tumor types where both receptors are expressed and may therefore prolong antitumor effects and improve the survival of patients with lethal malignancies. Citation Format: Nellie K. McDaniel, Christopher T. Cummings, Toni M. Brand, Mari Iida, Justus Hulse, Hannah E. Pearson, Rachel A. Orbuch, Olivia J. Ondracek, Kurtis D. Davies, Parkash Gill, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Randall J. Kimple, Paul M. Harari, Deborah DeRyckere, Douglas K. Graham, Deric L. Wheeler. MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A140.

Chemoradiation Combined with Phosphatidylserine-Targeting Antibody Enhances Systemic Anti-tumor Immune Responses

Recent advances in cancer immunotherapy have sparked significant interest in harnessing the body’s immune system to fight cancer. However, the response rates of cancer immunotherapies, including immune checkpoint blockade, remain low. Strategies to enhance anti-tumor immune responses by targeting different steps along the immune activation cascade that can complement T cell-based immunotherapies are clearly needed. Here, we examine whether the addition of an antibody that targets extracellular phosphatidylserine (PS), a molecule that is recognized by myeloid derived cells, can enhance anti-tumor immune responses of chemoradiation for non-small cell lung cancer (NSCLC). Chemoradiation (CRT) was combined with a murine monoclonal antibody (mch1N11). PS is highly expressed in both orthotopically and ectopically implanted 393P murine NSCLC models. Radiation dose was 2 Gy/day, given for 5 days. Chemotherapy consists of carboplatin and paclitaxel at a dose of 30 mg/kg and 10 mg/kg, respectively. PS-targeting antibody (mch1N11) was given at 3mg/kg for 2 weekly doses. For ectopic tumor models, bilateral tumors were established in the legs. Radiation was directed to the R leg and tumor on the L side was shielded. Tumor growth was measured either with CT imaging or digital caliper. Tumor infiltrating immune cell profiles were analyzed using immunohistochemistry. Survival analyses were performed using Kaplan-Meier method and compared using Log-rank test. CRT + mch1N11 treatment significantly inhibited growth and improved survival in mice implanted with orthotopic 393P tumors as compared to CRT alone (median: 21 vs. 15 days, HR: 2.77, P = 0.006). Tissue analyses showed that CRT significantly increased the expression of PD-L1 within the tumor and drastically reduced the number of tumor infiltrating CD8 T cells. For the CRT + mch1N11 group, a similar up-regulation of PD-L1 expression was noted. However, the addition of mch1N11 re-populated the infiltrating CD8 T cells within the tumor. In the bilateral tumor model, the addition of mch1N11 antibody to CRT resulted in tumor regression in ∼40% of the non-irradiated tumors in the contralateral side as compared to 0% in the CRT alone or chemotherapy + mch1N11 groups. We showed that PS-targeting combined with standard CRT can significantly prolong survival in preclinical models of NSCLC and generate enhanced systemic anti-tumor immunity against lesions outside of the irradiated field. CRT up-regulated PD-L1 expression within the tumor and depleted tumor infiltrating cytotoxic CD8 T cells. The addition of mch1N11 antibody to CRT, however, was able to re-populate this critical immune effector cell population. Together, our results demonstrate that PS-targeting antibodies may be combined with CRT to enhance intrinsic tumor immunogenicity, activate systemic anti-tumor immune responses, and act as a priming strategy to sensitize the tumor to immune checkpoint inhibition with PD-1 or PD-L1 antibodies.

Abstract 317: Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC)

Abstract ERK1/2, a key downstream effector of RAS mutations, is involved in the signaling network which drives cell proliferation, survival, metastasis and cancer resistance to drug treatment (including MEK and BRAF inhibitors). Lung cancer is a leading cause of cancer death worldwide. KRAS mutation present in up to 30% of NSCLC patients is associated with a poor prognosis and represents an unmet medical need. In KRAS mutant NSCLC, enhanced ERK activation cooperates with dysregulation of the cell cycle checkpoint (e.g., cyclin D, CDK4 and CDK6 complex), and contributes to tumor progression; thus, the simultaneous inhibition of ERK and the CDK4/6 pathway is hypothesized to augment tumor growth inhibition. LY3214996, a novel and highly selective small molecule inhibitor of ERK1 and ERK2, is currently in phase I clinical trial and has been shown to inhibit cell proliferation in RAS or BRAF mutant tumor cells in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4 and CDK6-selective inhibitor is currently in phase III studies for ER positive breast cancer and KRAS mutant NSCLC. In this study we explore the potential efficacy of combined inhibition of ERK1/2 and CDK4 and CDK6 in KRAS mutant NSCLC. The combination of LY3214996 and abemaciclib synergistically inhibited cell proliferation in 85% of KRAS mutant cells in an unbiased NSCLC panel. Combination treatment with LY3214996 and abemaciclib significantly decreased levels of phospho- p90RSK, phospho-Rb, phospho-S6 and Ki67; and synergistically inhibited cell proliferation and survival in KRAS mutant NSCLC cell lines including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). Subsequent in vivo studies showed that the combination treatment with LY3214996 and abemaciclib was well tolerated and led to more robust tumor growth inhibition or regression in all KRAS mutant NSCLC xenograft models (H2122, A549 and H441) compared with either single agent treatment (p≤0.002). Furthermore, in xenograft tumors the combination of LY3214996 and abemaciclib resulted in more significant reduction of phospho-p90RSK, phospho-Rb, phospho-S6 and Ki67 in H2122 tumors compared with either single agent. Overall, the combined inhibition of ERK1/2 and CDK4 and CDK6 was tolerated and enhanced antitumor efficacy in several KRAS mutant NSCLC preclinical models. These data support the feasibility of combining ERK inhibitor LY3214996 with CDK4 and CDK6 inhibitor abemaciclib as a promising strategy for the treatment of KRAS mutant NSCLC patients, and provides the rationale for the combination study in the on-going phase I LY3214996 clinic trial (NCT02857270). Citation Format: Wenjuan Wu, Shripad V. Bhagwat, Constance King, Susan Pratt, Xueqian Gong, Julie Stewart, Bonita Jones, Robert Flack, Richard Beckman, Beverly Falcon, Jason Manro, William T. McMillen, Ramon V. Tiu, Sheng-Bin Peng, Christoph Reinhard, Sajan Joseph, Sean Buchanan. Combination of a novel ERK1/2 inhibitor (LY3214996) with CDK4 and CDK6 inhibitor (abemaciclib) enhances antitumor efficacy in KRAS mutant non-small cell lung cancer (NSCLC) [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 317. doi:10.1158/1538-7445.AM2017-317

Small Molecule Inhibitor of NRF2 Selectively Intervenes Therapeutic Resistance in KEAP1-Deficient NSCLC Tumors.

Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ∼400 000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap 'N' Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.

Abstract 4834: Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma

Abstract Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancer (NSCLC) cases, primarily through loss of the endogenous Notch inhibitor, Numb, or via gain-of-function mutations in the Notch1 receptor. Notch activity is associated with poor overall survival among NSCLC patients whose tumors are wildtype for TP53. Here, we characterized the interaction between BMS-906024, a clinically relevant gamma secretase inhibitor (GSI) that inhibits Notch activation, and front-line chemotherapy in preclinical models of NSCLC. MTS drug synergy assays consisting of treatment with BMS-906024, cisplatin or paclitaxel, or the combination of GSI and chemotherapy were performed on a panel of human NSCLC cell lines, most of which were derived from adenocarcinomas. Analysis of the drug effects with CalcuSyn yielded significantly lower CI values for the GSI BMS-906024 combined with paclitaxel than with cisplatin (average CI = 0.54 vs 0.85, respectively; P = 0.001). The synergy between BMS-906024 and paclitaxel was significantly greater in Kras-wildtype than Kras-mutant cells (average CI = 0.39 vs 0.68, respectively; P = 0.009), while there was no correlation with EGFR or TP53 status. Treatment of lung adenocarcinoma xenografts in NOD scid gamma mice confirmed enhanced antitumor activity for the combination treatment of BMS-906024 and paclitaxel by mechanisms currently under investigation. These results are a step toward identification of the optimal combination of the GSI BMS-906024 with standard chemotherapies, as well as potential biomarkers that could be used to predict patient response to Notch-targeted therapy. Citation Format: Katherine M. Morgan, Francis Lee, Erin Michaud, Bruce S. Fischer, Sharon R. Pine. Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma. [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 4834.