Mutant Lung Cancer Cell Lines Research Articles

Abstract LB266: AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer

Abstract Introduction: Studies have shown that molecular targeted therapy can mediate Gasdermin-E (GSDME) dependent pyroptosis. However, GSDME expression is suppressed in many mutated patients, including those with KRAS-G12C-mutated non-small-cell lung cancer (NSCLC). The efficacy of AMG510 against KRAS-G12C inhibition (KRAS-G12Ci) is constrained by tumor recurrence. Pyroptosis is widely involved in the occurrence and development of tumors. However, the role and molecular mechanism of AMG510 in pyroptosis have not been well identified. Here, we demonstrate that inactivation of anti-apoptotic Bcl-2 family members during KRAS-G12Ci treatment enhances pyroptosis, which then promotes the sensitivity of AMG510 in NSCLC. Methods: We assessed pyroptosis using live cells imaging, LDH release assays, ELISA, Western blotting, and flow cytometry. In vitro assays were used to investigate the function of GSDME. Co-culture between peripheral blood mononuclear cells (PBMCs) and KRAS-G12C mutant lung cancer cell lines explored the anti-tumor immunity of cytotoxic T lymphocytes affected by knockdown of GSDME. The molecular mechanism was determined using bulk RNA sequencing, molecular cloning technology, and Western blotting. Results: We demonstrated that KRAS G12Ci treatment promotes cleavage of GSDME, release of high mobility group protein B1 (HMGB1), and LDH, markers of pyroptotic cell death. Additionally, GSDME was significantly downregulated in KRAS-mutated NSCLC compared with patients of KRAS wild type in TCGA cohort, while significantly upregulated after AMG510 treatment and downregulated after AMG510 resistance in vitro assay. We identified that KRAS-G12Ci resulted in the inactivation of BCL-xL, leading to caspase-3-dependent cleavage of gasdermin E. Conversely, amplification of BCL-xL downregulated the expression of GSDME, therefore suppressing the process of pyroptosis. GSDME knockdown in KRAS-G12C mutant lung cancer cell lines showed defective HMGB1 release, reduced caspase-3 viability, and decreased granzyme B and IFN-γ secretion by CD8+ T cells in response to KRAS-G12Ci, and more frequent tumor cell growth. Conclusion: Our findings indicate that GSDME-dependent pyroptosis is a critical determinant of KRAS-G12C-targeted inhibitor AMG510, and through the modulation exerted by the Bcl-2 family protein BCL-xL upon GSDME. These findings suggest that activation of GSDME may provide an effective strategy to sensitize cancer cells to KRAS-G12Ci treatment. Citation Format: Linfeng Luo, Anlin Li, Wei Du, Maojian Chen, JIng Li, Yixin Zhou, Li Zhang, Shaodong Hong. AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB266.

Abstract 4663: Establishing the role of PRMT5 in lung cancer tumorigenicity and modulation of expression in smokers

Abstract Studies show that PRMT5 (protein arginine methyl transferase 5) is responsible for increased tumorigenicity by activation of the PI3K/Akt signaling pathway promoting increased cancer cell proliferation. However, the role of PRMT5 in modulating EGFR TKI resistance is minimally studied. We hypothesize that over-expression of PRMT5 could promote tumorigenesis and TKI resistance by activating PI3K-Akt-mTOR signaling pathways. Hence, modulating this key biomarker may help reduce tumorigenicity and overcome TKI resistance. We performed qPCR to study the expression of PRMT5 in normal lung cells and NSCLC cell lines. We also compared the expression of these biomarkers in erlotinib resistant (ER) and Osimertinib resistant (OR) cells using Western blotting. PRMT5 was inhibited using GSK591 (PRMT5i) to evaluate its sensitivity to EGFR TKIs (ER and OR). PRMT5 expression in normal lung cells and NSCLC cells treated with Cigarette smoke extract (CSE) were also studied and correlated with immunostaining results from smoker and non-smoker lung tumor tissue samples. Changes in PRMT5 expression were analyzed in WT-EGFR lung cancer cell lines (H358, H2170), mutant EGFR lung cancer cell lines (H3255, H1975, PC9) and normal lung fibroblast cells (HLF). PRMT5 expression was upregulated by 1.6-2.1 fold in HLF, H2170 and H358 cells after 1 week of treatment with CSE. CSE treatment also upregulated expression of inflammatory cytokines (IL-1β, IL-8 and TNF-α) in H358 cells by 1.6-7 fold as analyzed by qPCR. PRMT5 expression was upregulated in EGFR-TKI resistant cell lines, by 1.5-1.9 fold in PC9ER, PC9OR and H2170ER cells, compared to parental cells. Similar results were found using qPCR, with 1.6-2.3 fold increase in PRMT5 mRNA expression in H2170OR, PC9ER/OR and H3255OR cells compared to parental cells. An MTT assay was done to assess percentage viability of cells after treatment of PRMT5i. While 2μM PRMT5i treatment of PC9OR cells decreased cell viability by 42%, an inhibitory effect that was more than additive was seen with a combination of 2μM of PRMT5i and 21nM of OR. Similar results were also observed in H2170ER cells. Migratory properties of OR resistant cells, were studied by wound healing assays. Percentage wound area in PRMT5i treated PC9OR cells was 29.3% as compared to 6.1% and 10.5% in diluent and OR treated cells respectively after 48 hrs. Combination treatment of PRMT5i and OR further inhibited cell migration with higher wound area of 41.7%. Immunostaining of lung tumor sections from smokers and non-smokers showed high expression of PRMT5 localized in the cytoplasm and nucleus. Microscopic analysis of 20 tumor sections from smokers showed higher expression of PRMT5 (p<0.05) compared to non-smokers. We can conclude that PRMT5 is upregulated in TKI resistant cells causing tumorigenesis. Modulation of PRMT5 can prove to be groundbreaking and revolutionary, as it may increase the efficacy of EGFR TKIs. Citation Format: Syed N. Ali, Meet Patel, Bamby Dieng, Cody Lund, Neelu Puri. Establishing the role of PRMT5 in lung cancer tumorigenicity and modulation of expression in smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4663.

Abstract 1078: Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines

Abstract KRAS is the most frequently mutated oncogene in lung adenocarcinoma (50%) and it is associated with a poor response to antineoplastic treatments, as well as poorer survival rates. KRAS activates the MAPK pathway, promoting cell survival and increasing protein synthesis. Novel inhibitors of KRAS G12C mutation (sotorasib) have demonstrated short-lasting responses due to resistance mediated by the AKT-mTOR-P70S6K pathway. In this context, metformin is a promising candidate to avoid this inconvenience by inhibiting mTOR and P70S6K. Therefore, this project aimed to explore the effects of the combination of sotorasib and metformin on cytotoxicity, apoptosis, and the activity of the mTOR pathway in lung cancer cell lines with or without KRAS mutations. Methods: We performed dose-effect curves to determine the IC50 concentration of sotorasib, and IC10 of metformin. Then, these doses were used to treat lung cancer cell lines A549 (KRAS G12S), H522 (without KRAS), and H23 (KRAS G12C). Cellular cytotoxicity was evaluated by MTT assay, apoptosis induction through flow cytometry with Annexin V and 7AAD, and the activity of P70S6K and AKT proteins by western blot. Main outcomes and measures: cell survival, apoptosis, expression, and phosphorylation of intermediaries belonging to the mTOR pathway. Results: The combined treatment showed a synergic effect on cytotoxicity, increased apoptosis induction, and notable inhibition of AKT and P70S6K1 proteins, compared to sotorasib alone or to control groups in all the cell lines, but predominantly in KRAS-mutated cells (H23 and A549). Conclusions: The combination of metformin with sotorasib showed synergic effects on cytotoxicity, increased apoptosis induction, and a remarkable inhibition of downstream proteins involved in the signaling of growth factor receptors in all tested cells. Moreover, these results suggest a sensitizing effect of metformin to sotorasib treatment in cells without KRAS mutations, such as H522. Citation Format: Norma Yanet Hernandez-Pedro, Pedro Barrios-Bernal, José Lucio-Lozada, Maritza Ramos-Ramirez, Oscar Arrieta. Sotorasib and metformin combination enhances cytotoxicity and apoptosis in KRAS mutant lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1078.

Abstract 412: Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer

Abstract Background: Lung cancer is one of the most common tumors, accounting for approximately 1.8 million deaths worldwide in 2020. KRAS is a frequently mutated oncogene, with mutations reported in roughly 20-25% of non-small cell lung cancer cases. Specifically, KRAS mutations at amino acid 12, resulting in a glycine to cysteine (G12C) substitution, occur in approximately 13% and 3% of lung and colon cancers, respectively, and less frequently in other solid tumors. Recently, the development of covalent KRASG12C inhibitors has shown meaningful anticancer activity in patients. However, many patients with KRASG12C do not respond and/or develop resistance to single-agent treatment. Here we investigate combination therapies that may overcome resistance and broaden patient response to KRASG12C inhibitors. Methods: In vitro preclinical KRASG12C models including lung and colon cancer cell lines were treated with a panel of small molecule and covalent KRASG12C inhibitors. The activity of single-agent versus combination treatments was measured in cell viability assays, and screen hits were validated by in vitro mechanism of action studies. Results: A curated set of 152 compounds was used in combination with screen monitoring cell-viability assays across 12 KRAS mutant lung and colon cancer cell lines. Compounds tested included kinase inhibitors targeting oncogenic signaling pathways, epigenetics modifiers, regulators of apoptosis, chemotherapeutics, and other anticancer agents. The screen identified synergy between covalent KRASG12C and several other inhibitors including those for FGFR, EGFR, and SHP2. Notably, pemigatinib, a potent and selective FGFR1-3 inhibitor, had a significantly high Bliss synergy score. Selectivity and siRNA knockdown experiments were performed to identify the specific FGFR isoform involved in synergistic anticancer activity. Inhibition of FGFR1 activity was shown to be essential, whereas an FGFR2-3 specific inhibitor demonstrated only modest activity in combination with KRASG12C inhibitors. Additionally, FGFR1 knockdown combined with KRASG12C inhibition resulted in increased drug sensitivity; in contrast, knockdown of other FGFR family members did not demonstrate similar increase in sensitivity to KRASG12C inhibition. Conclusions: Cell-viability combination screening identified strong synergy between KRASG12C covalent inhibitors and the FGFR1-3 inhibitor pemigatinib in KRASG12C cancer cell lines. The synergistic effect of pemigatinib and KRASG12C inhibition was notably strongest in mesenchymal cancer cell lines. This effect was further validated in in vitro selectivity and siRNA studies. Together, our results support pemigatinib as a promising agent for combination therapy with KRAS inhibitors in lung cancer. Citation Format: Angela Abdollahi, Margaret Favata, Darlise DiMatteo, Sean Schuette, Erika Boarder, Mark Rupar, Ricardo Macarron, Aidan Gilmartin, Hui Wang, Alejandro Amador-Arjona. Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 412.

Accommodation of ring C expanded deoxyvasicinone in the HDAC inhibitory pharmacophore culminates into a tractable anti-lung cancer agent and pH-responsive nanocarrier

A fragment recruitment process was conducted to pinpoint a suitable fragment for installation in the HDAC inhibitory template to furnish agents endowed with the potential to treat lung cancer. Resultantly, Ring C expanded deoxyvasicinone was selected as an appropriate surface recognition part that was accommodated in the HDAC three-component model. Delightfully, fused quinazolinone 6 demonstrating a magnificent anticancer profile against KRAS and EGFR mutant lung cancer cell lines (IC50 = 0.80–0.96 μM) was identified. Results of the mechanistic studies confirmed that the cell growth inhibitory effects of compound 6 stems for HDAC6 (IC50 = 12.9 nM), HDAC1 (IC50 = 49.9 nM) and HDAC3 inhibition (IC50 = 68.5 nM), respectively. Compound 6 also suppressed the colony formation ability of A549 cells, induced apoptosis, and increased autophagic flux. Key interactions of HDAC inhibitor 6 within the active site of HDAC isoforms were figured out through molecular modeling studies. Furthermore, a pH-responsive nanocarrier (Hyaluronic acid - fused quinazolinone 6 nanoparticles) was designed and assessed using a dialysis bag approach under both normal and acidic circumstances that confirmed the pH-sensitive nature of NPs. Delightfully, the nanoparticles demonstrated selective cell viability reduction potential towards the lung cancer cell lines (A549 lung cancer cell lines) and were found to be largely devoid of cell growth inhibitory effects under normal settings (L929, mouse fibroblast cells).

Abstract 1300: Targeted therapies prime lung cancer cells for macrophage-mediated destruction

Abstract The CD47/SIRPa axis is an immune checkpoint that regulates macrophage anti-tumor function. Therapies that block CD47 on cancer cells show promise in clinical trials for solid and hematologic malignancies, particularly when combined with other anticancer agents. However, the best combination strategies for using CD47-blocking therapies remain unknown. In this study, we developed a novel in vitro screening platform to identify drugs that render cancer cells more vulnerable to macrophage attack. We performed an unbiased screen of 800 FDA-approved drugs using primary human macrophages and PC9 cells, a human EGFR mutant lung cancer cell line. We identified EGFR tyrosine kinase inhibitors (TKIs) as drugs that act on the cancer cells and specifically enhance macrophage-mediated cytotoxicity (>4-fold enhancement, p<0.01). In contrast, conventional chemotherapy drugs either showed no significant enhancement or abrogated macrophage activation. The combination of EGFR TKIs with anti-CD47 antibodies elicited maximal phagocytosis across a range of cell lines and conditions. In long-term co-culture assays with macrophages, the combination of EGFR TKIs and anti-CD47 antibodies eliminated persister cells to prevent TKI resistance. Importantly, these findings extended to lung cancers with other RTK-MAPK pathway mutations, such as ALK-rearranged cancers (treated with lorlatinib, alectinib, or crizotinib) or KRAS G12C mutant cancers (treated with sotorasib or adagrasib). In xenograft and syngeneic mouse models, the combination of targeted therapies with CD47 ablation was able to dramatically reduce tumor burden. To understand the mechanism of synergy, we generated 8 different lung cancer cell lines resistant to EGFR, ALK, or KRAS inhibitors. The resistant lines significantly upregulated CD47 and concomitantly became more sensitive to macrophage attack in vitro and in vivo. By RNA sequencing, we identified multiple mechanisms contributing to vulnerability, including secretion of the cytokine MIP-3 by the cancer cells and alteration of other immunoregulatory molecules. Overall, we have identified a novel therapeutic strategy to enhance the efficacy of RTK-MAPK pathway inhibitors by combining them with anti-CD47 therapies. Our findings suggest cross-sensitivity occurs such that lung cancer cells that become resistant to targeted therapies also become more sensitive to macrophage attack. Our findings provide rationale for testing this combination approach in patients with lung cancers bearing driver mutations. Citation Format: Kyle Vacarro, Juliet Allen, Asaf Maoz, Sarah Reeves, Aaron Hata, Kipp Weiskopf. Targeted therapies prime lung cancer cells for macrophage-mediated destruction [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 1300.

Abstract 3277: SWI/SNF inactivation vulnerability

Abstract Genetic inactivation in specific SWI/SNF complex members induce sensitivity to the inhibition of KDM6A/UTX and KDM6B/JMJD3 in cancer. Despite the genetic inactivation of the tumor suppressor SMARCA4 is frequently found in cancer, there are no therapies that effectively target SMARCA4-deficient tumors. In our previous work, we reported that unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. In this context, SMARCA4 deficient cells showed an impaired transactivation and significantly reduced levels of the histone H3K27me3 specific demethylases, KDM6A/UTX and KDM6B/JMJD3, and their inhibition compromises cell viability specifically in SMARCA4 mutant cells. Furthermore, the in vivo administration of KDM6s inhibitor (GSK-J4) to mice orthotopically implanted with SMARCA4 mutant lung cancer cell lines or primary tumors of small cell carcinoma of the ovary, hypercalcemic type (SCCOHT), showed a strong anti-tumor effect, highlighting the vulnerability of the SMARCA4 deficient tumors to KDM6s inhibition as a biomarker that could be exploited for treating SMARCA4-mutant cancer patients (Romero OA, Vilarrubi A, et al. SMARCA4 deficient tumors are vulnerable to KDM6A/UTX and KDM6B/JMJD3 blockade. Nat Comm 12, 4319, 2021). Considering our previous observations, and due to the biological role of SMARCA4 as a core component of the SWI/SNF chromatin-remodeling complex, whose members are genetically inactivated in approximately 20% of all human cancers, we wanted to determine the potential response to KDM6s inhibitors in a context of genetic alterations in genes encoding for the other subunits of this remodeling complex. For this propose, we have integrated state of the art technology like genome-wide chromatin modification analysis (ChIP-seq) and transcriptome analysis (RNA-seq), using human cancer cell lines and preclinical models of different cancer types with genetic inactivation at different SWI/SNF-complex members, including mouse models such as orthoxenografts, to design a personalized epigenetic treatment based on the genetic background. Our results showed that, like SMARCA4 deficient cells, specific mutations at some, but not all, SWI/SNF components induce refractoriness to SAHA, aberrant increase of H3K27me3 mark and sensitizes cancer cells to KDM6 inhibitor accompanied with significantly reduced levels of KDM6A and KDM6B expression. These results suggest a strong functional relationship between KDM6A/6B activity and the SWI/SNF-complex, in the control of gene expression and cancer development. These results will be of great value for the stratification of tumors according to their genetic background for tailored treatments, opening the possibility to use SWI/SNF mutations as potential biomarkers for personalized epigenetic-based therapeutics in cancer. Citation Format: Andrea Vilarrubi, Fernando Setien, Eva Pros, Pedro P. Medina, Antonio Gomez, Alberto Villanueva, Octavio A. Romero, Montse Sanchez-Cespedes. SWI/SNF inactivation vulnerability [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 3277.

Abstract 2975: RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in NSCLC

Abstract Background: RAS is the most commonly mutated oncogene in cancer, with KRAS mutated in ~30% of non-small cell lung cancer (NSCLC). RAS is a small GTPase cycling between GTP-bound ‘ON’ state and GDP-bound ‘OFF’ state. KRAS oncoproteins cycle between these states via hydrolysis and nucleotide exchange with the aid of GAPs, including NF1, and GEFs. Given the ’inactive state’ inhibition of recently developed KRAS G12C inhibitors on the GDP-bound state, this has highlighted the continued reliance of KRAS-mutants upon cycling, and the potential for mutants to retain dependence upon upstream influences, including NF1, for pathogenicity. Methods: 474 patients with advanced RAS- and/or NF1-mutant NSCLC were retrospectively identified from four tertiary cancer centers between 2008 -2021. DNA from archival FFPE samples, serum or combination underwent targeted NGS panels to identify mutations. Molecular, clinical, pathological and treatment outcome data were collected. Online resources including cBioPortal and Project Achilles were used to assess the functional role of any findings. Results: KRAS mutations were identified in 416/474 patients and NF1-mutations in 63/474 patients, eight of whom harbored two NF1-mutations. 24/63 (38%) of NF1-mutant cancers had a concomitant KRAS-mutation. We identified that KRAS G13D was more prevalent in NF1 mutant cancers vs. NF1 wildtype (NF1 MT: 6/24, 25%; vs. NF1 WT: 4/281, 1.4%; p<0.0001). KRAS G12C was identified in 11/24 (45.8%) of the double mutants vs. 109/282 (38.7%) of the NF1 WT patients (p=0.52). Those with G13D/NF1 co-mutation had a predicted pathogenic NF1-mutation in 5/6 (83%) of cases. Functional analysis of NF1 KO in G13D mutant lung cancer cell lines identified that NF1 was more essential in G13D mutant cell lines, with median Chronos score -0.26 G13D vs. -0.04 G12C (p=0.02). mRNA expression data identified a range of mRNA expression Z-scores relative to all samples for NF1-mutant cancers, ranging from lowest expression at -4.54 to +1.63, median -0.51. A higher mRNA expression was identified for NF1 missense mutations of unknown significance compared to truncating mutations with likely pathogenic, loss of function. Conclusions: These results highlight the co-mutational landscape of KRAS G13D with NF1 in NSCLC, suggesting functional importance conferred by NF1 loss, and highlight NF1 mutations as an additional pathogenic even when combined with KRAS mutation. The genomic landscape of KRAS and NF1 mutant NSCLC will be explored further through Whole Genome Sequencing data from the 100,000 Genomes Project (Genomics England). Citation Format: Helen M. Adderley, Mihaela Aldea, Jaqueline Aredo, Mathew Carter, Matthew Church, Pantelis Nicola, Jamie Weaver, Aisha Ghaus, Damien Vasseur, Matthew Krebs, Nicola Steele, Fiona Blackhall, Heather Wakelee, Benjamin Besse, Colin Lindsay. RAS precision medicine transatlantic partnership: Exploration of RAS and NF1 co-mutations in 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 2975.

Abstract 5848: Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein

Abstract Mutant KRAS is found in >30% of lung adenocarcinomas, and tumors with mutant KRAS usually have poor outcomes with current therapies. The rationale for the new treatment approach described here for tumors with mutant KRAS arises from the critical antitumor role that we have uncovered for reactivating the DLC1 tumor suppressor protein, which is frequently downregulated in a wide range of human cancers. Although several mechanisms account for DLC1 downregulation, here we have focused on a subset of mutant KRAS lung tumor lines that express wild type DLC1 mRNA but lack detectable DLC1 protein. We have recently reported that cytoplasmic EZH2 protein directly methylates Lysine-678 of DLC1 protein leading to its ubiquitination by CUL-4A and consequently, proteasomal degradation. In this study, we have found a specific inhibitor of CRM1 protein (Chromosomal Region Maintenance 1, also known as Exportin 1 [XPO1]) substantially reduces cytoplasmic EZH2 level in several mutant KRAS lung cancer cell lines demonstrating that cytoplasmic EZH2 depends on its nuclear export by CRM1. CRM1, which is the major mammalian nuclear export regulator, is frequently overexpressed and/or mutated in human cancer, including lung adenocarcinomas, and these phenotypes are associated with poor prognosis. Endogenous CRM1 and EZH2 protein colocalized and formed a complex in several lung cancer lines and patient-derived tumor xenografts implying the cancer relevance of this interaction. In addition to blocking the cytoplasmic export of EZH2, pharmacologic inhibition of CRM1 increased the steady-state level of DLC1 protein in several mutant KRAS lung cancer lines and patient-derived xenografts. The tumor suppressor activity of the post-translationally stabilized DLC1 protein was further increased by kinase inhibitors of AKT and SRC, which reverse the direct phosphorylation and attenuation of DLC1 tumor suppressor functions by these kinases. The three-drug combination of inhibitors of CRM1 (Selinexor), AKT (MK-2206), and SRC (Saracatinib), which is well-tolerated in mice, induced potent inhibition of tumor xenograft growth and was associated with induction of markers of apoptosis and senescence. Although each of these inhibitors has multiple targets, CRISPR/Cas9 knockout of DLC1 abolished most of the tumor growth inhibitory activity of the three-drug combination, strongly suggesting that DLC1 is the predominant target for the antitumor activity of the drug combination. Interestingly, KRAS siRNA knockdown phenocopied CRM1 inhibition, as it reduced cytoplasmic EZH2, DLC1 methylation, and DLC1 degradation. Our findings suggest CRM1 inhibitors, when given in conjunction with AKT and/or SRC kinase inhibitors, may have clinical efficacy against lung tumors with mutant KRAS. Citation Format: Brajendra K. Tripathi, Nicole Hirsh, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Fernando J. de Miguel, Katerina Politi, James H. Doroshow, Douglas R. Lowy. Inhibition of the CRM1 nuclear export protein can treat mutant KRAS tumors through stabilizing the DLC1 tumor suppressor protein [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 5848.

Abstract 422: Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications

Abstract Introduction: Mutant p53 drives several hallmarks of cancer through a gain-of-function oncogenic program. The oncogenicity of mutant p53 includes sustained proliferation, increased survival and resistance to therapy, increased invasion and metastasis, and genomic instability. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. We and others observe that proteasome inhibitors cause paradoxical depletion of mutant p53 in lung cancer cell lines. Objectives: In this project, we sought to determine the mechanism(s) of mutant p53 depletion by proteasome inhibition and its therapeutic implications in lung cancer. Methods: Human lung cancer cell lines bearing mutant p53 and wildtype p53 (WTp53) were used in this study. An isogenic panel of cell lines with mutant p53, WTp53 and a vector control was also used to determine mutant p53 dependency of the observed effects. Western blotting, immunoprecipitation, siRNA technology, DNA transfection were used for protein detection, protein-protein interactions, gene knock-down and overexpression respectively. Cycloheximide chase assay was used to determine protein degradation. The cancer genome atlas (TCGA) analysis was performed using the cBioPortal platform. Results: Proteasome inhibitors resulted in depletion of mutant p53 in the lung cancer cell line H1975. This effect was also confirmed using the isogenic model described above. Depletion of mutant p53 occurred through a degradation mechanism and was time dependent. While WTp53 accumulated at initial timepoints of proteasome inhibition, mutant p53 did not accumulate but instead depleted with time. This suggested that the proteasome is not the primary mechanism for mutant p53, in contrast to WTp53. Since autophagy is an alternative mechanism of protein degradation in eukaryotes, we evaluated for, and found evidence of autophagy upon proteasome inhibition. We further showed that autophagic degradation of mutant p53 could involve the heat shock protein 70. Our TCGA data analysis showed proteasome genes amplification in mutant p53 lung cancers, suggesting a possible functional dependency. Furthermore, RNA sequencing analysis showed upregulation of most proteasome genes upon stable expression of the R273H mutant in p53-null lung cancer cell line. Finally, in vitro cell viability assays using two different proteasome inhibitors showed increased sensitivity of the lung cancer cell line with mutant p53 compared to WTp53. Conclusion: The cumulative evidence of proteasome hyperactivity, degradation of mutant p53 and in vitro evidence of increased susceptibility of mutant p53 lung cancer cell lines suggests that proteasome inhibitors warrant a closer attention for therapeutic targeting of mutant p53 in lung cancer. Citation Format: Eziafa I. Oduah, Larisa Litovchick, Steven R. Grossman. Novel approach to destabilization of oncogenic mutant p53 and therapeutic implications [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 422.

Abstract 3187: Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome

Abstract Background: KRAS is commonly mutated in solid tumors. Cancer-associated fibroblasts (CAFs) are a key tumor stroma component, which can affect cancer progression and drug response. We hypothesized that defining the differential secreted proteins between CAFs and KRAS mutant-cancer cells could reveal potential mechanisms by which CAFs influence cancer progression and drug resistance. Methods: We studied 12 cell lines: 3 KRAS mutant-colorectal cancer cell lines (H747, LIM2099 and SW620), 3 KRAS mutant-lung cancer cell lines (H1792, H2030 and H23), 3 KRAS mutant-pancreatic cancer cell lines (CAPAN1, DANG, MIAPACA2) and 3 CAF cell line models (colorectal and lung CAF and pancreatic stellate cells). We quantified proteins in cell lines and serum-free media after 24 hour incubation in cells using mass spectrometry in a multiplexed manner (12plex) in triplicates to characterize the proteome and the profile of secreted proteins (secretome), respectively. Prediction software (signalP, secretomeP, TMHMM) and databases (surfaceome and cell surface protein atlas) were used to annotate secreted or transmembrane proteins. Results: Our proteome analysis quantified 9307 proteins. T-test between the cell types (CAF vs cancer) identified 1770 differentially expressed proteins, where 712 proteins were CAF-enriched. This included known CAF markers (e.g α smooth muscle actin) but also proteins not known to be CAF markers such as heat shock protein beta-5/6 (HSPB5/6). The secretome analysis identified 2330 secreted or transmembrane proteins where 202 were differentially expressed between cancer cells and CAFs. 190 proteins were CAF-enriched with the highest ranking being syndecan-2 (SDC2), collagen α-28(I) chain (COL28A1), decorin (DCN) and wnt family member 5B (WNT5B). Interestingly, basal proteome analysis of KRAS mutant-cancer cells showed expression of receptors corresponding to the CAF-enriched ligands, such as receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2) which respond to WNT5B. This suggests that CAF-enriched ligands may activate certain downstream pathways, including non-canonical WNT signaling, in KRAS mutant-cancer cells. Conclusion: We undertook deep quantitative proteomics where we identified potential novel CAF markers HSPB5/6. We have also found proteins in the CAF secretome, such as WNT5B, which may drive drug resistance in KRAS mutant-cancer cells. Citation Format: Rachel Lau, Lu Yu, Theodoros Roumeliotis, Lisa Pickard, Adam Stewart, Jyoti Choudhary, Udai Banerji. Understanding the role of cancer-associated fibroblasts (CAFs) on pathogenesis of KRAS mutated cancers by defining differences in the basal state proteome and secretome [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 3187.

Sensitization of Epidermal Growth Factor Receptor (EGFR) Inhibitors Induced by Radiotherapy Combined with Programmed Death Ligand-1 (PD-L1) Inhibitors

To explore the effect of radiotherapy combined with programmed death ligand-1 (PD-L1) inhibitors on the sensitization of epidermal growth factor receptor (EGFR) inhibitors, 76 patients with nonsmall cell lung cancer (NSCLC) were rolled into group A (lung adenocarcinoma, 55 cases) and group B (lung squamous carcinoma, 21 cases). Another 63 healthy volunteers were set as controls (group C). Patients in group A were rolled into mutation group (15 cases) and wild group (22 cases) regarding the presence of EGFR mutations. The sPD-L1 protein in serum samples was determined via enzyme-linked immunosorbent assay (ELISA). Expressions of PD-L1, EGFR, and immune interferon (IFN-γ) in lung cancer cell lines (LCCL) mutant PC9 and HCC827, and wild-type A549 and H1299 were analyzed. After separation of T lymphocytes, four LCCLs and T lymphocytes were co-cultured to detect the proliferation and apoptosis of T lymphocytes. The results showed that PD-L1 level in EGFR-sensitive mutant LCCLs PC9 and HCC827 after X-ray irradiation was obviously inferior to controls (P < 0.05). The proliferation of T cells in mutant LCCLs PC9 and HCC827 was substantially superior to co-culture system (co-CS) (P < 0.05). After the PC9 co-CS was treated with X-rays, PD-L1 inhibitors, and X-rays combined with PD-L1 inhibitors, the secretion of IFN-γ was markedly increased versus controls (P < 0.05). In short, radiotherapy combined with PD-L1 inhibitors can enhance the proliferation of T cells and inhibit their apoptosis, and greatly increase the secretion of IFN-γ by T cells.

Abstract P066: Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer

Abstract Drug resistance is ultimately the cause of death for most cancer patients – even initially strong responses to treatment are usually followed by the emergence of resistance over time. This suggests the existence of residual or persistent cancer cells, creating a reservoir that ultimately gives rise to stable resistance. These drug tolerant persisters (‘the deadly survivors’) have been described for over a decade in numerous studies; they are often present as a minor fraction of the total tumour population and may exploit non-genetic (transcriptional) programs to allow the cells to survive drug treatment. 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. We carried out parallel genome-wide CRISPR gain and loss of function screens in EGFR mutant lung cancer cell lines treated with EGFR inhibitors, to identify the genes and pathways that may be important in enabling the survival of persister cells. We observed recurrent resistant genes in previously identified resistance pathways including PI3K (PTEN, TSC1, TSC2), MAPK (KRAS, NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, KEAP1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockout demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, key activators of the Hippo pathway. A closer review of the CRISPR screen data confirmed that many resistance hits are members of this pathway - upstream regulators (NF2, AMOTL2), core signalling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signalling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and increased expression of canonical Hippo transcriptional targets. We therefore reasoned that the Hippo pathway might be involved in maintaining the survival of drug tolerant persister cells in this setting. Acute treatment of EGFR mutant cell lines with osimertinib was associated with increased nuclear localisation of YAP1 and WWTR1 and increased expression of canonical Hippo transcriptional targets. Furthermore, the combination of osimertinib and a TEAD inhibitor (K-975) almost completely abolished the survival of drug tolerant persister cells following treatment, indicating that this pathway is an important survival mechanism following drug treatment. Consequently, we propose Hippo signalling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Ercia Banks, Nin Guan, Shanade Dunn, Maria Lisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer [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 P066.

Abstract 1100: Gain and loss of function genome-wide CRISPR screens identify Hippo signaling as an important driver of resistance in EGFR mutant lung cancer

Abstract 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. In many cases the molecular resistance mechanisms remain unknown. A systematic identification of the involved genes and pathways is critical to overcome osimertinib resistance. To define the resistance landscape of EGFR kinase inhibition we performed genome-wide gain and loss of function CRISPR screens in EGFR mutant lung cancer cell lines treated with osimertinib. Resistance hits were enriched for genes in previously identified resistance pathways including PI3K (PTEN, TSC2), MAPK (NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, LZTR1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockouts demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, indicating transcriptional activation of the Hippo pathway. According to our screening data, many resistance hits mapped onto the Hippo signaling axis - upstream regulators (NF2, AMOTL2), core signaling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signaling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and elevated activity of a TEAD reporter system, indicating activation of Hippo transcriptional programs. To maintain osimertinib resistance, expression of both YAP1 and WWTR1 was necessary, suggesting non-redundant roles for both Hippo main effectors in mediating osimertinib resistance. The combination of osimertinib and a TEAD inhibitor (MYF-01-37) reversed the resistance phenotype in NF2 KO cell line models in long-term proliferation assays and also substantially repressed the emergence of drug-tolerant persister cells in PC-9, HCC827 and HCC4006 cell lines following osimertinib treatment. These cells exhibited an enhanced apoptotic response when treated with combination of osimertinib and TEAD inhibitor. Consequently, we propose Hippo signaling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan S. Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Erica Banks, Nin Guan, Shanade Dunn, Maria Luisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signaling as an important driver of resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1100.

Targeting histone deacetylase enhances the therapeutic effect of Erastin-induced ferroptosis in EGFR-activating mutant lung adenocarcinoma.

BackgroundIntrinsic or acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) is common, thus strategies for the management of EGFR-TKIs resistance are urgently required. Ferroptosis is a recently discovered form of cell death that has been implicated in tumorigenesis and resistance treatment. Accumulating evidence suggests that ferroptosis can be therapeutically exploited for the treatment of solid tumors; however, whether ferroptosis can be targeted to treat EGFR mutant lung cancer and/or overcome the resistance to EGFR-TKIs is still unknown.MethodsThe effect of ferroptosis inducers on a panel of EGFR mutant lung cancer cell lines, including those with EGFR-TKI intrinsic and acquired (generated by long-term exposure to the third-generation EGFR-TKI osimertinib), was determined using cytotoxicity assays. Further, drug candidates to enhance the effect of ferroptosis inducers were screened through implementing WGCNA (weighted gene co-expression network analysis) and CMAP (connectivity map) analysis. Flow cytometry-based apoptosis and lipid hydroperoxides measurement were used to evaluate the cell fates after treatment.ResultsCompared with EGFR-TKI-sensitive cells, those with intrinsic or acquired resistance to EGFR-TKI display high sensitivity to ferroptosis inducers. In addition, Vorinostat, a clinically used inhibitor targeting histone deacetylase, can robustly enhance the efficacy of ferroptosis inducers, leading to a dramatic increase of hydroperoxides in EGFR mutant lung cancer cells with intrinsic or acquired resistance to EGFR-TKI. Mechanistically, Vorinostat promotes ferroptosis via xCT downregulation.ConclusionsFerroptosis-inducing therapy shows promise in EGFR-activating mutant lung cancer cells that display intrinsic or acquired resistance to EGFR-TKI. Histone deacetylase inhibitor (HDACi) Vorinostat can further promote ferroptosis by inhibiting xCT expression.

Genomic and pathological heterogeneity in clinically diagnosed small cell lung cancer in never/light smokers identifies therapeutically targetable alterations.

Small‐cell lung cancer (SCLC) occurs infrequently in never/former light smokers. We sought to study this rare clinical subset through next‐generation sequencing (NGS) and by characterizing a representative patient‐derived model. We performed targeted NGS, as well as comprehensive pathological evaluation, in 11 never/former light smokers with clinically diagnosed SCLC. We established a patient‐derived model from one such patient (DFCI168) harboring an NRAS Q61K mutation and characterized the sensitivity of this model to MEK and TORC1/2 inhibitors. Despite the clinical diagnosis of SCLC, the majority (8/11) of cases were either of nonpulmonary origin or of mixed histology and included atypical carcinoid (n = 1), mixed non‐small‐cell lung carcinoma and SCLC (n = 4), unspecified poorly differentiated carcinoma (n = 1), or small‐cell carcinoma from different origins (n = 2). RB1 and TP53 mutations were found in four and five cases, respectively. Predicted driver mutations were detected in EGFR (n = 2), NRAS (n = 1), KRAS (n = 1), BRCA1 (n = 1), and ATM (n = 1), and one case harbored a TMPRSS2‐ERG fusion. DFCI168 (NRAS Q61K) exhibited marked sensitivity to MEK inhibitors in vitro and in vivo. The combination of MEK and mTORC1/2 inhibitors synergized to prevent compensatory mTOR activation, resulting in prolonged growth inhibition in this model and in three other NRAS mutant lung cancer cell lines. SCLC in never/former light smokers is rare and is potentially a distinct disease entity comprised of oncogenic driver mutation‐harboring carcinomas morphologically and/or clinically mimicking SCLC. Comprehensive pathologic review integrated with genomic profiling is critical in refining the diagnosis and in identifying potential therapeutic options.

Effect of long non-coding RNA long stress-induced noncoding transcript 5 on erlotinib resistance to lung cancer cells and the underlying mechanisms.

To explore the relationship between long non-coding RNA (lncRNA) long stress-induced noncoding transcript 5 (LSINCT5) and erotinib resistance to lung cancer cells and the potential mechanisms. Human lung cancer cell line A549, H520, H358, H1299, SPCA1, and PC9 were collected and cultured. Epidermal growth factor receptor (EGFR) mutant lung cancer cell line PC9 was divided into a control group, a resistance group, a interference group I and II. The control group was treated with dimethylsulfoxide (DMSO) for 10 weeks and then was transfected with control target sequence expression vector. The resistant group was treated with erlotinib at gradient concentration (0.1, 0.2, 0.4, 0.8, and 1.6 μmol/L, respectively) for 2 weeks and then transfected with control target sequence expression vector. Interference group I and II were treated with erlotinib at gradient concentration (0.1, 0.2, 0.4, 0.8, and 1.6 μmol/L, respectively) for 2 weeks and then transfected with the shRNA targeting expression vectors 1 and 2. 50% inhibitory concentration (IC50) of erlotinib was detected by cell counting kit-8 (CCK-8) assay. The mRNA expressions of LSINCT5, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were measured by real-time PCR. The protein levels of PI3K, Akt, and phospho-Akt (p-Akt) were detected by Western blotting. The divergences of Akt and IgG binding to LSINCT5 were detected by RNA immunoprecipitation (RNA-IP) experiment. The expression of LSINCT5 in PC9 cells was significantly higher than that in other lung cancer cell lines (all P<0.05). Compared with the control group, the IC50 of erotinib and the expression of LSINCT5, PI3K, and Akt mRNA and protein in the resistance group were significantly higher (all P<0.05), and the IC50 of erotinib and the expression of LSINCT5, Akt, and p-Akt in the interference group I and II were significantly lower (all P<0.05). Compared with IgG, LSINCT5 binding to Akt was increased significantly (P<0.05). The expression of LSINCT5 is high in the erlotinib-resistant cells. Interference with LSINCT5 may inhibit the expression and activity of Akt and promote the cell sensitivity to erlotinib.

Abstract 3410: Adaptive resistance mechanisms to KRASG12C specific inhibitors

Abstract Covalent inhibitors of KRASG12C mutants allow specific targeting of KRAS driven signaling yet early studies suggest bypass signaling as a mechanism of resistance. Understanding adaptive signaling mechanisms enabling cells to survive KRASG12C inhibition could identify combination therapies and associated predictive biomarkers. We employed KRASG12C mutant lung cancer cell lines and evaluated their sensitivity in 2D cell culture to mimic the resistant state. We combined small molecule targeted agents (N=292 compounds) screens alongside KRASG12C inhibition as well as global mass spectrometry based phosphoproteomics (pY/S/T) to examine adaptive signaling changes. KRASG12C mutant lung cancer cell lines have differential sensitivity to KRASG12C inhibition with ARS-1620. Signaling analysis indicates inhibition of p-ERK and to some degree pAKT after 6 hours with evidence of rebound by 24 hr. To expand our understanding of cell type specific response and pathway reactivation upon KRASG12C inhibition, TMT-based quantitative phosphoproteomics was performed after phosphotyrosine (pY) enrichment in 3 cell lines (H358, Calu1 and H1792) after ARS-1620 treatment (6h and 24h). We identified 2275 human phosphosites in total, corresponding to 1153 proteins in 987 protein groups, out of which 433 phosphosites were differentially expressed (1.5-fold change and p-value &amp;lt; 0.05) between any treatment time point and control. Preliminary analysis revealed some common and cell line specific response to ARS-1620. MAPK1, MAPK3, MET, CTNND1, and VTI1B exhibit the strongest down-regulation, across all time points in all cell lines. We observed 84 phosphosites corresponding to 77 proteins that were more abundant in ARS-1620 treated cells indicating a potential for adaptive resistance signaling. Results from the small molecule screens indicate compounds targeting ERBB/HER family of kinases and PI3K pathway showed better synergistic effects with ARS-1620. Signaling analysis in H358, H23 and H2122 cells revealed more abundant tyrosine phosphorylated and total HER2 and HER3 upon ARS-1620 treatment. Further suppression of pERK and pAKT was observed when afatinib was added to ARS-1620 resulting in more combined effects on cell viability. When compared to more specific EGFR inhibition (erlotinib), afatinib enhanced inhibition of pERK and pAKT by ARS-1620 and had more effects on cell viability. However, in H1792 cells, phosphoproteomics identified increased tyrosine phosphorylation of FGFR1 following ARS-1620 and this correlated with combination effects on cell viability of ARS-1620 with the FGFR inhibitor AZD-4547. Lastly, our results suggest that cells sensitive to combination with either HER TKI or FGFR TKI with ARS-1620 are also demonstrating combination effects with SHP2 inhibition (SHP099). Our combined chemical biology and phosphoproteomics results suggest widespread adaptive signaling changes, some of which are contributing to adaptive resistance. Results suggest a more important role of pan-HER inhibition compared with EGFR inhibition. These data may guide biomarker development strategies, especially those that reflect adaptive signaling responses, to predict clinically effective combination strategies in diverse group of KRASG12C mutant lung cancer. Citation Format: Hitendra Singh Solanki, Bin Fang, Eric A. Welsh, Aik C. Tan, Fumi Kinose, Uwe Rix, Eric B. Haura. Adaptive resistance mechanisms to KRASG12C specific inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3410.

Abstract 923: Pulsatile MEK inhibition improves anti-tumor immunity and T cell function in Kras mutant lung cancer

Abstract KRAS is one of the most commonly identified driver oncogenes in non-small cell lung cancer (NSCLC) and is frequently associated with disease refractory to currently available modalities of treatment. Targeted therapy to inhibit MEK signaling has shown promising tumor growth control in preclinical models, but is followed by quick rebound in tumor growth. Recently, immune checkpoint blockade has demonstrated clinical activity by dis-inhibiting T cells suppressed in the tumor microenvironment. Thus, we sought to identify the most effective therapy for the treatment of KRAS mutant NSCLC patients by targeting cancer cells and activating immune infiltrating cell populations concurrently. To achieve this, we focused on studying the impact of pulsatile MEK inhibition on the immune microenvironment utilizing Kras mutant lung cancer cell lines and Kras mutant transgenic lung cancer mouse models. We found that pulsatile MEK inhibition maintained T cell activation better than continuous treatment ex vivo and in vivo. Treatment with two different MEK inhibitors (selumetinib and trametinib) each resulted in higher CTLA-4 and PD-1 expression in T cells using pulsatile treatment, compared to continuous treatment ex vivo. In addition, the pulsatile schedule showed superior anti-tumor effects and delayed drug resistance compared to continuous administration in vivo. Further, a combination of pulsatile MEK inhibitor treatment and CTLA-4 blockade resulted in the most prolonged survival of Kras tumor-bearing mice. Similar experiments in immune-deficient mice confirmed that this prolonged survival is conferred by adaptive immunity. Our findings set the foundation for a combinatorial therapeutic strategy using pulsatile targeted therapy together with immune checkpoint blockade in patients to optimally enhance tumor apoptosis and promote long-term immune response simultaneously. We are now continuing our studies using a KRAS G12C inhibitor to define its immune modulating properties and will then rationally combine with immune based therapy. Citation Format: Hyejin Choi, Jiehui Deng, Shuai Li, Tarik Silk, Lauren Dong, Elliott J. Brea, Sean Houghton, David Redmond, Hong Zhong, Jonathan Boiarsky, Esra A. Akbay, Paul D. Smith, Taha Merghoub, Kwok-Kin Wong, Jedd D. Wolchok. Pulsatile MEK inhibition improves anti-tumor immunity and T cell function in Kras mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 923.

Abstract LB-122: Combinations of SHP2 inhibitor to overcome RAS activation by receptor tyrosine kinases in response to ERK inhibition

Abstract Introduction: The RTK-RAS-MAPK pathway is frequently activated in cancers due to a variety of mechanisms including mutations or amplifications in RTK, KRAS or BRAF. The effectiveness of inhibitors targeting those oncogenic drivers is often limited by the pathway feedback activation originated at the RTK level in response to ERK inhibition. The non-receptor protein tyrosine phosphatase SHP2 mediates RAS activation downstream of various receptor tyrosine kinases. Potent and selective allosteric SHP2 inhibitors such as TNO155 are in clinical development and offer an appealing one-size-fits-all approach to overcome RTK-mediated feedback activation of RAS and enhance the efficacy of inhibitors targeting RTK, BRAF or KRASG12C. We sought to study the combination efficacy and mechanism in pre-clinical models for prioritization in clinical trials. Experimental procedures: The combination efficacy and synergy of TNO155 with EGF816 (a 3rd generation EGFR inhibitor), dabrafenib+trametinib or a tool KRAS G12C inhibitor (G12Ci) were respectively assessed in a number of EGFR mutant lung cancer models, BRAFV600E colorectal cancer models and KRASG12C lung and colorectal cancer models. The effect of the combinations on ERK inhibition was also studied. Results: TNO155 has varying single agent activity in vitro in EGFR mutant lung cancer cell lines and is not affected by clinically relevant resistance mechanisms to EGFR inhibitors such as secondary mutations in EGFR (T790M and C797S) or MET amplification. In addition, the combination of TNO155 and EGF816 is synergistic across cell lines, coincident with sustained ERK inhibition. In BRAFV600E colorectal cancer cell lines with feedback activation of EGFR, MET or FGFR respectively in response to treatment with dabrafenib+trametinib, TNO155 synergistically enhanced the efficacy of dabrafenib+trametinib in all three cell lines, phenocopying respective RTK inhibitors. In KRASG12C lung cancer cell lines, quick rebound of p-ERK was observed as early as 24 hour post treatment with G12Ci and cannot be blocked by increasing concentrations of G12Ci, suggesting feedback activation of wild-type KRAS or other RAS isoforms. In contrast, TNO155 effectively blocked the p-ERK rebound and enhanced the efficacy of G12Ci. Similar observations were made in KRASG12C colorectal cancer cell lines. Conclusions: Our findings suggest that SHP2 inhibition is an effective strategy to block the feedback activation of wild type and G12C KRAS, as well as NRAS and HRAS, by a variety of RTKs, in response to ERK inhibition. Given the mutant selective properties of those inhibitors we studied, the tolerability of their combinations with TNO155 is highly expected. Our data provide pre-clinical rationale to explore those TNO155 combinations in the corresponding cancer indications in clinic. Citation Format: Huaixiang Hao, Chen Liu, Hongyun Wang, Hengyu Lu, Scott Delach, Matthew LaMarche, Jeffrey Engelman, Peter Hammerman, Giordano Caponigro, Susan Moody, Morvarid Mohseni. Combinations of SHP2 inhibitor to overcome RAS activation by receptor tyrosine kinases in response to ERK inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-122.