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Mutation calls from cell line samples. (Supplementary Table 6)

The efficacy of molecularly targeted anti-cancer therapies may be limited by the presence of co-occurring mutations within a tumor.1-3 Conversely, these alterations may confer collateral vulnerabilities that can be leveraged for the development of novel therapeutic approaches. KRAS mutant lung cancers are distinguished by recurrent mutations in tumor suppressor genes such as TP53 and STK11/LKB1.4 However, clinically actionable targets associated with these alterations are largely undefined. BH3 mimetics targeting distinct pro-survival BCL-2 family proteins have been proposed to enhance the efficacy of targeted therapies, but no biomarkers exist that can predict which patients are most likely to respond. Here we show that inhibition of oncogenic signaling in KRAS-LKB1 mutant lung cancer cells creates a specific dependency on the anti-apoptotic protein MCL-1 for survival. We find that loss of the LKB1-NUAK signaling axis derepresses a JNK-mediated stress response to KRAS or MEK inhibition, allowing inhibitory phosphorylation of BCL-XL that alters BH3-protein interactions and makes cells vulnerable to concurrent MCL-1 inhibition. These results uncover a previously unknown role for LKB1 in regulating the mitochondrial apoptotic response of cancer cells independent of its tumor suppressor activity mediated by AMPK5-7 and SIK8,9 kinases and suggest a fundamental role for LKB1-NUAKs in suppressing the cellular stress response. Additionally, our study reveals a therapeutically targetable vulnerability in KRAS-LKB1 lung cancer cells and suggests a genotype-informed strategy for improving the efficacy of KRAS-targeted therapies.

There is a longstanding desire to take therapeutic advantage of dysregulated metabolic states in cancer. While it has been appreciated that lung tumors rewire their cellular metabolic networks to support unrestrained proliferation, metabolic vulnerabilities have largely not been explored in the context of specific onco-genotypes. This represents a major gap in our understanding of how different oncogenic drivers in non-small cell lung cancer (NSCLC) confer reliance on discrete metabolic networks to sustain tumor growth. The goals of this project are (1) to investigate metabolic dependencies in distinct molecular subtypes of lung cancer and (2) to elucidate how metabolic reprogramming drives resistance to targeted therapy. Using patient-derived cell culture models and tumor specimens collected from patients with ALK-positive (ALK+) NSCLC, we identified that lung tumors with ALK rearrangements harbor a unique metabolic signature marked by reliance on anabolic nucleotide pathways. A phosphoproteomic screen in ALK+ patient-derived cells identified a novel metabolic target of ALK signaling, GUK1, the only known enzyme responsible for GDP synthesis. We show that ALK binds to and phosphorylates GUK1 and that ALK-mediated GUK1 phosphorylation augments GDP/GTP nucleotide biosynthesis. Steady-state and tracing metabolomic studies demonstrate that ALK inhibition and GUK1 phosphomutant are epistatic in guanine nucleotide production. Molecular dynamic modeling suggests that phosphorylation of GUK1 alters the dynamics of active site closure to enhance substrate processivity and protects GUK1 from a non-catalytic confirmation. Introduction of phosphomutant GUK1 into ALK+ patient-derived cell lines results in decreased tumor proliferation in vitro and in vivo in xenograft models. Spatially resolved mass spectrometry imaging of tumor specimens from ALK+ patients demonstrates significant enrichment of guanine nucleotides in ALK+ and phospho-GUK1+ tumor cells. We identified that other oncogenic fusion proteins regulate GUK1 phosphorylation, highlighting the need to further characterize GUK1 as a metabolic liability in NSCLC. Furthermore, a subset of patient-derived cell lines with resistance to ALK tyrosine kinase inhibitors (TKIs) exhibits increased expression and phosphorylation of GUK1, indicating that regulation of this metabolic enzyme may play a role in mediating acquired resistance. We anticipate these studies will pave the way for the development of new therapeutic approaches by exploiting metabolic vulnerabilities in oncogene-driven lung cancers. Citation Format: Jaime Laurel Schneider, Kiran Kurmi, Ishita Dhiman, Roberta Colapietro, Shakchhi Joshi, Christian Johnson, Satoshi Yoda, Joao Paulo, Daniela Ruiz, Sylwia Stopka, Gerard Baquer, Jessica Lin, Kevin Haigis, Nathalie Agar, Steven Gygi, Aaron Hata, Marcia Haigis. GUK1 is a novel metabolic liability in oncogene-driven 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 1156.

Acquired drug resistance to even the most effective anti-cancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified, the underlying molecular mechanisms shaping tumor evolution during treatment are incompletely understood. We recently demonstrated that lung cancer targeted therapies commonly used in the clinic induce the expression of cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug tolerant cancer cells persisting during therapy. Preventing therapy-induced A3A mutagenesis by gene deletion decreased the accumulation of chromosomal aberrations and delayed the emergence of drug resistance. Thus, we hypothesize that inhibition of A3A may represent a potential therapeutic strategy to halt the evolution of resistant clones and prevent acquired resistance to lung cancer targeted therapies. Understanding the molecular mechanism of A3A induction during targeted therapy is a crucial step to develop targeting A3A therapies.Using a targeted drug combination screen, we found that DNA methyltransferase (DNMT) inhibitors induce expression of APOBEC3A in non-small cell lung cancer cells, phenocopying the effects of targeted therapies. RNA-seq profiling revealed that both targeted therapies and DNMT inhibitors increase expression of non-coding repeat RNAs. Repeatome analysis identified distinct classes of non-codding RNAs including endogenous retrovirus elements (ERV) in drug tolerant persister cells. Activation of endogenous intracellular viral sensing pathways by exogenous nucleic acids mimicking viral infection induced APOBEC3A, and genetic perturbation of RIG-I or MAVS reduced APOBEC3A induced by targeted therapy. These findings suggest that epigenetic derepression of retroviral repeat elements may underly APOBEC3A mutagenic activity and tumor evolution during lung cancer targeted therapy. Citation Format: Hideko Isozaki, Ramin Sakhtemani, Naveed Nikpour, Susanna Monroe, Jessica Lin, Lecia Sequist, Zofia Piotrowska, Justin Gainor, Rémi Buisson, Michael Lawrence, Aaron Hata. Epigenetic regulation of APOBEC3A mutagenesis and tumor evolution during targeted therapy in non-small cell 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 137.

Cancer cells gain drug-tolerant states and evade therapy. In response to KRAS G12C inhibitor (G12Ci), KRAS mutant non-small cell lung cancer (NSCLC) cells maintain a drug-tolerant state by aurora kinase A (AURKA). AURKA can phosphorylate c-Raf to maintain new KRAS signaling, however, how AURKA becomes activated to cause KRAS G12Ci resistance is unclear. We show here that KRAS G12C + AURKA inhibition cause synthetic lethality in KRAS G12C NSCLC cells. LY3499446 (KRAS G12Ci) and LSN3321213 (aurora kinase A inhibitor) induced apoptosis that is independent of inhibited MAPK reactivation. Using high-content imaging that tracks single-cell fate during treatment, we observed that single-agent KRAS G12Ci induces G1 arrest in a sub-population of cells. Upon co-treatment with G12C + AURKAi, cells that are halted in G1 phase undergo early apoptosis, while those that initially evade G1 arrest and proceed through G2/M undergo apoptosis in subsequent G1. These data suggest the hypothesis that AURKA inhibition may increase the probability of G1 checkpoint-induced apoptosis by facilitating chromosomal misalignment and genomic instability. In summary, we provide clinical rationale for clinical testing of KRAS G12C + AURKA inhibitors. We also suggest a novel mechanism explaining the dependency of KRAS G12C resistant subpopulations on AURKA, leading to the opportunity to investigate the role of genomic instability in conferring KRAS G12Ci adaptive resistance. Citation Format: Chendi Li, Jeremy Chang, Mohammed U. Syed, Anahita Nimbalkar, Yi Shen, Steve Altschuler, Lani Wu, Xueqian Gong, Aaron Hata. Aurora kinase A inhibition overcome adaptive resistance to KRAS G12C inhibitor by G1-checkpoint induced apoptosis in KRAS non-small cell 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 3868.

BACKGROUND: Drug-loaded meshes offer a promising delivery strategy for the prevention of local recurrence. Patient-derived xenograft (PDX) models are representative of individual patient tumors and predictive of clinical outcomes. METHODS: A patient-derived xenograft model was established in NSG mice using tumor tissue from a patient with aggressive lung adenocarcinoma. Paclitaxel-loaded meshes (PGA+PTX) were electrospun. Tumor-bearing mice were randomized into four groups after macroscopic complete resection: 1) no treatment (n = 10); 2) intraperitoneal paclitaxel (IP PTX) at 20 mg/kg (n = 10); 3) PGA mesh without drug (n = 14); and 4) PGA+PTX mesh at 12 mg/kg (n = 14). A 1 cm(2) mesh was placed onto the tumor resection beds. Groups were observed for local recurrence for 120 post-operative days. RESULTS: PDX mice treated with PGA+PTX meshes post-resection exhibit a greater than 5-fold increase in recurrence-free survival (p

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

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 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.

The recent approval of the KRAS G12C inhibitor sotorasib (AMG 510) for non-small cell lung cancer (NSCLC) marked a milestone in the development of targeted therapies for KRAS mutant cancers. While sotorasib and other KRAS G12C inhibitors have demonstrated rapid and durable responses in the clinic, some patients do not achieve responses. The identification of specific vulnerabilities conferred by recurrent co-occurring mutations may enable the development of biomarker-driven combination therapies with enhanced activity in distinct subsets of patients. We screened a panel of KRAS-mutant NSCLC cell lines as well as patient-derived xenograft (PDX) mouse models and observed that loss of the tumor suppressor STK11/LKB1 is associated with increased sensitivity to combined MAPK (either the KRAS G12C inhibitor sotorasib or MEK inhibitor trametinib) and MCL-1 inhibition (AMG 176). Restoration of LKB1 expression in LKB1-deficient cell lines and PDX tumors blunted the apoptotic response to MAPK + MCL-1 inhibition; conversely, deletion of LKB1 in LKB1 wild-type models increased sensitivity. Mitochondrial apoptotic cell death is regulated by interactions between pro- (e.g., BIM) and anti-apoptotic (e.g., MCL-1, BCL-XL) BCL-2 family members. MAPK inhibition increases BIM, while MCL-1 inhibition prevents BIM sequestration by MCL-1, resulting in apoptosis. LKB1 deficient cells exhibit increased association of BIM and MCL-1 upon MAPK inhibition, effectively priming cells for death upon inhibition of MCL-1. Mechanistically, LKB1 deficiency and associated loss of NUAK phosphorylation leads to hyperactivation of the JNK phospho-kinase network. JNK phosphorylates MCL-1 at S64 and T163, which enhances BIM: MCL-1 protein-protein interaction. Conversely, JNK phosphorylates BCL-XL at S62 and prevents sequestration of BIM. This series of phosphorylation events increases MCL-1 dependence and creates a specific vulnerability of KRAS-LKB1 tumors to MAPK + MCL-1 inhibition. Consistent with this mechanism, ex vivo treatment of tumor tissue from a KRAS-LKB1 mutant NSCLC patient with sotorasib or trametinib increased MCL-1 dependent priming. These results reveal a novel link between LKB1 and the regulation of BCL-2 family proteins and provide preclinical rationale for evaluation of combined KRAS G12C + MCL-1 inhibitors for KRAS-LKB1 mutant NSCLC. Citation Format: Chendi Li, Mohammed Usman Syed, Yi Shen, Audris Oh, Cameron Fraser, Johannes Kreuzer, Christopher Nabel, Kaitlyn Webster, Robert Morris, Sean Caenepeel, Anne Y. Saiki, Karen Rex, J. Russell Lipford, Wilhelm Hass, Kristopher Sarosiek, Paul E. Hughes, Aaron Hata. LKB1 loss rewires JNK-induced apoptotic protein dynamics through NUAKs and sensitizes KRAS-mutant non-small cell lung cancers to combined KRAS G12C + MCL-1 blockade [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 2150.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR04) of the Conference Proceedings. Citation Format: Haichuan Hu, Lecia Sequist, Zofia Piotrowska, Hillary Mulvey, Sundus Noeen, Patricia Hare, David Kodack, Aaron Hata, Matt Niederst, Cyril Benes, Jeff Engelman. Decoding tumor microenvironment to enhance NSCLC targeted therapy [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B25.

Background: Tyrosine kinase inhibitors (TKI) have yielded great responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations, however these and other targeted therapies are limited by intrinsic and acquired drug resistance. Previous study from our group was looking into tumor autonomous resistance mechanisms by developing patient-derived cancer models (PDCs). In this study, we aimed to decipher the non-autonomous resistance mechanisms via tumor microenvironment by developing patient-derived fibroblast (PDF) cell lines. Method: Cancer-associated fibroblast cells are isolated directly from EGFR mutant and ALK translocated NSCLC biopsies. Over 30 PDFs models have been established, which represent different clinical features and response profiles. Result: By co-culturing the PDCs with PDFs, we found that there is considerable variability in both models for their magnitude and mechanism by which the TKI treatment is desensitized. Both HGF dependent and HGF independent resistance mechanisms can be overcome by specific therapeutic combinations. Conclusion: Together, our results indicate that PDFs are clinically relevant models for deciphering non-autonomous resistance mechanisms, that they are heterogeneous in protecting cancer cells from TKI treatment, and that the resistance mediated by PDFs can be overcome by specific therapeutic combinations. Citation Format: Haichuan Hu, Hillary Mulvey, Sundus Noeen, Kodack David, Aaron Hata, Matthew Niederst, Cyril Benes, Jeffrey Engelman. Patient-derived tumor microenvironment models uncover nonautonomous TKI resistance mechanisms in 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 1012. doi:10.1158/1538-7445.AM2017-1012