Abstract Approximately 30% of lung cancers are driven by mutationally activated KRAS. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, a major obstacle in treating lung cancer is resistance to current therapeutic treatments. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagy in response to treatment with sotorasib. Currently, the only therapeutics in clinical use to inhibit autophagy are lysosomal inhibitors (hydroxy)chloroquine. High concentrations of these compounds are needed to achieve modest inhibition of autophagy, suggesting that the potency of these compounds may limit clinical responses. ULK kinases are serine/threonine kinases that are necessary for the initiation of autophagy. DCC-3116 is a potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy. Treatment with sotorasib and DCC-3116 led to superior anti-tumor effects in preclinical models of lung cancer. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in the regulation of nutrient sensing and autophagy. In other KRAS-driven cancers, the LKB1-AMPK-ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data demonstrates that LKB1 is dispensable for sotorasib-induced autophagy, suggesting the mechanism(s) of autophagy induction in KRASG12C-driven lung cancer may be different from those detected in other KRAS mutated cancers. Consequently, we have generated genetically engineered mouse models (GEMMs) of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. We have treated KRASG12C-driven GEMMs with sotorasib and/or DCC-3116 to test the sensitivity of lung tumors to these treatment options. Citation Format: Phaedra Ghazi, Kayla O'Toole, Sanjana Boggaram, Michael Scherzer, Madhumita Bogdan, Bryan Smith, Dan Flynn, Conan Kinsey, Martin McMahon. Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer [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 4322.

Mutational activation of KRAS occurs commonly in lung carcinogenesis and, with the recent U.S. Food and Drug Administration approval of covalent inhibitors of KRASG12C such as sotorasib or adagrasib, KRAS oncoproteins are important pharmacological targets in non-small cell lung cancer (NSCLC). However, not all KRASG12C-driven NSCLCs respond to these inhibitors, and the emergence of drug resistance in those patients who do respond can be rapid and pleiotropic. Hence, based on a backbone of covalent inhibition of KRASG12C, efforts are underway to develop effective combination therapies. Here, we report that the inhibition of KRASG12C signaling increases autophagy in KRASG12C-expressing lung cancer cells. Moreover, the combination of DCC-3116, a selective ULK1/2 inhibitor, plus sotorasib displays cooperative/synergistic suppression of human KRASG12C-driven lung cancer cell proliferation in vitro and superior tumor control in vivo. Additionally, in genetically engineered mouse models of KRASG12C-driven NSCLC, inhibition of either KRASG12C or ULK1/2 decreases tumor burden and increases mouse survival. Consequently, these data suggest that ULK1/2-mediated autophagy is a pharmacologically actionable cytoprotective stress response to inhibition of KRASG12C in lung cancer.

Data collection and refinement statistics

Abstract KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor–induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. Significance: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties. See related commentary by Hobbs and Der, p. 696. This article is highlighted in the In This Issue feature, p. 681

Supplementary Materials and Methods

GSEA analysis of pancreata expressing WT or mutant K-Ras.

GSEA analysis of spleens expressing WT or mutant K-Ras.

Proteomics data from mouse tissues.

GSEA analysis of colons expressing WT or mutant K-Ras.

Approximately 25% of lung cancers are driven by mutationally-activated KRAS. A major obstacle in treating lung cancer is resistance to current therapeutic treatments. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, the twin problems of primary or acquired resistance to direct pharmacological inhibition of KRASG12C remain major obstacles in sustaining the deepest and most durable patient responses. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. Moreover, combined inhibition of RAS pathway signaling plus autophagy had potent anti-tumor effects in preclinical models of melanoma or pancreatic cancer. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagic flux in response to treatment with KRASG12C inhibitors. Moreover, combined treatment of such cells with a specific, selective and potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy, led to superior anti-tumor effects in human cell line xenograft models. Previous work to understand why inhibiting autophagy sensitizes KRAS mutant cancer cells to targeted therapy has been confounded by the use of non-specific lysosomal inhibitors such as hydroxychloroquine. Using selective autophagy inhibitors, such as ULK1/2i, provides a unique opportunity to investigate the precise mechanism as to why inhibition of autophagy sensitizes cells to KRASG12C inhibition. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in regulation of nutrient sensing and autophagy. Patients with KRAS-mutated lung cancer whose tumors also lack LKB1 expression are characterized by aggressive behavior and resistance to standard treatment. In other KRAS-driven cancers, the LKB1>AMPK>ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data suggests that LKB1 is dispensable for sotorasib-induced autophagy in KRASG12C-driven lung cancer cells. To this end, KRASG12C-driven lung cancer cell lines with loss of LKB1 expression significantly increased autophagic flux after treatment with either a KRASG12C inhibitor or inhibitors of RAF>MEK>ERK signaling suggesting that the mechanism(s) of autophagy induction in KRASG12C-driven may be different to those detected in other KRAS mutated cancers. Consequently, we have generated new genetically engineered mouse models of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. Citation Format: Phaedra C. Ghazi, Conan Kinsey, Madhumita Bogdan, Bryan D. Smith, Daniel L. Flynn, Martin McMahon. Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR03.

The recent approval of KRAS G12C inhibitors has provided novel treatment options for lung, colorectal and other cancer patients harboring this mutation and has changed the landscape of research for these cancers. Current priorities in the field include characterizing the mechanism of action of KRAS G12C inhibitors and the mechanisms underlying resistance to KRAS inhibition. Utilizing access to pre and post-treatment patient samples from KRYSTAL-1, preliminary gene expression analyses from baseline and post-adagrasib (Cycle 1, Day 8) treated patient samples demonstrated multiple oncogenic pathway gene signatures, including MYC, mTOR, cell cycle, EMT and inflammation-related signatures, were significantly regulated by adagrasib. KRAS/MAPK pathway and cell cycle-related genes including ETV4, CCND1, DUSP6, TOP1, and CENPA, as well as the Singh KRAS dependency signature were significantly downregulated in Cycle1, Day 8 compared to baseline tumor biopsies. Genes implicated in inflammation and the immune response were significantly upregulated in Cycle 1, Day 8 compared to baseline tumor biopsies. In agreement with the gene expression data as well as findings from syngeneic mouse models, preliminary immunohistochemical analysis on matched baseline, post-adagrasib treated (Cycle 1, Day 8), and end of treatment FFPE patient samples revealed marked changes in tumor cell mechanistic biomarkers and immune cell types following adagrasib treatment. These observations included decreased tumor Ki67 and increased tumor PD-L1, as well as marked alterations in tumor immune cell composition, including increased CD8+ T cells and decreased MDSCs following adagrasib treatment in several patients. Finally, flow cytometry and TCRb sequencing data from adagrasib and pembrolizumab-treated patient blood samples demonstrated an increase in several activated CD8+ T cell populations and the emergence of new T cell clones in a subset of patients after combination treatment. Together, these data suggest robust evaluation of patient biopsies pre and post adagrasib treatment may better predict outcomes that can be achieved in patients harboring KRASG12C mutant cancers and can guide early clinical development strategies, including support for combining adagrasib with immune checkpoint inhibitors. Citation Format: Jill Hallin, Laura Hover, Julio Fernandez-Benet, Adam Pavlicek, Kenna Anderes, Pasi A. Jänne, Gregory J. Riely, Alexander I. Spira, Jun Zhang, Peter Olson, James G. Christensen. Effects of adagrasib on oncogenic signaling, immune cell regulation and biomarkers of response in preliminary clinical analyses [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B012.

Supplementary Figures S1-S18

YAP activation in OMM1 cells

Proteomics data from human cell lines.

Abstract KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor–induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. Significance: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties. See related commentary by Hobbs and Der, p. 696. This article is highlighted in the In This Issue feature, p. 681

Mutationally-activated RAS oncoproteins are detected in approximately 19% of newly diagnosed human cancers and have been targets for drug discovery for over 40 years. Recently, the FDA approved sotorasib, a covalent inhibitor of KRASG12C, for the treatment of non-small cell lung cancer (NSCLC). Although sotorasib demonstrated clear clinical benefits, the emergence of drug resistance has led to drug combination approaches with the goal of deepening and sustaining the durability of patient responses. One potential mechanism of drug resistance is the induction of autophagy, which cancer cells use to survive during periods of stress such as treatment with pathway-targeted therapies. Indeed, KRAS-mutated cancer cells have been shown to exhibit constitutive autophagy for survival. Additionally, such cells further increase autophagy when treated with pathway-targeted inhibitors of RAS>RAF>MEK>ERK MAP kinase signaling as a resistance mechanism. Hence, the combination of a KRASG12C inhibitor with a specific and potent autophagy inhibitor could lead to deeper and more sustained clinical responses. DCC-3116 is an investigational, potent and selective pharmacological inhibitor of the protein kinases ULK1 and ULK2, which are critical initiating components of the autophagy pathway. Treatment of KRASG12C non-small cell lung cancer (NSCLC) cell lines with sotorasib induced autophagy by ~2-fold via activation of ULK kinases as measured by an increase in ULK-mediated phosphorylation of ATG13. Sotorasib-mediated ULK kinase activation, and resulting autophagic flux, was inhibited by DCC-3116 in a dose-dependent manner with IC50 values of 81-160 nM in NSCLC cell lines. These effects translated to in vivo efficacy. In the Calu-1 and H358 KRASG12C NSCLC xenograft models, the combination of DCC-3116 and sotorasib resulted in tumor regression whereas single treatment arms afforded only inhibition of tumor growth. A NSCLC KRASG12C patient derived xenograft (PDX) model further supported the DCC-3116 and sotorasib combination with increased tumor growth inhibition compared to sotorasib alone. These data demonstrate a compelling rationale to study DCC-3116 in combination with KRASG12C inhibitors such as sotorasib in NSCLC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors with documented KRAS, NRAS or BRAF mutations (NCT04892017). Citation Format: Martin McMahon, Madhumita Bogdan, Mary J. Timson, Hikmat Al-Hashimi, Phaedra Ghazi, Yu Zhan, Bryan D. Smith, Conan G. Kinsey, Daniel L. Flynn. DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with the KRASG12C inhibitor sotorasib resulting in tumor regression in KRAS mutant NSCLC xenograft models [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 3600.

NTRK1 gene fusions are actionable drivers of numerous human malignancies. Here, we show that expression of the TPR-NTRK1 fusion kinase in immortalized mouse pancreatic ductal epithelial (IMPE) (pancreas) or mouse lung epithelial (MLE-12) cells is sufficient to promote rapidly growing tumors in mice. Both tumor models are exquisitely sensitive to targeted inhibition with entrectinib, a tropomyosin-related kinase A (TRKA) inhibitor. Initial regression of NTRK1-driven tumors is driven by induced expression of BIM, such that BIM silencing leads to a diminished response to entrectinib in vivo. However, the emergence of drug-resistant disease limits the long-term durability of responses. Based on the reactivation of RAF>MEK>ERK signaling observed in entrectinib-treated tumors, we show that the combination of entrectinib plus the MEK1/2 inhibitor cobimetinib dramatically forestalls the onset of drug resistance in vivo. Collectively, these data provide a mechanistic rationale for rapid clinical deployment of combined inhibition of TRKA plus MEK1/2 in NTRK1-driven cancers.

Abstract GNAQ and GNA11 (GNAQ/11) mutations are found in less than 2% of all melanoma, but more than 80% of uveal melanoma. Mutations in these Gα proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF->MEK1/2->ERK1/2) and YAP signaling. Metastatic uveal melanoma is refractory to all forms of pharmacologic treatment, such as FDA-approved targeted therapies inhibiting MEK1/2 (i.e. trametinib and binimetinib). We show that combining MEK1/2 inhibitors with 4-aminoquinoline antimalarials, chloroquine or hydroxychloroquine, resulted in synergistic and apoptosis-mediated cytotoxicity in GNAQ/11 mutant uveal melanoma cell lines. Interestingly, in contrast to our previous work in pancreatic and other RAS-driven cancers, the lysosomotropic role of chloroquine was not sufficient to promote cytotoxicity with MEK1/2 inhibitors, as neither lysosome inhibition with Bafilomycin A1 nor autophagy-specific and macropinocytosis-specific inhibition yielded enhanced cell death in combination with MEK1/2 inhibition. We then found that chloroquine prevented nuclear localization of the transcriptional coactivator, YAP, suggesting a novel mechanism of chloroquine. YAP inhibition combined with MEK1/2 inhibition enhanced cell death only in the presence of Bafilomycin A1. Gα-specific inhibition (inhibiting YAP and MAPK) combined with Bafilomycin A1 yielded similar results. This implies that the ability of chloroquine to inhibit both YAP signaling and lysosome function is required for promoting cell death in the presence of MEK1/2 inhibition. For in vivostudies, we utilized a hepatic colonization model using luciferized human metastatic uveal melanoma cell lines, OMM2.5 and OMM1. Daily treatment of trametinib with hydroxychloroquine in combination resulted in delayed tumor growth and increased overall survival compared to either treatment as monotherapy or chemotherapy. These findings were also recapitulated in an immunocompetent mouse model in which immortalized mouse melanocytes (Melan-A) with either a GNAQ or GNA11 activating mutation were implanted into syngeneic C57BL/6 mice. Our findings identify a novel mechanism of chloroquine and suggest a potentially effective strategy combining two FDA-approved drugs for the treatment of metastatic uveal melanoma. Citation Format: Amanda Truong, Michael Scherzer, Conan Kinsey, John Michael Sanchez, Jae Hyuk Yoo, Jackson Richards, Donghan Shin, Phaedra Ghazi, Michael Onken, Kendall Blumer, Shannon Odelberg, Martin McMahon. Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma [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 1890.

AbstractPurpose: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAPK and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of patients with metastatic uveal melanoma. Experimental Design: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAPK pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity. Results: Inhibition of GNAQ/11-mediated activation of MAPK signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death. Conclusions: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.