MAP kinase and autophagy pathways cooperate to maintain RAS mutant cancer cell survival

Significance Currently, there is no effective targeted therapy for oncogenic KRAS-driven cancer. We set out to identify RAS effector and stress response genes that critically support KRAS addiction, and therefore could serve as potential targets for KRAS mutant cancer. Using a combinatorial siRNA platform, we systematically interrogated the patterns of oncoeffector dependency in KRAS mutant and WT colorectal and pancreatic cancer cell lines and in normal cell lines. We found that RAF kinases are the major KRAS oncoeffectors and that cotargeting BRAF and CRAF kinases together with the autophagy E1 ligase ATG7 could efficiently eliminate KRAS mutant cells while minimizing toxicity in normal cells. Our work thus establishes a framework for the rational selection of target combinations for cancer treatment. Oncogenic mutations in the small GTPase KRAS are frequently found in human cancers, and, currently, there are no effective targeted therapies for these tumors. Using a combinatorial siRNA approach, we analyzed a panel of KRAS mutant colorectal and pancreatic cancer cell lines for their dependency on 28 gene nodes that represent canonical RAS effector pathways and selected stress response pathways. We found that RAF node knockdown best differentiated KRAS mutant and KRAS WT cancer cells, suggesting RAF kinases are key oncoeffectors for KRAS addiction. By analyzing all 376 pairwise combination of these gene nodes, we found that cotargeting the RAF, RAC, and autophagy pathways can improve the capture of KRAS dependency better than targeting RAF alone. In particular, codepletion of the oncoeffector kinases BRAF and CRAF, together with the autophagy E1 ligase ATG7, gives the best therapeutic window between KRAS mutant cells and normal, untransformed cells. Distinct patterns of RAS effector dependency were observed across KRAS mutant cell lines, indicative of heterogeneous utilization of effector and stress response pathways in supporting KRAS addiction. Our findings revealed previously unappreciated complexity in the signaling network downstream of the KRAS oncogene and suggest rational target combinations for more effective therapeutic intervention.

[1]  Joshua M. Korn,et al.  Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF. , 2018, Cancer research.

[2]  R. Stephens,et al.  Differential Effector Engagement by Oncogenic KRAS. , 2018, Cell reports.

[3]  M. Barbacid,et al.  c-RAF Ablation Induces Regression of Advanced Kras/Trp53 Mutant Lung Adenocarcinomas by a Mechanism Independent of MAPK Signaling. , 2018, Cancer cell.

[4]  Pooja Shah,et al.  Probing the roles of SUMOylation in cancer cell biology by using a selective SAE inhibitor. , 2017, Nature chemical biology.

[5]  J. Tabernero,et al.  A Multi-Arm Phase I Study of the PI3K/mTOR Inhibitors PF-04691502 and Gedatolisib (PF-05212384) plus Irinotecan or the MEK Inhibitor PD-0325901 in Advanced Cancer , 2017, Targeted Oncology.

[6]  Antoine de Weck,et al.  Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening , 2017, Cell.

[7]  Phillip G. Montgomery,et al.  Defining a Cancer Dependency Map , 2017, Cell.

[8]  S. Elledge,et al.  A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells. , 2017, Cell reports.

[9]  M. P. Dillon,et al.  Design and Discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers. , 2017, Journal of medicinal chemistry.

[10]  L. Larue,et al.  RAF proteins exert both specific and compensatory functions during tumour progression of NRAS-driven melanoma , 2017, Nature Communications.

[11]  P. Jänne,et al.  Selumetinib Plus Docetaxel Compared With Docetaxel Alone and Progression-Free Survival in Patients With KRAS-Mutant Advanced Non–Small Cell Lung Cancer: The SELECT-1 Randomized Clinical Trial , 2017, JAMA.

[12]  E. White,et al.  Autophagy and Tumor Metabolism. , 2017, Cell metabolism.

[13]  Eric S. Lander,et al.  Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras , 2017, Cell.

[14]  K. Westover,et al.  Rationale for RAS mutation-tailored therapies. , 2017, Future oncology.

[15]  Ji Luo,et al.  One-step immortalization of primary human airway epithelial cells capable of oncogenic transformation , 2016, Cell & Bioscience.

[16]  Chang S. Chan,et al.  Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells , 2016, Genes & development.

[17]  J. Grilley-Olson,et al.  A phase Ib dose-escalation study of the MEK inhibitor trametinib in combination with the PI3K/mTOR inhibitor GSK2126458 in patients with advanced solid tumors , 2016, Investigational New Drugs.

[18]  Yi Liu,et al.  Selective Inhibition of Oncogenic KRAS Output with Small Molecules Targeting the Inactive State. , 2016, Cancer discovery.

[19]  Neal Rosen,et al.  Allele-specific inhibitors inactivate mutant KRAS G12C by a trapping mechanism , 2016, Science.

[20]  Gregory McAllister,et al.  Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy , 2015, Proceedings of the National Academy of Sciences.

[21]  D. Planchard,et al.  A randomized phase II study of the MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer (NSCLC)†. , 2015, Annals of oncology : official journal of the European Society for Medical Oncology.

[22]  E. White,et al.  Atg7 Overcomes Senescence and Promotes Growth of BrafV600E-Driven Melanoma. , 2015, Cancer discovery.

[23]  L. Rubinstein,et al.  Biomarker-driven phase 2 study of MK-2206 and selumetinib (AZD6244, ARRY-142886) in patients with colorectal cancer , 2015, Investigational New Drugs.

[24]  E. Van Cutsem,et al.  A Phase Ib Dose-Escalation Study of the Oral Pan-PI3K Inhibitor Buparlisib (BKM120) in Combination with the Oral MEK1/2 Inhibitor Trametinib (GSK1120212) in Patients with Selected Advanced Solid Tumors , 2014, Clinical Cancer Research.

[25]  M. Loda,et al.  Combined MEK and PI3K Inhibition in a Mouse Model of Pancreatic Cancer , 2014, Clinical Cancer Research.

[26]  S. Fesik,et al.  Drugging the undruggable RAS: Mission Possible? , 2014, Nature Reviews Drug Discovery.

[27]  O. Maertens,et al.  Defining key signaling nodes and therapeutic biomarkers in NF1-mutant cancers. , 2014, Cancer discovery.

[28]  Ji Luo,et al.  Development of siRNA payloads to target KRAS-mutant cancer. , 2014, Cancer discovery.

[29]  Gerald C. Chu,et al.  Autophagy is critical for pancreatic tumor growth and progression in tumors with p53 alterations. , 2014, Cancer discovery.

[30]  Chang S. Chan,et al.  Autophagy is required for glucose homeostasis and lung tumor maintenance. , 2014, Cancer discovery.

[31]  N. Caplen,et al.  Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening , 2014, Breast Cancer Research.

[32]  D. Esposito,et al.  Dragging ras back in the ring. , 2014, Cancer cell.

[33]  Michael Peyton,et al.  Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non-Small-Cell Lung Cancer , 2013, Cell.

[34]  E. White,et al.  Autophagy is required for mitochondrial function, lipid metabolism, growth, and fate of KRASG12D-driven lung tumors , 2013, Autophagy.

[35]  D. Morrison,et al.  Effects of Raf dimerization and its inhibition on normal and disease-associated Raf signaling. , 2013, Molecular cell.

[36]  Travis J Cohoon,et al.  Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models. , 2013, Cancer cell.

[37]  Eugen C. Buehler,et al.  C911: A Bench-Level Control for Sequence Specific siRNA Off-Target Effects , 2012, PloS one.

[38]  Michael J. Emanuele,et al.  Evolutionarily conserved protein ERH controls CENP-E mRNA splicing and is required for the survival of KRAS mutant cancer cells , 2012, Proceedings of the National Academy of Sciences.

[39]  G. Stamp,et al.  Genetic Deletion of RALA and RALB Small GTPases Reveals Redundant Functions in Development and Tumorigenesis , 2012, Current Biology.

[40]  A. Tolcher,et al.  The Clinical Effect of the Dual-Targeting Strategy Involving PI3K/AKT/mTOR and RAS/MEK/ERK Pathways in Patients with Advanced Cancer , 2012, Clinical Cancer Research.

[41]  D. Bar-Sagi,et al.  RAS oncogenes: weaving a tumorigenic web , 2011, Nature Reviews Cancer.

[42]  P. Mazur,et al.  Early requirement of Rac1 in a mouse model of pancreatic cancer. , 2011, Gastroenterology.

[43]  D. Tuveson,et al.  C-Raf is required for the initiation of lung cancer by K-Ras(G12D). , 2011, Cancer discovery.

[44]  M. Barbacid,et al.  c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma. , 2011, Cancer cell.

[45]  S. Elledge,et al.  Functional identification of optimized RNAi triggers using a massively parallel sensor assay. , 2011, Molecular cell.

[46]  H. Coller,et al.  Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. , 2011, Genes & development.

[47]  M. Barbacid,et al.  Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival , 2010, The EMBO journal.

[48]  W. Sellers,et al.  MEK1 mutations confer resistance to MEK and B-RAF inhibition , 2009, Proceedings of the National Academy of Sciences.

[49]  K. Kinzler,et al.  Glucose Deprivation Contributes to the Development of KRAS Pathway Mutations in Tumor Cells , 2009, Science.

[50]  Ben S. Wittner,et al.  Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1 , 2009, Nature.

[51]  Michael J. Emanuele,et al.  A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene , 2009, Cell.

[52]  Amanda Doucette,et al.  An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer , 2009, Nature.

[53]  Ji Luo,et al.  Principles of Cancer Therapy: Oncogene and Non-oncogene Addiction , 2009, Cell.

[54]  Ralph Weissleder,et al.  Effective Use of PI3K and MEK Inhibitors to Treat Mutant K-Ras G12D and PIK3CA H1047R Murine Lung Cancers , 2008, Nature Medicine.

[55]  C. Der.  Faculty Opinions recommendation of Differential effects of oncogenic K-Ras and N-Ras on proliferation, differentiation and tumor progression in the colon. , 2008 .

[56]  A. Sweet-Cordero,et al.  Requirement for Rac1 in a K-ras induced lung cancer in the mouse. , 2007, Cancer research.

[57]  P. Khavari,et al.  Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia. , 2007, Developmental cell.

[58]  S. Fesik,et al.  Survivin depletion preferentially reduces the survival of activated K-Ras-transformed cells , 2007, Molecular Cancer Therapeutics.

[59]  C. Sousa.  Faculty Opinions recommendation of RalB GTPase-mediated activation of the IkappaB family kinase TBK1 couples innate immune signaling to tumor cell survival. , 2006 .

[60]  M. White,et al.  RalB GTPase-Mediated Activation of the IκB Family Kinase TBK1 Couples Innate Immune Signaling to Tumor Cell Survival , 2006, Cell.

[61]  M. Baccarini,et al.  Raf-1 sets the threshold of Fas sensitivity by modulating Rok-α signaling , 2005, The Journal of cell biology.

[62]  F. Watt,et al.  Stem Cell Depletion Through Epidermal Deletion of Rac1 , 2005, Science.

[63]  Sheila M. Thomas,et al.  Role of Phosphoinositide 3-Kinase Regulatory Isoforms in Development and Actin Rearrangement , 2005, Molecular and Cellular Biology.

[64]  Walter Kolch,et al.  Role of the Kinase MST2 in Suppression of Apoptosis by the Proto-Oncogene Product Raf-1 , 2004, Science.

[65]  J. Cedarbaum.  Survival , 2004 .

[66]  Jing Chen,et al.  Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[67]  C. Pritchard,et al.  MEK kinase activity is not necessary for Raf‐1 function , 2001, The EMBO journal.

[68]  U. Rapp,et al.  Overlapping and specific functions of Braf and Craf-1 proto-oncogenes during mouse embryogenesis , 2000, Mechanisms of Development.

[69]  R. Herrera,et al.  A role for the MEK/MAPK pathway in PMA-induced cell cycle arrest: modulation of megakaryocytic differentiation of K562 cells. , 1998, Experimental cell research.

[70]  K. Pumiglia,et al.  Cell cycle arrest mediated by the MEK/mitogen-activated protein kinase pathway. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[71]  J. Stone,et al.  RAS signalling is abnormal in a c-raf1 MEK1 double mutant , 1995, Molecular and cellular biology.

[72]  R. Seger,et al.  The Nuclear Translocation of ERK. , 2017, Methods in molecular biology.

[73]  Taebo Sim,et al.  Covalent Guanosine Mimetic Inhibitors of G12C KRAS. , 2017, ACS medicinal chemistry letters.

[74]  A. Tolcher,et al.  Phase I study of the MEK inhibitor trametinib in combination with the AKT inhibitor afuresertib in patients with solid tumors and multiple myeloma , 2014, Cancer Chemotherapy and Pharmacology.

[75]  S. Elledge,et al.  Principles of Cancer Therapy: Oncogene and Non-oncogene Addiction , 2009, Cell.