Non-oncogene addiction in cancer cells

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BACKGROUND. We define NOA as increased dependency of cancer cells on stress-response pathways for survival. The malignant state of the cancer cell is associated with a unique set of oncogenic stress phenotypes involving DNA damage, metabolic stress, proteotoxic stress, inflammatory microenvironment, and immune surveillance. Oncogenic stress renders cancer cells more dependent on stress-response pathways for survival and consequently, more sensitive to the disruption of these stress-response pathways. In contrast, normal cells in the body do no experience oncogenic stress, therefore normal cells are much less sensitive to the perturbation of stress-response pathways. This fundamental difference in cancer vs. normal cell's dependency on stress-response pathways forms the theoretical basis of NOA. NOA is distinct from oncogene dependency because stress response pathway genes are rarely mutated in the cancer genome. NOA represents a broad category of synthetic lethal and collateral dependency mechanisms in cancer cells, and targeting NOA could offer orthogonal therapeutic approaches to current targeted treatment of tumors. OBJECTIVES: The objectives of this project are 1.) Identify NOA genes and genetic pathways in cancer cells using genetic screen and hypothesis-based approaches; 2) Investigate the molecular mechanism that underly a NOA phenomenon and understand its essentiality to the oncogenic state; and 3) Explore the therapeutic implication of NOA as potential cancer drug targets. We will carry out these objectives primarily using cancer cells harboring the KRAS oncogene as our model system as KRAS mutant tumors have generally lacked effective therapies. MAJOR ACTIVITIES, SIGNIFICANT RESULTS AND KEY OUTCOMES. 1.) NOA of KRAS mutant cells to the RNA splicing factor ERH. Through a synthetic lethal RNAi screen in KRAS mutant cells, we have identified the ERH gene as a synthetic lethal partner in KRAS mutant cells. ERH is an evolutionarily conserved protein with poorly understood function. Using mass-spectrometry, we identified ERH associates with the spliceosome protein SNRPD3 and is therefore a component of the RNA splicing machinery. We showed that ERH is required for the proper splicing and expression of a subset of mitotic genes including CENPE that are critical for maintaining chromosomal stability in KRAS mutant cells. This work defined a new function for the ERH protein uncovered a previously unknown NOA in KRAS mutant cells to the RNA splicing machinery. This work suggests that selective perturbation of RNA splicing to disrupt the balance of mitotic proteins could be a potential approach to target KRAS mutant cancer cells. This work has been completed and published. 2.) NOA of KRAS mutant cells to the SUMO pathway for transformed growth. Through a synthetic lethal RNAi screen in KRAS mutant cells, we have identified the SUMO pathway, particularly the SUMO E2 ligase UBE2, to play an important role in supporting the viability and transformation growth of KRAS mutant cancer cells. Using mass-spectrometry, we identified multiple proteins whose SUMOylation are disrupted in a KRAS-dependent manner. Using gene rescue approaches, we showed that several SUMO target proteins, including KAP1, CHD1 and EIF3L, are critical for the viability of KRAS mutant cells under anchorage independent conditions. This work identifies a new role of the SUMO pathway in KRAS-driven oncogenesis and suggests the SUMO pathway as a potential drug target for KRAS mutant tumors. This work has been published. We are currently exploring the effect of small-molecule SUMO inhibitors in preclinical models of KRAS mutant cancer. 3) NOA of KRAS mutant cells to autophagy. Through a combinatorial RNAi analysis of the gene dependency landscape in KRAS mutant cells, we identified critical OA and NOA that components in the Ras signaling network and in stress-response pathways, respectively. We found CRAF/RAF1 as the major onco-effector of mutant KRAS and the autophagy E1 ligase ATG7 as a major NOA. This work identifies the autophagy pathway as a co-target with CRAF as a rational combination for KRAS mutant cells. This work has been published. We are currently investigating the mechanism by which autophagy contributes to the growth and survival of KRAS mutant tumors.