Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9

Abstract

The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.he Ras family of small GTPases are signal transduction molecules downstream of growth factor receptors. Ras activates a number of downstream effector pathways to regulate cell proliferation, survival and motility, these effectors include the MAP kinase (MAPK) pathway, the PI3-kinase (PI3K) pathway, the small GTPases RalA, RalB, and Rho, and phospholipase-Ce (1). Activating mutations in Ras are frequently found in human malignancies, with mutations in the KRAS gene being particularly prevalent. KRAS mutations occur in ∼60% of pancreatic ductal carcinomas, 26% of lung adenocarcinomas, and 45% of colorectal carcinomas, as well as a significant fraction of ovarian, endometrial, and biliary track cancers (2, 3). A salient hallmark of the Ras oncogene is its ability to transform cells to enable anchorage-independent 3D colony growth in vitro and tumor growth in vivo. Consequently, Ras mutant cancer cells often exhibit oncogene addiction to Ras such that extinction of the Ras oncogene leads to either a reversion of the transformed phenotype or loss of viability (4, 5). Therapeutically, the Ras oncoprotein has proven pharmacologically intractable thus far: intensive drug screening efforts have not yielded high-affinity, selective Ras inhibitors. Farnesyltransferase inhibitors that aimed to block Ras membrane localization are ineffective against KRAS because of its alternative geranylgeranylation. Inhibitors targeting Ras effector kinases, including MEK, PI3K, and Akt, are currently undergoing clinical evaluations, but they have yet to demonstrate clear clinical benefits (6). Thus, KRAS mutant tumors represent a class of "recalcitrant cancer" with urgent, unmet therapeutic needs.To gain new insight into the genetic dependencies of Ras mutant cancers and discover new therapeutic targets, we and others have previously carried out genome-wide synthetic lethal screens in KRAS mutant and WT cells to identify genes whose depletion leads to greater toxicity in KRAS mutant cells. In our screen we found a wide array of genes, many of which are involved in cellular stress response, that are required to maintain the viability of KRAS mutant cells (7). We proposed the concept of "nononcogene addiction" to explain the heig...

Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9

“…Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme.A nX -rayc rystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at as ite distal to its catalytic cysteine.T hese fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm.Mechanistic and biophysical analyses,c oupled with molecular dynamics simulations,p oint toward ligand-induced rigidification of Ubc9 as amechanism of inhibition.The posttranslational modification of protein substrates with as mall ubiquitin-like modifier (SUMO) tag occurs through at ightly regulated E1/E2/E3 enzymatic cascade and modulates ab road variety of cellular functions.[1] Ubc9 is the only E2 enzyme involved in the conjugation of all three SUMO isoforms to many diverse substrates within the proteome.As acentral enzyme in the SUMOylation cycle,Ubc9 is required for normal embryonic development, and it is dysregulated in av ariety of disease states such as cancer [2] and ischemia.[3]Ubc9 has been suggested as ap otential anticancer target for small-molecule inhibitors,n otably for MYC-driven [4] and RAS/Raf-driven [5] cancers,a sw ell as multiple myeloma.[6]Despite its relevance to disease,i th as been challenging to identify small molecules that modulate the function of Ubc9.In related ubiquitin-like signaling pathways,inhibitors of E1 [7] and E3 [8] enzymes are now clinically used as anticancer chemotherapeutics.Bycontrast, very few reversible inhibitors of any of the approximately 40 known ubiquitin and ubiquitin-like E2 conjugating enzymes have been discovered, [9] only one of which has been characterized in complex with the protein.[10] Thus,n ew experimentally validated chemical inhibitors of Ubc9 would provide substantial insight into targeting this important enzyme,a nd potentially E2 enzymes in general.One powerful approach to identify ligands for challenging protein targets is fragment-based inhibitor discovery.[11] In general, fragment-based approaches leverage sensitive techniques such as NMR, thermal shift, surface plasmon resonance (SPR), or X-ray crystallography to identify lowmolecular-weight ligands that bind weakly but specifically to target proteins.[12] Weak leads identified through fragmentbased approaches can provide excellent starting points for the development of highly active inhibitors,e ven in cases where the targets are considered to be challenging,such as proteinprotein interactions or so-called "undruggable" targets. [13] As part of al arger program aimed toward identifying chemical inhibitors of Ubc9, we elected to pursue an X-ray crystallographic fragment screening strategy (see the Supporting Information).…”

Streptonigrin Inhibits SENP1 and Reduces the Protein Level of Hypoxia-Inducible Factor 1α (HIF1α) in Cells

“…25 Because angiogenesis plays a critical role in the growth of solid tumors, it is a target for developing anti-cancer therapies. Thus, our identification of the SENP1- mediated effects of SN on both HIF1α stability and SUMOylation suggest a potential for using SN against cancer types that respond to angiogenesis inhibitors or are dependent on SUMOylation/de-SUMOylation cycles, 26–28 including cancer stem cells. 29, 30 The findings provided here, along with progress in the total synthesis of SN, 11–13 also provide a basis for further modification of SN to improve its efficacy and reduce toxicity for future clinical use.…”

Targeting nuclear thymidylate biosynthesis

“…Reduced Ubc9 expression suppresses growth of KRAS mutant colorectal cancer cells 117 , and Spectinomycin B1, an inhibitor of Ubc9, inhibits proliferation of MCF-7 breast cancer cells 118 . Reduced expression of the SUMO-activating enzyme subunit 2 (SAE2) and Ubc9 impair cell proliferation in cancer cells as does reduced expression of SAE2 alone in U2OS osteosarcoma cells.…”