Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite tremendous progress in its molecular characterization. Indeed, PDA tumors harbor four signature somatic mutations1–4, and a plethora of lower frequency genetic events of uncertain significance5. Here, we used Sleeping Beauty (SB) transposon-mediated insertional mutagenesis6,7 in a mouse model of pancreatic ductal preneoplasia8 to identify genes that cooperate with oncogenic KrasG12D to accelerate tumorigenesis and promote progression. Our screen revealed new candidates and confirmed the importance of many genes and pathways previously implicated in human PDA. Interestingly, the most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumors. Although prior work had attributed a pro-survival role to USP9X in human neoplasia9, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and mRNA expression in PDA correlates with poor survival following surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2′-deoxycytidine elevates USP9X expression in human PDA cell lines to suggest a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with KrasG12D to rapidly accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. Therefore, we propose USP9X as a major new tumor suppressor gene with prognostic and therapeutic relevance in PDA.

Purpose Genetic alterations of KRAS, CDKN2A, TP53 and SMAD4 are the most frequent events in pancreatic cancer. We determined the extent to which these four alterations are coexistent in the same carcinoma, and their impact on patient outcome. Experimental Design Pancreatic cancer patients who underwent an autopsy were studied (n=79). Matched primary and metastasis tissues were evaluated for intragenic mutations in KRAS, CDKN2A and TP53 and immunolabeled for CDKN2A, TP53 and SMAD4 protein products. The number of altered driver genes in each carcinoma was correlated to clinicopathologic features. Kaplan-Meier estimates were used to determine median disease free and overall survival, and a Cox proportional hazards model used to compare risk factors. Results The number of genetically altered driver genes in a carcinoma was variable, with only 29 patients (37%) having an alteration in all four genes analyzed. The number of altered driver genes was significantly correlated with disease free survival (p=0.008), overall survival (p=0.041) and metastatic burden at autopsy (p=0.002). On multivariate analysis, the number of driver gene alterations in a pancreatic carcinoma remained independently associated with overall survival (p=0.046). Carcinomas with only one to two driver alterations were enriched for those patients with the longest survival (median 23 months, range 1–53). Conclusions Determinations of the status of the four major driver genes in pancreatic cancer, and specifically the extent to which they are coexistent in an individual patients cancer, provides distinct information regarding disease progression and survival that is independent of clinical stage and treatment status.

Purpose Pancreatic cancer (PC) is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for PC might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs. Experimental Design Using a panel of genetically defined human PC cell lines, we tested gemcitabine (anti-metabolite), docetaxel (anti-microtubule), mitomycin C (alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug) and artemisinin (control). Results All PC cell lines were sensitive to triptolide and docetaxel. Most PC cells were also sensitive to gemcitabine and MMC. The vast majority of PC cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized PC cells to cisplatin and irinotecan by 2–4 fold, but they were modestly less sensitive to gemcitabine. PC cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A inactivated PC cells were 3–4 fold less sensitive to gemcitabine and MMC. Conclusions Chemosensitivity of PC cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of PC in the future. Further work will need to confirm these responses and determine their magnitude in vivo.