Over the last several years there has been a remarkable convergence of research related to oncogenic aberrations of the spliceosome, which has linked the identification of recurrent spliceosome mutations in tumors to medicinal chemistry. We designed synthetic natural product analogs (the sudemycins) that modulate the activity of the spliceosome and show potent and selective antitumor activity. We hypothesized that splicing gene mutations cause alterations in splice isoforms, which provide a selective advantage to tumor cells, and that perturbation of this environment with spliceosome modulators may cause a selective disadvantage for tumors. To test this hypothesis, we generated MSCV-based retroviruses encoding the wildtype or S34F mutant U2AF1 alleles. We infected c-kit+ primary C57BL/6J murine bone marrow cells with these recombinant retroviruses and cultured them in vitro with cytokine-supplemented media. Vehicle-treated cells expressing the mutant allele had reduced growth over 5 days, compared to cells expressing the wildtype allele or empty vector (P<0.0001, n=5 technical replicates, two experiments). At doses of 10-150 nM, sudemycin D1 (but not the inactive analog sudemycin E-OH) induced a reproducible dose-dependent decrease in cell growth and increase in apoptosis, as measured by Annexin V, in cells expressing the mutant, compared to the wildtype allele or empty vector. To study the effect of the U2AF1 mutation and sudemycin exposure on splicing, we analyzed RNA isoforms of Fmr1, a gene known to undergo alternative splicing. After retroviral transduction, GFP+ cells were sorted and RNA was isolated for RT-PCR analysis at baseline or 2h-6h after exposure to sudemycin D1 (75 nM) or vehicle. The U2AF1 S34F mutation increased the abundance of Fmr1 transcripts utilizing a cryptic splice acceptor, compared to cells transduced with empty vector or wildtype U2AF1 (P<0.05, n=3 technical replicates). These experiments provide proof-of-concept that cells expressing mutant U2AF1 have increased sensitivity to spliceosome modulators. The sudemycins show a short half-life (<15 min) when dosed IV via a bolus tail vein injection. So we have developed a jugular continuous infusion model to achieve therapeutic drug levels, and found that sudemycin D6 is tolerated at doses up to 200 mg/kg/h in mice. Additionally we have investigated numerous drug combinations and found significant cytotoxic co-treatment synergism with the experimental dual mTOR and PI3K inhibitor BEZ235, which suggests opportunities for multi-agent animal studies as a prelude to the design of human clinical trials. In this presentation we will also report in vitro and in vivo data on new sudemycin analogs along with additional work on the pharmacology, pharmacokinetics, toxicity and in vivo efficacy. Our results indicate a maturing therapeutic potential for sudemycin D6, which is in preclinical development for the treatment of subsets of MDS, melanoma and numerous other human cancers.
Citation Format: Timothy A. Graubert, Sharyn Baker, Philip M. Potter, Manorama Tripathi, Matthew Walter, Gustavo Palacios, Thomas R. Webb. Spliceosome-targeted antitumor drug discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1125. doi:10.1158/1538-7445.AM2013-1125