Abstract 343: Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia

Abstract

Abstract Recently, deep sequencing technology identified SF3B1, a gene encoding a splicing factor, is recurrently mutated in chronic lymphocytic leukemia (CLL). Moreover, its mutation was associated with rapid disease progression and resistance to treatment. SF3B1 is a critical component of the spliceosome, leading to the potential of splicing regulation in CLL pathogenesis. However, the mechanism by which SF3B1 mutations affect CLL disease progression is not known. CLL cells are characterized by their resistance to apoptosis, driven mostly by the overexpression of the Bcl-2 family anti-apoptotic proteins. Many members of the family have multiple splicing variants, sometimes encoding proteins with totally opposite functions. For example, the alternatively spliced, shorter variant of the anti-apoptotic protein Mcl-1, Mcl-1S, resembles BH3-only proteins and induces apoptosis. Using herboxidiene, an inhibitor of SF3B1, we explored the activity of SF3B1 in splicing control of Mcl-1 in primary CLL cells. Primers and probes were designed to specifically detect the Mcl-1 pre-mRNA, the anti-apoptotic Mcl-1 and pro-apoptotic Mcl-1S by real-time RT-PCR. We found that inhibition of SF3B1 reduced the level of Mcl-1 in a concentration dependent manner, which was accompanied by the simultaneous surge of Mcl-1S mRNA by over 10 fold. Immunoblots confirmed reduction of Mcl-1L and concurrent increase of Mcl-1S protein. These results indicated a major role of SF3B1 in Mcl-1 splicing control toward the anti-apoptotic variant. In contrast, although also depleting the Mcl-1 mRNA, the Cdk9 inhibitor SNS-032 reduced both the long and short forms of Mcl-1, as well as its pre-mRNA, consistent with its inhibition on transcription. Uridine incorporation measurements showed inhibition of total RNA synthesis by herboxidiene at concentrations above 30 nM, but dramatic reversion of Mcl-1 splicing was observed at 10 nM, which did not affect RNA synthesis, suggesting that the major mechanism of herboxidiene in CLL cells was modification of Mcl-1 splicing. As CLL cells are critically dependent on Mcl-1 for survival, reducing its level induced mitochondrial membrane permeabilization and apoptosis, which was further accelerated by the accumulation of Mcl-1S. Apoptosis occurred in a time- and concentration- dependent manner, with IC50 of less than 100 nM after 24 hr of incubation. Herboxidiene toxicity was selective to CLL B cells, while sparing the normal lymphocytes from healthy donors. Herboxidiene was equally toxic to CLL cells harboring SF3B1 mutations compared to cells carrying the wild-type SF3B1 (n=5 each). Real-time RT-PCR showed similar increase in the ratio of short to long forms of Mcl-1 by herboxidiene in these two groups. Together, these studies identified SF3B1 mediated splicing control as a novel target for CLL therapy. Further investigations are needed to understand the mechanism of SF3B1 mutations on the control of mRNA splicing. Citation Format: Rong Chen, Yuling Chen, Emanuela M. Ghia, Laura Rassenti, Thomas Kipps, Michael J. Keating, William G. Wierda, William Plunkett. Targeting SF3B1 mediated splicing control of Mcl-1 in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2014-343