Activation of checkpoint arrest and homologous DNA repair are necessary for maintenance of genomic integrity during DNA replication. Germ-line mutations of the ataxia telangiectasia mutated (ATM) gene result in the well-characterized ataxia telangiectasia syndrome, which manifests with an increased cancer predisposition, including a 20% to 30% lifetime risk of lymphoid, gastric, breast, central nervous system, skin, and other cancers. Somatic ATM mutations or deletions are commonly found in lymphoid malignancies, as well as a variety of solid tumors. Such mutations may result in chemotherapy resistance and adverse prognosis, but may also be exploited by existing or emerging targeted therapies that produce synthetic lethal states.

Expression of cyclic adenosine monophosphate-specific phosphodiesterase 7B (PDE7B) mRNA is increased in patients with chronic lymphocytic leukemia (CLL), thus suggesting that variation may occur in the PDE7B gene in CLL. As genetic variation in other PDE family members has been shown to associate with numerous clinical disorders (reviewed in this manuscript), we sought to identify single-nucleotide polymorphisms (SNPs) in the PDE7B gene promoter and coding region of 93 control subjects and 154 CLL patients. We found that the PDE7B gene has a 5′ non-coding region SNP –347C>T that occurs with similar frequency in CLL patients (1.9%) and controls (2.7%). Tested in vitro, –347C>T has less promoter activity than a wild-type construct. The low frequency of this 5′ untranslated region variant indicates that it does not explain the higher PDE7B expression in patients with CLL but it has the potential to influence other settings that involve a role for PDE7B.

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

We have found that B‐cell chronic lymphocytic leukemia (CLL) have a several‐fold increase in the mRNA of phosphodiesterase 7B (PDE7B), which catalyzes cAMP hydroysis. In order to assess the molecular basis for this increase, we tested for the presence of single nucleotide polymorphisms (SNPs) in the promoter and coding regions of the gene (chromosome 6q23‐q24). We thus undertook SNP discovery and expression using DNA isolated from peripheral blood mononuclear cells of 100 control subjects and 151 CLL patients and analyzed differences by Chi square testing. We didn't identified any SNPs in coding region but one in non‐coding, −347G>A. This SNP had a similar frequency in CLL (3%) and controls (2%, P = 0.4669); however, it was found more frequently in males vs. females. A potential explanation for this gender difference is that position −347 is a putative binding site for SRY (sex‐determining region of the Yp11.3 chromosome), which encodes a transcriptional factor that initiates male sex determination and thus, may be related to the predisposition of males to CLL. Using a luciferase assay reporter, we found that −357G>A significantly lowered the promoter activity compared to wildtype (p<0.05). Thus, the presence of −357G>A SNP in the promoter region of PDE7B, although not associated with CLL, can alter promoter activity and thus may contribute to inter‐individual and gender differences in PDE7B expression and activity.