Alternative splicing plays a fundamental role in human biology and its relevance in cancer is rapidly emerging. Recent advances in oligonucleotide sequencing demonstrate that alternative splicing and gene mutations involved in the spliceosome can play a role in tumorigenesis and cancer progression in haematologic malignancies, including chronic lymphocytic leukemia (CLL). However, there are few, if any, well-defined molecular probes that can be used to modulate splicing events in vitro or in vivo. As such, it is uncertain whether the spliceosome system can serve as a molecular target in cancer.
We have addressed these questions using FD-895 and Pladienolide B, two bioactive polyketides that target the splicing factor SF3B. Primary leukemia cells from CLL patients were incubated with these two agents and the samples were analyzed for an early evidence of mRNA intron retention (splicing inhibition) and apoptosis.
We observed that primary leukemia CLL cells incubated with FD-895 and Pladienolide B (10–1,000 nM) showed evidence of mRNA intron retention as early as 4 hours after incubation with increasing ratios of unspliced/spliced mRNA in several genes tested (DnaJ homolog subfamily B member 1, DNAJB1; RIO kinase 3, RIOK3; and X-box binding protein 1, XBP1). Contrary to that, neither Bendamustine (10–100 μM) nor Fludarabine (F-Ara-A, 10–100 μM) induced accumulation of unspliced mRNAs.
CLL cells incubated with FD-895 and Pladienolide B (10–1,000 nM) underwent apoptosis with evidence of changes in mitochondrial transmembrane potential detected by DiOC6. Apoptosis was observed as early as 24 hours after incubation and required 3 2 hours of incubation with FD-895 and Pladienolide B. The IC50 for both agents was in the 100 nM range. Importantly, FD-895 and Pladienolide B both were able to induce apoptosis in CLL cells that were protected from spontaneous apoptosis using co-culture conditions with stromal cell support and despite the presence of high-risk prognostic factors such as ZAP-70 expression, unmutated IGHV and Del(17p), or the presence of SF3B1 mutation.
In conclusion, we have shown for the first time that FD-895 and Pladienolide B induce early mRNA intron retention (splicing inhibition) in primary leukemia cells from patients with CLL and apoptosis at nanomolar concentrations in all patient samples tested (n=20). In vitro apoptosis was observed regardless of the presence of poor prognostic factors such as Del(17p) and SF3B1 mutations. In addition, the pro-apoptotic activity of FD-895 and Pladienolide B was present in CLL-stromal cell co-culture conditions that may resemble the protective tumor microenvironment. Our data presents evidence that the spliceosome system is a valid target in CLL and provides the rationale for the development of inhibitors that target this pathway.
Kipps:Abbott: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.