A Cyclopropane-Derived Stable Analog Of Fd-895 Induces Apoptosis and Inhibition Of mRNA Splicing In Lymphoma and Chronic Lymphocytic Leukemia: A Novel Therapeutic Approach

Abnormalities in RNA splicing have been implicated in tumorigenesis and cancer progression. Recent studies have defined RNA splicing as in attractive target for development of effective anticancer therapy. However, significant hurdles need to be solved before spliceosome inhibitors are ready for clinical trial applications. The work presented here addresses a major obstacle, specifically the design of active and metabolically stable spliceosome / RNA splicing inhibitors.

We previously reported that FD-895 and Pladienolide B (PLAD-B) induce apoptosis viathe inhibition of RNA splicing in chronic lymphocytic leukemia (Kashyap et al, Blood 2012; 120: 3890). However, FD-895 and PLAD-B hydrolyze relatively rapidly in aqueous solution. To address this issue, we applied semi-synthesis and total synthesis to reveal key structure activity relationships (SARs) regulating the RNA splicing inhibition of FD-895. This information was applied to synthesize a panel of cyclopropane analogs (CypB) that showed improved biochemical stability and potent pro-apoptotic activity in a panel of lymphoma cell lines and primary lymphoma - leukemia cells from patients with mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL).

A series of cyclopropane analogs and FD-895 were tested for hydrolysis and stability using a combination of NMR and mass spectral methods. Based on stability, we selected a first generation cyclopropane analog, CypB, to conduct our experiments. Primary leukemia - lymphoma cells as well as cell lines were incubated with increasing concentrations of FD-895, and CypB over a gradient of 10-1,000 nM. We observed rapid development of intron retention within 15 min after incubation at concentrations ranging from 10-100 nM using a series of spliceosome related genes (DNAJB1, RIOK3, and BRD2). Contrary to that, intron retention was not observed after treatment with chemotherapy controls (fludarabine and etoposide). By using qRT-PCR, we were able to quantify unspliced mRNA forms induced by FD-895, and CypB but not by chemotherapy agents.

In addition, we observed a close correlation between the level of intron retention and development of apoptosis induced by FD-895 and CypB in all cell lines and primary leukemia - lymphoma samples (IC₅₀ range: 3-1,000 nM). Apoptosis induced by these compounds was also observed in chemotherapy resistant patient samples independently of TP53 function, deletion or mutational status.

To understand the molecular mechanism of FD-895 and CypB induced apoptosis, we analyzed the expression profile of alternative spliced forms of genes related to apoptosis (mcl-1 and Bcl-X) by RT-PCR. Interestingly, we observed that FD-895, and CypB induced the expression of small alternative spliced forms that are known to be pro-apoptotic. This was not observed after incubation with control chemotherapy agents.

In conclusion, using a SAR strategy we were able to optimize novel FD-895 based cyclopropane analogs. These compounds induce early mRNA intron retention in different lymphoma cell lines and primary lymphoma - leukemia samples derived from patients. Furthermore, these analogs induced apoptosis in a TP53 independent manner and induce the expression of pro-apoptotic alternatively spliced RNA genes. Our studies showed that combining medicinal chemistry and cancer biology, we were able to discover labile functional epoxide groups present in FD-895 that can be removed enhancing its stability and preserving the pro-apoptotic activity of this compound. This synthesis and optimization strategy provides new avenues for analog design in an effort to develop spliceosome inhibitors that can be used for cancer treatment. Current efforts are underway to complete these translational studies.