Synergistic apoptotic effect by the combination of clk inhibitor CTX-712 and BCL-2 inhibitor ABT-199 with or without azacytidine in AML and MDS

Abstract Alternative splicing (AS) plays a critical role in myeloid malignancy progression through abmormal expression or mutation of splicing factors. Splicing factors mutations, including SF3B1, SRSF2, U2AF1 and ZRSR, are among the most frequest alterations in myelodysplastic symdromes (MDS) and a subset of acute myeloid leukemia (AML). Serine/arginine (SR) domains are rich in many splicing factors, including SRSF2, U2AF1 and ZRSR. CDC2-like kinases (CLKs) are key regulators of AS through phosphorylation of SR-rich splicing factors (SRSFs). Therefore, CLK inhibitors might have a potential role in MDS and AML therapy by targeting SRSFs function. The CLKs family consists of four proteins (CLK1-4). We first investigated the roles of CLKs in MDS and AML. By RNA sequencing analysis in CD34+ bone marrow stem and progenitor cells from 69 MDS and 31 CMML patients without prior treatment, significantly increased (p0.05) CLK4 expression was observed in MDS patients compared with 20 healthy donors. No methylation was detected in CLKs promotor regions in 32 MDS and CMML patients by using reduced representation bisulfite sequencing analysis. CLK family mutations was rare in myeloid hematological malignancies (Cancer Genome Atlas (TCGA) database). By western blot analysis, we detected protein expression of all four CLKs in leukemia cell lines MOLM13 and THP1. CTX-712 is a potent pan-CLK inhibitor. To investigate the therapeutic potential of CTX-712 in MDS and AML, 4 AML cell lines (U937, THP1, MOLM13 as well as MOLM13-AZA (azacytidine resistant derivatives), 3 MDS patients and 2 healthy donors were studied. Flow cytometry analysis was performed to detect cell proliferation and apoptosis. Western blot analysis was used to study the SR protein phosphorylation. CTX-712 inhibited the proliferation of all AML cell lines studied with 72 hours of treatment. The IC-50 for U937, THP1, MOLM13 as well as MOLM13-AZA were 71.3±12.5 nM, 147±16.1 nM, 35.5±2.3 nM and 34.3±4.5 nM separately. CTX-712 induced apoptosis was observed in all these cell lines with a dose dependent manner. No different effect was observed between MOLM13 and MOLM13-AZA. In MOLM13 cells, decreased phosphorylation of SRSF2, SRSF4 and SRSF6 was observed in a dose dependent manner at 6 hours of CTX-712 treatment. For the 3 MDS patients, primary CD34+ cell population was selected using CD34 Microbead kit UltraPure (Miltenyi Biotec). The cells were cultured in expansion medium for 7 days, differential medium for 4 days, then CTX-712 was added to treat for 72 hours. The IC-50 for the 3 patients were: pt1 (CREBBP, SF3B1, SMC3) 26.8±15.21 nM; pt2 (TERT, IKZF1) 119.9±16.5 nM; pt3 (ASXL1, KRAS, SRSF2, TET2, STAG2) 45±8.5 nM. Apoptosis induction effect was observed in all three patients with a dose dependent manner. We further did the same test in 2 healthy donor controls, the IC-50s were 89.5±13.6 nM and 37.25±6.7 nM separately. Finally, we studied the apoptosis induction effect of CTX-712 in combination with BCL-2 inhibitor ABT-199 in MOLM13 and THP1 cell lines with 72 hours of treatment. Synergistic apoptotic induction effect was observed in both cell lines. For MOLM13, 40% of apoptosis was induced with 60nM of CTX-712 treatment, 41% and 56% of apoptosis with 3nM and 10nM of ABT-199 treatment alone. 86% and 94% of apoptosis were observed for the combination of 60nM CTX-712 with 3nM or 10nM of ABT-199 treatment separately. THP1 was resistant to both CTX-712 and ABT-199 compared with MOLM13. For THP1, 30% of apoptosis was induced with 300nM CTX-712 treatment, 26% and 50% of apoptosis with 1uM and 3uM of ABT-199 treatment alone. 86% and 93% of apoptosis were observed for the combination of 300nM CTX-712 with 1uM or 3uM of ABT-199 treatment separately. Synergistic apoptosis induction effect was also observed using CTX-712 combined azacytidine with or without ABT-199 in MOLM13 cell line with 72 hours of treatment. 48% of apoptosis was induced with 60nM CTX-712 treatment, 27% with 300nM of azacitidine, 31% with 3nM of ABT-199 treatment. 80% of apoptosis was observed for the combination of 60nM CTX-712 and 300nM azacitidine treatment. 96% of apoptosis was observed for the combination of all the three drugs. Taken together, our results suggested that the CLK inhibitor is a noval therapeutic approach for the treatment of MDS and AML; the combination of CTX-712 with ABT-199 and /or azacytidine could have significant antileukemia activity.