CDC2-like Kinases (CLKs) Inhibition As a Novel Targeted Therapeutic Strategy in Myelodysplastic Syndromes (MDS)

Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by bone marrow failure, dysplasia of myeloid blood cell lineages, and increased risk of developing acute myeloid leukemia (AML). The growth and spread of a somatically mutated clone represents the pathogenesis of MDS. Alternative splicing is a fundamental mechanism to generate protein diversity in human, splicing factors mutations, including SF3B1, SRSF2, U2AF1 and ZRSR, occur in more than 50% of the MDS patients, represent the most frequent somatic mutation and suitable therapeutic targets in MDS. Serine/arginine-rich (SR) domains are rich in many splicing factors, including SRSF2, U2AF1 and ZRSR. CDC2-like kinases (CLKs) are a group of protein kinases involved in regulating mRNA splicing through phosphorylating various SR proteins. Expression or activity changes of CLKs are associated with cancer development and progression. It was reported that depletion or inhibition of CLKs could change alternative splicing events through reduction the phosphorylation levels of SR proteins. The aim of this study is to test the role of CLKs in the pathogenesis of MDS, as well as the therapeutic potential of CLK inhibitor in MDS.

The CLKs family consists of four proteins (CLK1-4). By RNA sequencing, we first evaluated the mRNA expression levels of CLKs in CD34+ bone marrow stem and progenitor cells (BM-HSPCs) from 69 MDS patients, median age of 71 (42-87), and 31 CMML patients, median age of 73 (47-87) without prior treatment. Compared with 20 heathy donor controls, significantly increased (p<0.05) CLK4 expression was observed in MDS patients. CLK1- 4 genes are located in chromosome 2q33.1, 1q22, 15q24.1 and 5q35.3 separately. They all contains promoter CpG island. We evaluated the promoter methylation of CLK genes by using RRBS (reduced representation bisulfite sequencing) in 32 MDS and CMML patients, there was no methylation detected. We then investigated the CLKs 1-4 mutations using the Cancer Genome Atlas (TCGA) database. The CLKs family mutation was rare in myeloid hematological malignancies: only low impact CLK1 mutations were found in 2 cases, and 1 moderate impact CLK3 mutation in 1 case.

CLKs inhibitors have been developed and showed anti-tumor effect. We then further investigated the therapeutic potential of CLK inhibitor in MDS. CTX-712 is a potent pan-CLK inhibitor, it is highly selective against CLK1-4 through inhibits phosphorylation of SRSFs. 4 AML cell lines (U937, THP1, MOLM13 as well as MOLM13 hypomethylating agent (HMA)-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 (Chemietek, Indianapolis, IN) 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 hypomethylating agent (HMA)-resistant derivatives 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. No different effect was observed between MOLM13 and its HMA-resistant derivatives. In MOLM13 cells, decreased phosphorylation of SRSF2, SRSF4 and SRSF6 was observed in a dose dependent manner at 6 hours of CTX-712 treatment. We then studied the CTX-712 effect on 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 were added into differential medium 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. 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.

Our study suggests that CLKs may have roles in MDS pathogenesis, the inhibition of CLKs have therapeutic potential, more specific CLKs inhibitor development is needed.

Montalban-Bravo:Rigel: Research Funding; Takeda: Research Funding. Chien:AbbVie: Consultancy; Rigel Pharmaceuticals: Consultancy. Garcia-Manero:Astex: Research Funding; Bristol Myers Squibb: Other: Personal fees, Research Funding; Curis: Research Funding; Genentech: Research Funding; Aprea: Research Funding; Helsinn: Other: Personal fees; Astex: Other: Personal fees; Forty Seven: Research Funding; Amphivena: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Merck: Research Funding; Onconova: Research Funding; AbbVie: Research Funding; Helsinn: Research Funding; H3 Biomedicine: Research Funding; Genentech: Other: Personal fees.