Abstract
Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML), designated as chromatin/spliceosome-mutated AML, where major SFs mutated include SF3B1, SRSF2, U2AF1, and ZRSR2. SF mutations are largely mutually exclusive and except for ZRSR2 mutations, heterozygous, suggesting cancer vulnerability to the excessive splicing abnormality caused by homozygous or multiple mutations. Thus, SF functions might be a plausible target of therapy for MDS/AML.
Of potential interest in this regard is serine/arginine-rich (SR) domains ubiquitously shared by many SFs, including U2AF1, SRSF2, and ZRSR2, which need to be phosphorylated for their nuclear translocation by evolutionally conserved kinases, known as the CLK family of proteins. CLK family kinases regulate mRNA splicing by phosphorylating various SR proteins, and inhibition of CLK family kinases result in reduction of phosphorylation levels of SR proteins, induction of splicing alterations, and protein depletion for multiple genes. In addition, a recent report showed that CLK inhibition can induce skipped exons, cell death, and cell growth suppression. Therefore, CLK inhibitors might have a role in the therapeutics of SF-mutated MDS/AML, by further compromising RNA splicing.
To show the proof-of-concept for this hypothesis, we have recently developed an orally available and highly potent CLK inhibitor, CTX-712, and evaluated its anti-leukemic activities both in vitro and in vivo. CTX-712 potently suppressed phosphorylation of major SR domain-containing SFs. When tested in human myeloid cell lines (K562 and MV-4-11), it showed a strong inhibitory effect on cell proliferation with IC50 of 0.15 and 0.036 μM, respectively. The anti-leukemic effect was also confirmed by survival assay using a total of 79 patient-derived primary AML cells (the average of IC50 was 0.078 μM). In addition, CTX-712 suppressed phosphorylation of multiple SR proteins including SRSF2/3/4/6. RNA-seq analysis revealed that CTX-712 induced global splicing changes, which typically resulted in exon exclusion (exon skipping) of cassette exon. When measured by the degree of spliced vs. un-spliced reads (percent spliced-in value) in cassette exon events, inhibition of RNA splicing induced by the drug was positively correlated with the sensitivity to the drug (IC50) in primary AML cells (N=32, R=0.54, P=0.0015), suggesting that the degree of splicing changes after drug treatment could be an indicator predicting the efficacy of CTX-712.
To further investigate the effect of CTX-712 on tumor growth in vivo, we established 13 subcutaneous models of MDS/AML-derived xenografts (PDX), which were treated with varying doses of CTX-712. Consistent with in vitro observations, PDX with larger splicing changes after CTX-712 treatment showed a stronger regression than those with smaller splicing changes. We observed the degree of splicing changes induced by CTX-712 tended to correlate with the drug sensitivity as measure by tumor growth inhibition (TGI) although it was not statistically significant (N=13, R=-0.47, P=0.1). In particular, CTX-712 induced the regression of PDX in a dose-dependent manner where the degree of tumor regression correlated with the size of splicing changes. Of note, the PDX with the largest splicing changes showed that 4 out of 5 mice treated using a high dose protocol (12.5 mg/kg) achieved complete remission (the tumor shrank completely to unmeasurable size). Two weeks after treatment, tumor volumes (mm3) were 762 ± 147 (vehicle), 331 ± 64 (low dose of CTX-712: 6.25mg/kg, P=0.028), and 39 ± 39 (high dose, P=0.0014) (N=5 each, mean ± SEM). Median survival time (days) was 34.5 (vehicle) and 93.5 (high dose) (N=2). Overall, 12 out of 13 PDX MDS/AML models showed anti-tumor effect of CTX-712 with a trend of better effects for larger splicing changes.
In conclusion, our results demonstrated a potent effect of CTX-712 on both RNA splicing and tumor regression for MDS/AML-derived cells, providing mechanistic insight into CLK inhibition and its effect on RNA splicing and anti-leukemic effects and also a rationale for further investigation of CTX-712 in MDS/AML.
Disclosures
Yoda:Chordia Therapeutics inc.: Research Funding. Morishita:Chordia Therapeutics lnc: Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties. Mizutani:Chordia Therapeutics inc.: Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties. Tozaki:Honda Motor Co., Ltd.: Current equity holder in publicly-traded company; Chordia Therapeutics inc.: Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties; Kyowa Kirin Co., Ltd.: Current Employment, Patents & Royalties: Japanese Patent Application No.2002-078819. Satoh:Chordia Therapeutics inc.: Current Employment, Current holder of stock options in a privately-held company. Nannya:Fuji Pharma: Honoraria; Pfizer: Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Speakers Bureau; Sumitomo Pharma: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical Company: Speakers Bureau; Filgen: Speakers Bureau; Kyowa-Kirin: Speakers Bureau; Asahi Kasei Pharma: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Janssen Pharmaceutical: Speakers Bureau; Otsuka Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Daiichi Sankyo RD Novare: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding. Miyake:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company Ltd.: Current equity holder in publicly-traded company, Patents & Royalties: Chordia has royalty bearing rights to execute Takeda's CLK inhibitor etc. patent., Research Funding. Ogawa:Astellas: Speakers Bureau; MSD: Speakers Bureau; Otsuka Pharmatheutical: Research Funding; Clinical Research Support Center Kyushu: Research Funding; Sysmex: Honoraria; Kirin/Chugai: Speakers Bureau; Nanpu Hospital: Research Funding; 62/187386 (US01): Patents & Royalties; The Mitsubishi foundation: Honoraria; The Chemo-Sero-Therapeutic Research Institute: Speakers Bureau; Astrazeneca: Speakers Bureau; DaiichiSankyo: Speakers Bureau; Pfaizer: Speakers Bureau; ASAHI Genomics: Current equity holder in publicly-traded company; 2014-191287: Patents & Royalties; Chordia Threapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding; Novartis: Honoraria, Speakers Bureau; Esai Pharmatheutical: Consultancy; 2013-526957 (JP02): Patents & Royalties; 2013-096582 (JP01): Patents & Royalties; 2015-239547: Patents & Royalties; PCT/JP2014/062112 (WO01): Patents & Royalties; 15/353395 (US03): Patents & Royalties.
Author notes
Asterisk with author names denotes non-ASH members.