Abstract 5494: CTX-712, a novel splicing modulator targeting myeloid neoplasms
Authors: Yoda Akinori , Morishita Daisuke , Ochi Yotaro , Mizutani Akio , Mori Takuto , Takeda June , Tozaki Hirokazu , Satoh Yoshihiko , Nannya Yasuhito , Makishima Hideki , Miyake Hiroshi , Ogawa Seishi 
Published: June 2022

Abstract
Splicing factors (SFs) are among the most frequent mutational targets in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), where major SFs mutated include SF3B1, SRSF2, U2AF1 and ZRSR2. SF mutations are heterozygous and largely mutually exclusive, suggesting synthetic lethality of multiple SFs mutations and also providing a rationale of targeting SFs for new drug development, particularly in SF-mutated cancers. To show proof-of-concept for this, 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 (IC50=0.15 and 0.036 μM, respectively). The anti-leukemic effect was also confirmed by survival assay using a total of 79 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. We observed that the degree of splicing (percent spliced-in value) in cassette exon events induced by the drug was positively correlated with the sensitivity to the drug (IC50) in primary AML cells (N=32, R=0.61, P=0.00018), suggesting that the degree of splicing changes after drug treatment could still be a biomarker of 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. Interestingly, PDX with larger splicing changes showed a stronger tumor inhibition than those with smaller splicing changes. We observed a correlation between the degree of splicing changes induced by CTX-712 and the drug sensitivity (tumor growth inhibition) although it was not significant (N=13, R=-0.51, P=0.077). In particular, the PDX with the largest splicing changes showed a significant response to CTX-712 in a dose-dependent manner. Of note, 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. These results provide mechanistic insights of CLK inhibition and a rationale for further investigation of CTX-712 in MDS/AML.
Citation Format: Akinori Yoda, Daisuke Morishita, Yotaro Ochi, Akio Mizutani, Takuto Mori, June Takeda, Hirokazu Tozaki, Yoshihiko Satoh, Yasuhito Nannya, Hideki Makishima, Hiroshi Miyake, Seishi Ogawa. CTX-712, a novel splicing modulator targeting myeloid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5494.