Abstract 5944: Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers
Authors: Cui J. Jean , Jiang Ping , Deng Wei , Zhai Dayong , Ling Nancy , Li Danan , Wang Zhenping , Hu Yue , Darjania Levan , Whitten Jeff , Rogers Evan , Rui Eugene 
Published: March 2024

Abstract
Aberrant alternative splicing is a newly recognized hallmark of cancer, that has been shown to play a critical role in tumorigenesis, cancer progression, and therapeutic resistance via multiple mechanisms, including increased proliferation, decreased apoptosis, promoted migration, enhanced metastatic potential, and induced evasion of immune surveillance. Serine and arginine-rich splicing factors (SRSFs) are RNA-binding proteins (RBPs) that regulate constitutive and alternative splicing. SRSFs are often mutated or overexpressed in cancers, resulting in widespread alterations in splicing patterns. The Cdc2-like kinase (CLK) family and dual-specificity tyrosine-regulated kinase (DYRK) phosphorylate SRSFs, influencing the assembly of spliceosome machinery, exon recognition, and splicing. Therefore, targeting CLK/DYRK kinases can modulate cancer specific splicing isoforms, opening avenues for new therapeutic interventions. BH-30236 was designed as a novel orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLK with IC50s of 0.134, 0.165, and 0.446 nM against CLK1, CLK2, and CLK4, respectively in enzymatic kinase assays. At clinically relevant concentrations, BH-30236 also inhibited DYRK1A/1B/2, proviral insertion site of moloney murine leukemia virus kinase 3 (PIM3), and FMS-like tyrosine kinase 3 (FLT3) with IC50s of 0.111, 0.148, 0.562, 0.115 and 0.248 nM, respectively. In cancer cells, BH-30236 impaired the phosphorylation of SRSFs, Tau and 4EBP1, the direct downstream substrates of CLK, DYRK, and PIM kinases with IC50s of 40-60 nM, ~50 nM, and ~80 nM, respectively. Furthermore, BH-30236 potently inhibited the FLT3 phosphorylation with an IC50 of 0.16 nM. Overall, BH-30236 regulated alternative splicing by primarily inducing skipped exons in favor of anti-tumor isoforms, leading to cancer cell death and growth suppression in a broad panel of cancer cell lines and in vivo efficacy studies. For example, BH-30236 potently inhibited cell proliferation with an IC50 of 0.62 nM in FLT3-ITD positive MV-4-11 cells and achieved complete tumor regression in the MV-4-11 cell-derived xenograft tumor model, even after dose cessation for 4 weeks. In MV-4-11 cells, BH-30236 increased pro-apoptosis splicing variant BCLxS, downregulated RNA expression of BCL2, MCL1, and AML stem cell markers CD33 and CD123. In addition, BH-30236 has demonstrated good human ADME and preclinical safety profiles. Collectively, the preclinical study results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors, as a single agent or in combination with other therapies.
Citation Format: J. Jean Cui, Ping Jiang, Wei Deng, Dayong Zhai, Nancy Ling, Danan Li, Zhenping Wang, Yue Hu, Levan Darjania, Jeff Whitten, Evan Rogers, Eugene Rui. Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5944.