Abstract 5596: Novel multikinase CLK inhibitor BH-30236 targets hematological malignancies through alternative splicing regulation

Abstract RNA splicing dysregulation plays a critical role in tumorigenesis, cancer progression, and therapeutic resistance. Components of the spliceosome machinery are frequently mutated in myeloid malignancies, affecting 40-85% of patients with Myelodysplastic Syndrome (MDS), and 10‒25% of Acute Myeloid Leukemia (AML) cases. Recurrent aberrant alternative splicing (AS) patterns have also been detected in AML patients even in the absence of somatic spliceosome gene mutations. Furthermore, genetic screens have shown that the loss of splicing factors sensitizes AML to BCL2 inhibitor venetoclax. As such, splicing modulators are now acknowledged as a promising therapeutic target for hematologic malignancies. CDC2-like kinases (CLKs) play a pivotal role in regulating mRNA splicing via the phosphorylation of serine/arginine-rich splicing factor (SRSF) proteins which facilitate pre-mRNA splice site recognition. Consequently, inhibiting CLKs may offer a viable strategy for targeting cancers dependent on aberrant splicing. BH-30236 is a novel, orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLKs, proviral integration for the Moloney murine leukemia virus 3 kinase (PIM3), FMS-like tyrosine kinase 3 (FLT3), and dual-specificity tyrosine-regulated kinase (DYRK) 1/2. BH-30236 potently suppressed SRSF phosphorylation and demonstrated in vitro efficacy at nanomolar range across immortalized and patient-derived AML cells, including cells insensitive to venetoclax. To evaluate how BH-30236 impacts mRNA expression and alternative splicing, RNA sequencing was performed on 6 patient-derived AML cells before and after BH-30236 treatment. BH-30236 modulated AS in genes associated with DNA damage response, mRNA splicing, and chromosome remodeling. Gene ontology enrichment analysis of genes downregulated by BH-30236 included terms related to mitochondrial translation, respiratory chain complex assembly, and regulation of cytochrome c release. Specifically, BH-30236 decreased the expression of anti-apoptotic BCL2 family genes MCL1 and BCL2A1, whose expression correlates with venetoclax resistance. Consistent with this, BH-30236 enhanced the potency of venetoclax in AML cell lines, including those with TP53 mutations. The combination of BH-30236 and venetoclax also synergistically suppressed tumor growth or induced deep tumor regression in venetoclax-resistant AML cell-derived xenograft models (Kasumi-1, MOLM-13). These observations support further evaluation of BH-30236 in combination with venetoclax for hematologic malignancies. A Phase 1 study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary anti-leukemic activity of BH-30236 in adults with relapsed/refractory AML and higher-risk MDS is currently ongoing (NCT06501196). Citation Format: Nancy Ling, Gregory Sims, Ping Jiang, Wei Deng, Dayong Zhai, Danan Li, Zhenping Wang, Yue Hu, Joshua Choi, Eric Wang, Armin Graber, Jean Cui. Novel multikinase CLK inhibitor BH-30236 targets hematological malignancies through alternative splicing regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5596.