Novel Multikinase Clk Inhibitor BH-30236 Targets Hematological Malignancies through Alternative Splicing Regulation
Authors: Ling Nancy , Sims Gregory , Jiang Ping , Deng Wei , Zhai Dayong , Li Danan , Wang Zhenping , Hu Yue , Wang Eric , Graber Armin , Cui J. Jean 
Published: December 2024

RNA splicing dysregulation, a newly recognized hallmark of cancer, plays a critical role in tumorigenesis, cancer progression, and therapeutic resistance via multiple mechanisms, including increased proliferation, reduced apoptosis, enhanced metastatic potential, and induced evasion of immune surveillance. 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 alternative splicing (AS) patterns have also been detected in AML patients even in the absence of somatic spliceosomal gene mutations. As such, splicing modulators are now acknowledged as promising therapeutic targets for hematologic malignancies. The 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, CLK inhibition 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. RNA sequencing of AML cell lines and patient-derived AML cells were performed to directly evaluate how BH-30236 impacts alternative splicing. These cells were exposed to either DMSO (control) or BH-30236 (30 nM) for 8 hours and differential alternative splicing events (ASE) were quantified using multivariate analysis of splicing transcripts (rMATS). Among the tested cells, BH-30236-induced ASE were observed in genes involved in biological processes such as DNA damage response, mRNA splicing, and chromosome remodeling. ASE regulated by BH-30236 were also enriched in genes associated with mTOR and MYC signaling, pathways implicated in hematologic oncogenesis. Recent studies highlight the sensitivity of BCL2 family genes to splicing modulation. We demonstrate that BH-30236 decreases the expression of anti-apoptotic BCL2 family genes (MCL1, BCL2, BCL2A1), and increases the expression of pro-apoptotic BCLxS. Consistent with this, the combination of BCL2 inhibitor venetoclax with BH-30236 synergistically induced tumor growth inhibition and/or regression in highly resistant AML cell-derived xenograft models (Kasumi-1, MOLM-13). These observations support further evaluation of BH-30236 as a multikinase splicing modulator for hematologic malignancies, either as a single agent or in combination with other therapies. 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).