BH-30236, a Novel Macrocyclic Clk Inhibitor Modulating Aberrant RNA Splicing, Demonstrates Potent Anti-Cancer Activity Against Myeloid Malignancies
Authors: Deng Wei , Jiang Ping , Li Danan , Zhai Dayong , Ling Nancy , Wang Zhenping , Hu Yue , Rogers Evan , Darjania Levan , Whitten Jeff , Shao Jesse , Montalban Antonio , Rui Eugene , Cui J. Jean 
Published: December 2024

Alternative splicing (AS) is a primary mechanism for mRNA transcript diversification and protein expression regulation. In cancer, altered mRNA splicing promotes oncogenic transformation, induces metastasis, and confers resistance to cancer treatment. Splicing factor (SF) mutations represent a novel class of driver mutations in myeloid neoplasms, particularly in myelodysplastic syndromes (MDS) and a subset of acute myeloid leukemia (AML). Mutations or imbalanced expression/activity of splicing factors, such as serine-arginine-rich SFs (SRSFs), often result in dysregulation of RNA splicing, drug resistance and cancer progression. Aberrant alternative splicing events also occur in patients without any splicing factor mutations and are frequently associated with poor prognosis in AML patients. CDC2-like kinases (CLK) are key regulators of AS via phosphorylation of SRSFs.
BH-30236 is designed as a novel, orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLKs. In recombinant kinase assays, BH-30236 inhibited CLK1, CLK2, and CLK4 with IC50 values of 0.134, 0.165, and 0.446 nM, respectively. BH-30236 also inhibited dual-specificity tyrosine-regulated kinase (DYRK) 1A/1B/2, proviral insertion site of Moloney murine leukemia virus kinase 3 (PIM3), and FMS-like tyrosine kinase 3 (FLT3) with IC50 values of 0.110, 0.148, 0.562, 0.115 and 0.248 nM, respectively. In cellular assays, BH-30236 inhibited the phosphorylation of SRSFs, Tau, and 4EBP1, the direct downstream substrates of CLK, DYRK, and PIM kinases with IC50 values of 40-60 nM, ~50 nM, and ~80 nM, respectively. BH-30236 also regulated alternative splicing by primarily inducing skipped exons. Through AS, BH-30236 increased ratio of pro- to anti-apoptosis isoforms of apoptosis related proteins, suppressed the expression of DNA damage repair proteins, and down-regulated FLT3-ITD and RUNX1-RUNX1T1 oncogene protein expression. BH-30236 showed great potency against 26 hematological cancer cell lines (median IC50 23.34 nM) in an in vitro cell proliferation assay and against 31 patient-derived human AML models (median IC50 37.5 nM) in an ex vivo cell viability assay.
Furthermore, BH-30236 also potently inhibited FLT3 phosphorylation with an IC50 value of 0.16 nM in AML cell line with FLT3-ITD mutation. BH-30236 potently inhibited cell proliferation with an IC50 value of 0.98 nM in FLT3-ITD positive MV-4-11 cells and achieved complete tumor regression in MV-4-11 tumor model, even at 30 days after treatment termination. Moreover, BH-30236 suppressed leukemia growth in patient-derived xenograft AML models in mice in both FLT3-ITD positive and negative models.
Finally, BH-30236 showed great combination benefit when combined with BCL-2 inhibitor venetoclax, azacitidine, or other chemotherapeutic agents in both in vitro assays and in vivo models. BH-30236 and venetoclax synergistically inhibited cell proliferation of parental MOLM-13 cells. In addition, BH-30236 showed similar potency in both parental and venetoclax-resistant MOLM-13 cells with IC50 values of 4.6 nM and 2.2 nM, respectively. In MOLM-13 and other cell-derived xenograft mouse models, combinations of BH-30236 with venetoclax, azacitidine, or cytarabine resulted in marked tumor growth inhibition or deep tumor regression, showing better efficacy than either single agent treatment.
Collectively, these preclinical study results, together with the good human ADME and preclinical safety profiles of BH-30236, strongly support its clinical development. A Phase 1 study to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary anti-leukemic activity of BH-30236 in adults with relapsed/refractory acute myelogenous leukemia and higher-risk myelodysplastic syndrome is currently ongoing (NCT06501196).