Moloney Murine Leukemia Virus Kinases Research Articles

Abstract 5944: Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers

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.

Biological evaluation of carbazoyl hydrazine derivatives as potential Pim-1 kinase inhibitors for the treatment of human liver cancer

In this study, we investigated the anticancer activity of novel carbazoyl hydrazine derivatives as inhibitors of Proviral Integration site for Moloney murine leukemia virus kinase 1 (Pim-1) in liver cancer cells in vitro. The obtained compounds were characterized using 1H NMR, 13C NMR, HRMS, and FTIR. Through computer-assisted screening, antitumor activity testing, and Pim-1 inhibitory activity testing, most of the compounds exhibited significant inhibitory activity against liver cancer cell lines. Among them, compound N'-(2-(9H-carbazol-9-yl)acetyl)-4-fluorobenzohydrazide (4r) demonstrated a remarkable anticancer effect by inducing G2/M phase cell cycle arrest and apoptosis. It was identified as a highly efficient lead compound in our screening process. Overall, our study provides valuable insights into the anticancer activity of novel carbazoyl hydrazine derivatives as Pim-1 inhibitors in liver cancer cells. The identified lead compound 4r demonstrates promising potential for further exploration and development in future studies for liver cancer treatment.

Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases.

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC50 value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure–activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.

Effects of Pim-1 inhibitor on mouse model of inflammatory bowel disease induced by TNBS

To explore the role of Pim-1 in the pathology of inflammatory bowel disease and the potential effect of Pim-1 inhibitor on treating such disease. Methods: Forty-five BALB/c mice were randomly divided into 5 groups (n=9): A normal control group, a inflammatory bowel disease group, two different dose of Pim-1 inhibitor treatment groups, and steroidhormone treatment group. The model of inflammatory bowel disease was induced by intracolonic administration of 2, 4, 6-trinitrobenzenestdfonic acid (TNBS) and ethanol mixture. Mice were treated with Pim-1 inhibitor [intraperitoneal inject, 5 or 10 mg/(kg.d)] for 5 days and prednisone (intragastric administration, 0.1 mg/d) for 5 days. The DAI, colon length, gross score and pathological grade were evaluated. The expressions of T cell master transcription factors T-box expressed in T cells (T-bet), GATA binding protein 3 (GATA-3), RA orphan receptorγ (RORγt) and forkhead box P3 (Foxp3) were measured by Real-time PCR and Western blot, respectively. Results: Pim-1 inhibitor and prednisone showed therapeutic effect on acute TNBS colitis in vivo. GATA3 and RORγt were significantly up-regulated in acute TNBS colitis (P<0.05). In contrast, the expression of Foxp3 was suppressed in the inflammatory bowel disease group, whereas it did not cause any significant change in T-bet expression (P>0.05). Administration of Pim-1 inhibitor and prednisone resulted in suppression of GATA3, RORγt expression, and the increase of Foxp3 expression (P<0.05). Administration of Pim-1 inhibitor and prednisone resulted in inhibition of T-bet mRNA expression (P<0.05), but only prednisone could inhibit T-bet protein expression (P>0.05). Conclusion: Pim-1 inhibitor significantly suppresses Th2- and Th17-type immune responses. Furthermore, Pim-1 inhibitor could induce T-cell differentiation towards a Treg phenotype. Pim-1 inhibitor has therapeutic effect on acute TNBS colitis.

Discovery and Optimization of Quinazolinone-pyrrolopyrrolones as Potent and Orally Bioavailable Pan-Pim Kinase Inhibitors.

The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.

Synthesis of (Z)-2-((1H-indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one as a new potential PET probe for imaging of the enzyme PIM1

(Z)-2-((1H-Indazol-3-yl)methylene)-6-methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one is a potent and selective proviral integration site in moloney murine leukemia virus kinase 1 (PIM1) inhibitor with an IC50 value of 3nM. (Z)-2-((1H-Indazol-3-yl)methylene)-6-[11C]methoxy-7-(piperazin-1-ylmethyl)benzofuran-3(2H)-one, a new potential PET probe for imaging of the enzyme PIM1, was first designed and synthesized in 20–30% decay corrected radiochemical yield and 370–740GBq/μmol specific activity at end of bombardment (EOB). The synthetic strategy was to prepare a carbon-11-labeled Boc-protected intermediate followed by a quick acidic de-protection.

Rational Evolution of a Novel Type of Potent and Selective Proviral Integration Site in Moloney Murine Leukemia Virus Kinase 1 (PIM1) Inhibitor from a Screening-Hit Compound

Serine/threonine kinase PIM1 is an emerging therapeutic target for hematopoietic and prostate cancer therapy. To develop a novel PIM1 inhibitor, we focused on 1, a metabolically labile, nonselective kinase inhibitor discovered in our previous screening study. We adopted a rational optimization strategy based mainly on structural information for the PIM1-1 complex to improve the potency and selectivity. This approach afforded the potent and metabolically stable PIM1-selective inhibitor 14, which shows only a marginal increase in molecular weight compared with 1 but has a significantly decreased cLogP. The validity of our design concept was confirmed by X-ray structure analysis. In a cellular study, 14 potently inhibited the growth of human leukemia cell line MV4-11 but had a negligible effect on the growth of WI-38 (surrogate for general toxicity). These results demonstrate the effectiveness of our design strategy for evolving the screening-hit compound 1 into a novel type of PIM1 inhibitor, 14.