Abstract 3928: The pan-CLK/DYRK inhibitor cirtuvivint selectively disrupts alternative splicing and has broad anti-tumor activity in preclinical models
Authors: McMillan Elizabeth A. , Bossard Carine , Creger Emily , Merkwirth Carsten , Pippa Raffaella , Jarvis Matthew , Sriram Krishna , Pedraza Melinda , Ibanez Maureen , Bhat Deepti , Lai Carolyn , Stewart Josh , Eastman Brian , Mak Chi-Ching , White Michael A. , Beaupre Darrin 
Published: June 2022

Abstract
Dysregulated alternative pre-mRNA splicing (AS) is commonly associated with human malignancies, producing pathological proteomes that underlie disease initiation, progression, and therapeutic resistance. The CDC2-like kinases (CLKs) and dual-specificity tyrosine-regulated kinases (DYRKs) are thought to govern AS efficiency and specificity by directly phosphorylating splicing factors that influence pre-mRNA splice junction selection. Cirtuvivint (SM08502) is a first-in-class small molecule ATP-competitive inhibitor of CLK/DYRK kinases. To evaluate the contribution of these kinases to AS profiles, high-depth RNAseq analysis was performed across 39 tumor cell lines (representing 9 lineages) and compared to 6 non-cancerous cell lines after treatment with cirtuvivint. Both baseline and drug-induced changes in AS events (ASEs) were measured using a multivariate analysis of transcript splicing (rMATS). Pan-CLK/DYRK inhibition was found to affect a minority of baseline ASEs, leaving the majority of spliceosome activity intact in all samples; however, the magnitude and quantity of detected drug-induced alterations were larger in tumor compared to non-cancerous cell lines. The majority of ASEs resulting from pan-CLK/DYRK inhibition were enriched for exon skipping events and were tumor type-specific. Moreover, drug-induced ASEs were significantly associated with disease-promoting biology across lineage and oncogenic driver contexts, including perturbed splicing of the AR-V7 variant in treatment-resistant prostate cancer and MDM2 in p53 wild-type cancers.To assess the breadth and depth of CLK/DYRK dependencies in human cancers, the effects of pan-CLK/DYRK inhibition on growth and survival of 154 cancer cell line models, 46 PDX models, and 43 CDX models were tested. EC50s in cell viability assays ranged from 0.014 to 0.73 μM in culture with strong effects observed in subsets of cell lines across all lineages tested. Multiomic data integration revealed sensitivity to cirtuvivint was associated with alterations in splicing genes. In vivo, tumor growth inhibition studies testing cirtuvivint at exposures ~2X below the MTD resulted in significant disease control (≥50% TGI) in 38/46 PDX and 38/43 CDX models. These observations indicate broad cancer relevance, at least in the preclinical setting, and expose vulnerabilities to CLK-DYRK-regulated splicing that can potentially be therapeutically addressed with pan CLK/DYRK inhibitors.
Citation Format: Elizabeth A. McMillan, Carine Bossard, Emily Creger, Carsten Merkwirth, Raffaella Pippa, Matthew Jarvis, Krishna Sriram, Melinda Pedraza, Maureen Ibanez, Deepti Bhat, Carolyn Lai, Josh Stewart, Brian Eastman, Chi-Ching Mak, Michael A. White, Darrin Beaupre. The pan-CLK/DYRK inhibitor cirtuvivint selectively disrupts alternative splicing and has broad anti-tumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3928.