使用CLK抑制剂破坏剪接在恶性横纹肌样瘤临床前模型中显示活性

BACKGROUND. Malignant Rhabdoid Tumors (MRT) are rare lethal tumors affecting young children and characterized by genetic loss of SMARCB1 . SMARCB1 is a core subunit of the canonical BAF (cBAF) chromatin-remodeling complex and its loss disrupts canonical BAF assembly leading to dependence on alternative BAF complexes (not containing SMARCB1), notably the non-canonical BAF (ncBAF) complex, which contains BRD9 instead. MRT cells activate Wnt/beta-catenin signaling based on impaired repression of Wnt/beta-catenin target genes by TCF/LEF in the absence of a functional cBAF complex, representing a specific therapeutic vulnerability, as well as an overall dependence on the ncBAF complex which makes BRD9 another specific vulnerability. CLK inhibition via its effects on phosphorylation of splicing factors is known to inhibit Wnt/beta-catenin signaling by disrupting canonical splicing of TCF/LEF, and BRD9 has been reported to be downregulated in the context of splicing factor alterations. We hypothesized that the CLK/DYRK inhibitor SM09419, an analogue of the clinical stage CLK/DYRK inhibitor cirtuvivint/SM08502 (both developed by Biosplice Therapeutics, Inc./TenaRx, Inc.) could be active in MRT cells. METHODS. Established MRT cell lines and patient-derived xenograft (PDX) models were used for in vitro and in vivo experiments. Proliferation and growth were examined using Alamar Blue viability dye or colony forming assays. Cell cycle analysis was assessed by flow cytometry. mRNA expression and splice forms were evaluated by RNAseq, qPCR, RT-PCR, and long read sequencing. In vivo efficacy studies were conducted in NSG mice harboring MRT PDXs. RESULTS. SM09419 inhibited growth in vitro of six (A204, G401, JMRTK2, KYM-1, TM87-16, TTC549) MRT cell lines with IC 50 <200 nM for all cell lines. Cell cycle profiling showed increased arrest at G0/G1. SM09419 treatment resulted in upregulation of the cell cycle inhibitors p21 and p27, elevation of the pro-apoptotic markers cleaved PARP, BIM and PUMA and increased caspase 3/7 activity. RNAseq of MRT cell lines A204 and TTC549 treated with SM09419 showed downregulation of WNT /beta-catenin transcriptional targets and qPCR confirmed aberrant splicing of WNT pathway repressors LEF/TCF. Additionally, SM09419 significantly reduced BRD9 canonical transcripts in both cell lines and qPCR confirmed aberrant splicing of BRD9. In three MRT PDX models, SM09419 treatment (12.5 mg/kg and 25 mg/kg) for at least 40 days after implantation suppressed growth in a dose-dependent manner. CONCLUSION. The splicing inhibitor SM09419 exerts promising anti-tumoral effects in MRT models. Mechanistically, CLK inhibition as monotherapy may be hitting at least two known key vulnerabilities in MRT, namely Wnt/beta-catenin signaling and ncBAF function (via BRD9 misplicing). These findings provide a rationale for further early phase clinical trials of CLK inhibitors in MRT.