Abemaciclib in combination with venetoclax shows synergistically antiMCL efficacy in vitro and in vivo via downregulation of HSP27. (A) The combination of abemaciclib and venetoclax synergistically induced cytotoxicity after 24 hours of treatment in primary patient cells. (B) The combination of abemaciclib and venetoclax-induced cytotoxicity relative to a single agent after 72 hours of treatment in MCL cell lines. (C) Representative immunoblots indicate that proapoptotic markers were upregulated after treatment with single agents or in combination, as shown in panel B for 12 hours. (D) Mino-Ven-R CDX models treated with vehicle, abemaciclib (25 mg/kg per day, orally), venetoclax (5 mg/kg per day, orally), or their combination. The tumor burden indicated by the tumor volume is plotted. (E) Volcano plot indicating difference in gene expression by RNA-seq of tumors derived from mice treated with vehicle and the combination is shown. The significance with an inclusion level of >0.5 log fold change and adjusted P of <.01 is shown in red. Representative genes are labeled in blue. (F) Reduced mRNA expression of HSPB1 in the tumors of mice treated with vehicle, abemaciclib, venetoclax, or their combination. (G) Reduced HSP27 protein expression in the tumors of mice treated with vehicle, abemaciclib, venetoclax, or their combination. (H) Reduced protein levels of HSP27 and elevated proapoptotic marker cleaved PARP after combination treatment in JeKo-1 cells. (I) Ectopic overexpression of HSP27 protein in JeKo-1 with stable transduction of HSP27 cDNA. (J) Ectopic overexpression of HSP27 in JeKo-1 cells promotes cell growth and decreases sensitivity to abemaciclib and the combination of abemaciclib and venetoclax after 48 hours of treatment. (K) HSP27 KO in JeKo-1 using CRISPR/Cas9. (L-M) HSP27 KO in JeKo-1 cells leads to reduced cell growth (L) and increased sensitivity to venetoclax 48 hours after treatment (M). ABE, abemaciclib; DMSO, dimethyl sulfoxide (vehicle); Ven, venetoclax.