Page 115 - Haematologica May 2022

CDK12-transcription reprogramming in MCL and DLBCL
100 or 500 nM THZ531 for 12 h, we observed that gene signatures related to the MYC, mTOR and DDR pathways were significantly downregulated (Figure 3F, Online Supplementary Figure S3D). These results support the func- tional role of CDK12 in MCL survival and proliferation.
MDR1 upregulation drives resistance to THZ531 in mantle cell lymphoma and other aggressive B-celllymphomas
Despite the potency shown by THZ531 and other tran- scriptional CDK inhibitors in preclinical studies, resistance
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Figure 2. CDK12 sustains cell growth and survival through transcriptional activation of MYC and the mTOR-4EBP1-MCL-1 axis in mantle cell lymphoma and MYC- associated B-cell lymphomas. (A) Western blot analysis of phosphorylation of RNAPII in Ser2, 5 and 7, RNAPII, cleaved PARP, phosphor-p70S6K, p70S6K, phosphor- 4EBP1, 4EBP1, MCL-1, BCL-XL, BCL-2 and MYC in THZ531 sensitive cell lines and mantle cell lymphoma primary samples treated with the indicated doses of THZ531 at different time points. (B) Western blot analysis of MCL-1, MYC, phosphor-4EBP1 and 4EBP1 protein levels in HBL-2 cells with and without doxycycline-induced over- expression of 4EBP1. (C) Western blot analysis of cleaved PARP, MCL-1, BCL-XL, BCL-2, MYC and 4EBP1 protein expression in HBL-2 cells with and without doxycy- cline-induced overexpression of 4EBP1 treated with the indicated doses of THZ531 for 24 h. (D) Image-based cell-viability assays of HBL-2 cells with and without overexpression of MCL-1. Cells (1x106) were seeded in a 384-well plate with extracellular matrix. THZ531 was added at five serial diluted concentrations to the medi- um, and the plate was continuously imaged every 30 min for 96 h. All images were analyzed using a digital imaging analysis algorithm to detect cell viability based on membrane motion, and changes in viability were quantified by the area under the curve. (E) Western blot analysis of cleaved PARP, MYC, MCL-1 and BCL-XL in HBL-2 cells with and without MCL-1 overexpression treated with the indicated doses of THZ531 for 24 h. DOX: doxycycline; DMSO: dimethylsulfoxide.
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