Cells (1x10 6) were seeded in a 384-well plate with extracellular matrix. (D) Image-based cell-viability assays of HBL-2 cells with and without overexpression of MCL-1. (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 doxycycline- induced overexpression of 4EBP1 treated with the indicated doses of THZ531 for 24 h. (B) Western blot analysis of MCL-1, MYC, phosphor-4EBP1 and 4EBP1 protein levels in HBL-2 cells with and without doxycycline-induced overexpression of 4EBP1. (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. TPM: transcripts per kilobase million.ĬDK12 sustains cell growth and survival through transcriptional activation of MYC and the mTOR-4EBP1-MCL-1 axis in mantle cell lymphoma and MYCassociated B-cell lymphomas.
Data shown in (A) are representative of at least three independent experiments. (D) Correlation of MCL-1 (left), ATM (middle) and ATR (right) gene expression (log 2TPM) with CDK12 gene expression (log 2TPM) in primary patients’ samples. 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 area under the curve (AUC).
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THZ531 at five serial diluted concentrations was added to the medium, and the plate was continuously imaged every 30 min for 144 h. (C) Image-based cell-viability assays of primary mantle cell lymphoma samples 3x10 6 cells were seeded in a 384-well plate with extracellular matrix and lymphoma stromal cells. (B) Correlation of MCL-1 (left) or phosphor-4EBP1 protein level (right) with THZ531 IC 50 in B-cell lymphoma cell lines.
(A) Bar plot of log 10 of 50% inhibitory concentration (IC 50) of B-cell lymphoma cell lines treated with THZ531 for 72 h. Mantle cell lymphoma and other B-cell lymphoma cell lines and primary samples are exquisitely sensitive to CDK12 inhibition with THZ531, regardless of genetic background and drug resistance status. Our study indicates that CDK12 inhibitors, alone or together with EZH2 inhibitors, offer promise as novel effective approaches for difficult-to-treat DLBCL and MCL. Of note, EZH2 inhibitors reversed resistance to THZ531 by competitive inhibition of MDR1 and, in combination with THZ531, synergistically inhibited MCL and DLBCL growth in vitro. We also identified de novo and established acquired THZ531-resistant lymphoma cells conferred by over-activation of the MEK-ERK and PI3K-AKT-mTOR pathways and upregulation of multidrug resistance-1 (MDR1) protein.
By implementing pharmacogenomics and a cell-based drug screen, we found that THZ531 leads to inhibition of oncogenic transcriptional programs, especially the DNA damage response pathway, MYC target genes and the mTOR-4EBP1-MCL-1 axis, contributing to dramatic lymphoma suppression in vitro. Here, we report that both MCL and DLBCL are exquisitely sensitive to transcription-targeting drugs, in particular THZ531, a covalent inhibitor of cyclin-dependent kinase 12 (CDK12). Identification of novel targets and therapeutic strategies for these diseases represents an urgent need. Despite significant progress in the treatment of patients with diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), the prognosis of patients with relapsed disease remains poor due to the emergence of drug resistance and subsequent disease progression.
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