Page 121 - Haematologica May 2022

CDK12-transcription reprogramming in MCL and DLBCL
Discussion
MCL is an incurable form of lymphoma and the median overall survival of patients with this malignancy is only 4 to 5 years. Its clinical course usually involves sequential relaps- es, with no response to current standard therapies. MYC- associated large B-cell lymphoma is one of the most aggres- sive lymphomas. Patients often experience relapsed or refractory disease after an initial response to first-line ther- apy and the majority of those with MYC-associated B-cell lymphomas succumb to their disease.
CDK12 is a transcriptional CDK that complexes with cyclin K to mediate gene transcription by phosphorylating RNAPII. CDK12 has been demonstrated to specifically upregulate the expression of genes involved in response to DNA damage, stress and proliferation, as well as mRNA processing and cell survival17 by directly phosphorylating pre-mRNA processing factors, which induces premature cleavage and polyadenylation and a loss of expression of long genes that participate in the DDR.18,19 An increasing number of studies have highlighted CDK12 as a therapeutic target for cancer. Inhibition of transcriptional CDK could be an effective strategy to overcome resistance to targeted therapies, including erlotinib and crizotinib. Numerous other studies have identified specific genetic or cellular con- texts that confer enhanced sensitivity to CDK12 inhibition, including MYC dependency. In this study, we aimed to explore CDK12 as a novel vulnerability for MCL and MYC- associated B-cell lymphomas. We further determined the role of CDK12-mediated transcription activation and asso- ciated pathways in cell survival and growth of MCL and MYC-associated B-cell lymphomas. Here, we report that the aggressive B-cell lymphomas are exquisitely sensitive to transcription-targeting drugs, particular to the covalent CDK12 inhibitor THZ531. By implementing pharmacoge- nomics and a cell-based drug screen, we found that THZ531 leads to inhibition of oncogenic transcriptional programs, especially the DDR pathway, MYC target genes and the mTOR-4EBP1-MCL-1 axis, contributing to lym- phoma suppression ex vivo.
Importantly, we investigated molecular mechanisms involved in conferring resistance to THZ531 and examined whether combined inhibitors of CDK12 and EZH2 cooper- atively reprogram transcription repression to overcome resistance to THZ531, and, ultimately, inhibit lymphoma growth and survival in aggressive B-cell malignancies. We demonstrate that MDR1 is overexpressed in cell lines with de novo and acquired resistance to THZ531, and that MDR1 overexpression results in THZ531 resistance via MDR1- mediated export of THZ531. Our data implicate MDR1 as a therapeutic target to overcome THZ531 resistance. Additional testing with the chemically distinct CDK12 inhibitor SR-4835 revealed that MDR1 upregulation as an avenue of resistance in CDK12 inhibition is specific to THZ531 (data not shown). However, CDK12 inhibitors are
known functional substrates for drug transport molecules,31 and accordingly, it has been reported that MDR1 upregula- tion contributes to resistance against other compounds with the THZ chemical backbone, including the CDK7 inhibitor THZ1.29 We then identified GSK343 and UNC1999, two EZH2 inhibitors, as potent drugs in pre- venting MDR1-mediated export of THZ531 in lymphoma cells. The combination of GSK343 or UNC1999 with THZ531 exhibited synergistic efficacy against MCL and aggressive B-cell lymphomas. We further showed that GSK343 and UNC1999 inhibited MDR1 efflux by compet- ing with THZ531 for MDR1, consistent with previous reports suggesting that GSK343 and UNC1999 are potential MDR1 substrates.32 Thus, GSK343 and UNC1999 have a broad drug-sensitizing potential for lymphoma therapy. Furthermore, we also demonstrated that activation of MEK-ERK and PI3K-AKT-mTOR pathways contributes to MDR1 upregulation in THZ531-resistant cells. We identi- fied that de novo and established acquired THZ531-resistant lymphoma cells are associated with over-activation of MEK-ERK and PI3K-AKT-mTOR pathways, contributing to upregulation of MDR1 protein. Intriguingly, EZH2 inhibitors reversed THZ531 resistance by competitive inhi- bition of MDR1 and, in combination with THZ531, syner- gistically inhibited MCL and MYC-associated lymphoma growth in vitro. Our study indicates that CDK12 inhibitors, both alone and together with EZH2 inhibitors, offer prom- ise as a novel treatment strategy that can be an effective approach for MYC-dependent lymphomas and MCL. Furthermore, GSK343 and UNC1999 can also reverse drug resistance that has developed from MDR1 upregulation. Thus, GSK343 and UNC1999 can be used together with other MDR1-induced drugs such as doxorubicin and carfil- zomib to overcome drug resistance for greater and longer- lasting efficacy in patients with aggressive B-cell lym- phomas.
Disclosures
No conflicts of interest to disclose.
Contributions
JT and XZ conceived and designed the study; JG, MYW, YR, TLi, JCY and TLw performed experiments, and collected and assembled the data; JG, MYW, XZ, RY and JT analyzed and interpreted the data; JT, JG and MW wrote , reviewed and/or revised the manuscript; EMS, KHS, BDS, DRD and JT provided administrative, technical or material support.
Funding
This work was supported in part by grants from the National Cancer Institute CA241713, CA233601, and CA234519 (to JT), a grant from the Lymphoma Research Foundation (to JT) and by funds from Florida State Live Like Bella Pediatric Cancer Research Initiative to the H. Lee Moffitt Cancer Center & Research Institute.
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