Page 113 - Haematologica May 2022

CDK12-transcription reprogramming in MCL and DLBCL
cally significant. Data are shown with the mean ± standard devia- tion of at least three experiments. Analysis of variance (ANOVA) or the Kruskal-Wallis test was used for comparing data from mul- tiple groups.
Results
Mantle cell lymphoma and other B-cell lymphoma cell lines and primary samples are exquisitely sensitive to CDK12 inhibition regardless of genetic background and drug resistance status
We performed cell viability assays on over 40 B-cell lym- phoma lines for their response to the CDK12 inhibitor THZ531. These cell lines included MCL, DLBCL, double hit lymphoma and Burkitt lymphoma lines with a variety of genetic backgrounds. Established ibrutinib-resistant lines were also included for their vulnerability to CDK12 inhibi- tion. As shown in Figure 1A and Online Supplementary Figure S1A, most of these cell lines exhibited high sensitivity to THZ531 regardless of lymphoma type, genetic background (11q, p53 status) and drug resistance with all IC50 values in the nanomolar range except for REC-1. In parallel, we also performed western blotting on all tested lines to determine the relative protein abundances of MCL-1, 4EBP1 and its phosphorylation (Online Supplementary Figure S1B) and the correlation of protein abundances with THZ531 sensitivity. As shown in Figure 1B, THZ531 IC50 values were most cor- related to the protein levels of MCL-1 and phosphorylated 4EBP1. Furthermore, primary MCL samples were exquisite- ly sensitive to THZ531 (Figure 1C), implying that CDK12 has a functional role in aggressive B-cell malignancies. We therefore queried CDK12 gene expression from RNA- sequencing performed on 40 primary samples. Notably, the expression level of MCL-1 and DDR-related genes correlat- ed positively with CDK12 mRNA expression in B-cell lym- phomas (Figure 1D, Online Supplementary Figure S1C). Therefore, CDK12 expression is frequently elevated and is essential for cell growth and survival in MCL and aggressive B-cell lymphomas.
CDK12 sustains cell growth and survival through transcriptional activation of MYC, the mTOR-4EBP1-MCL-1 axis and DNA damage response pathway in mantle cell lymphoma and MYC-associated B-cell lymphomas
The strong correlations of sensitivity to THZ531 and MCL-1 protein level and 4EBP1 phosphorylation indicated the functional role of the mTOR-4EBP1-MCL-1 axis in the growth and survival of these aggressive lymphomas. We next examined the effect of CDK12 inhibition with THZ531 on cellular signaling molecules such as those involved in the PI3K-AKT-mTOR pathway in addition to MCL-1 in MCL (Z138, Jeko-1), double-hit lymphoma (DOHH-2), DLBCL (Val) cell lines and primary MCL sam- ples. Given that CDK12 is one of the transcriptional CDK considered to be a gatekeeper of transcriptional elongation, the effects of THZ531 on phosphorylation of Ser2 on the C-terminal domain of RNAPII, MYC, the AKT-mTOR- 4EBP1 pathway, BCL-2 family proteins, and apoptotic PARP cleavage were assessed by western blot. As shown in Figure 2A and Online Supplementary Figure S2A, THZ531 induced a dose-dependent inhibition of RNAPII-Ser2 phos- phorylation and variable degrees of inhibition of RNAPII- Ser5/7 phosphorylation at higher doses with associated
dramatic reductions of MYC and MCL-1 protein levels and triggered significant PARP cleavage in all the tested lym- phoma lines. In addition, phosphorylation of p70S6K and 4EBP1, the downstream targets of mTOR, were also markedly decreased after THZ531 treatment (Figure 2A, Online Supplementary Figure S2A). Together, these results, in conjunction with the correlative studies shown in Figure 1B, imply that the mTOR-4EBP1 pathway and subsequent cap-dependent translation targets, MCL-1 and MYC, medi- ate CDK12 function in these aggressive B-cell lymphomas. To confirm this notion, we employed the 4EBP1 mutant, 4EBP1, as a dominant cap-dependent translation initiation inhibitor.28 4EBP1 carries alanine substitutions at four ser- ine/threonine residues and thereby prevents dissociation of 4EBP1 from eIF4E induced by mTORC1. The abundance of MYC and MCL-1 proteins was decreased upon ectopic expression of doxycycline-induced 4EBP1 in HBL-2 cells (Figure 2B). Interestingly, when cells with ectopic expres- sion of doxycycline-induced 4EBP1 were treated with THZ531, we observed more dramatic reductions in MCL- 1, MYC, and cell viability with associated increased PARP cleavage relative to non-induced cells (Figure 2C, Online Supplementary Figure S2B). This enhanced effect of THZ531 is likely attributable to endogenous translation activity. Moreover, in line with these results, increased ectopic expression of MCL-1 attenuated THZ531-induced MCL-1 downregulation, apoptosis and colony formation (Figure 2D, E, Online Supplementary Figure S2C). Together, these results indicate that 4EBP1 and MCL-1 mediate, at least partially, the biological function of CDK12.
To determine the molecular mechanism and cellular pathways responsible for the activity of THZ531, RNA- sequencing was performed on THZ531-sensitive MCL lines (Z138, Jeko-1) that were treated or not with 100 nM THZ531 for 6 and 24 h. Transcriptomic analysis of these THZ531-treated cells revealed alteration of expression of a large set of genes, with a total of 2,405 genes downregulat- ed in common in the two cell lines after 6 h and 2,914 genes downregulated in common after 24 h, implying alteration of similar pathways (Figure 3A, Online Supplementary Figure S3A). Gene set enrichment analysis revealed that genes altered by THZ531 treatment for 24 h are associated with proliferation and survival pathways as well as both the MYC and mTOR-AKT pathways (Figure 3B, Online Supplementary Figure S3B). Further analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed several DDR pathways significantly negatively enriched by 24 h of treatment, including homologous recombination and the mismatch-repair pathways (Figure 3C, Online Supplementary Figure S3C). Overall, these data support THZ531-mediated transcriptional suppression.
In parallel, we performed drug screening in cells sensitive to THZ531 treatment and determined their drug response profiles. As shown in Figure 3D, the MCL and aggressive B-cell lymphoma lines were uniformly sensitive to inhibitors of the transcriptional machinery apparatus (CDK7, CDK9 and CDK12), AKT, mTOR, and PLK, as well as the chemotherapeutic drug doxorubicin. Intriguingly, each of these inhibitors and chemotherapeutic drugs is known to function by downregulating MCL-1, and ectopic overexpression of MCL-1 restored the potency of these agents (Figure 3E), thus supporting the role of MCL-1 in cell survival and drug response to CDK12 inhibition. In line with these cell line results, when we performed RNA- sequencing on primary MCL samples after treatment with
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