Page 112 - Haematologica May 2022

J. Gao et al.
of driving lymphoma progression, networks are rewired and, consequently, the signal rewiring must be targeted as a whole to obtain durable and improved clinical responses. In support of this concept, increasing evidence suggests that super-enhancers are required to maintain the expression of genes critical for cancer cell survival and proliferation. Given the biological importance of super-enhancers in the regulation of global transcriptome landscaping, super- enhancers have emerged as enticing therapeutic targets. Indeed, blocking super-enhancer-driven transcription result- ed in the concurrent disruption of multiple oncogenic machineries to which aggressive lymphoma cells are addicted, regardless of their mutational profiles.6,7 Thus, small-molecule inhibitors targeting transcriptional regula- tors and downstream global transcriptome and kinome reprogramming represent a promising approach to the treatment of aggressive lymphomas.
Transcriptional cyclin-dependent kinase 7 (CDK7) and 9 (CDK9), catalytic subunits of the transcription elongation factor P-TEFb, are considered to be gatekeepers of the tran- scriptional machinery,8 and clinical trials with small-mole- cule inhibitors of these kinases are underway.9 CDK7- and CDK9-mediated phosphorylation of serine on the C-termi- nal domain of RNA polymerase II (RNAPII) is known to be linked to transcriptional initiation and elongation. Phosphorylation of Ser2 on the C-terminal domain is cat- alyzed by P-TEFb and is associated with a large complex of proteins coined the super elongation complex.10,11 Moreover, CDK7 directly phosphorylates Ser5 and Ser7 of the C-termi- nal domain of RNAPII for RNAPII activity. Interestingly, ChIP-sequencing data demonstrated that CDK7 densely occupies super-enhancers that drive high levels of transcrip- tion of oncogenes, such as MYC, in a wide variety of can- cers, including T-cell acute lymphoblastic leukemia, non- small cell lung cancer, neuroblastoma and triple-negative breast cancer.12-14 Importantly, treatment of cancer with a selective, small-molecule CDK7 inhibitor, THZ1, leads to rapid loss of these super-enhancer-driven oncogenic tran- scripts,15 suggesting that super-enhancer-driven genes are especially vulnerable to inhibition of transcriptional machin- ery such as through inhibition of CDK7.16
CDK12 belongs to the transcriptional CDK family of ser- ine/threonine protein kinases that regulate transcriptional and post-transcriptional processes, thereby modulating multiple cellular functions. CDK12 modulates transcription elongation by phosphorylating the Ser2 on the C-terminal domain of RNAPII and was demonstrated to specifically upregulate the expression of genes involved in the DNA damage response (DDR), mRNA processing, stress and heat shock.17 Other studies indicated that CDK12 phosphory- lates pre-mRNA processing factors directly, thereby induc- ing premature cleavage and polyadenylation and a loss of expression of long genes (>45 kb), a substantial proportion of which participate in the DDR.18,19 In addition, an increas- ing number of studies point to CDK12 inhibition as an effective strategy to inhibit tumor growth, and synthetic lethal interactions have been described with MYC, EWS/FLI and PARP/CHK1 inhibition.20,21 Therefore, CDK12 has emerged as an appealing therapeutic target.
MCL is a B-cell malignancy in which the disruption of the DDR pathway and activation of cell survival mechanisms contribute to oncogenesis.22 MYC activation engages the replication stress and DNA damage pathway to allow not only robust cellular proliferation, but also limited clonal expansion and avoidance of cytotoxic DNA damage accu-
mulation in aggressive B-cell lymphomas.23,24 These data implicate CDK12 as a potential novel vulnerability for MCL and MYC-associated large B-cell lymphomas.
In this study, we determined the role of CDK12-mediated transcriptional activation and its associated pathway in cell survival and growth in MCL and MYC-associated large B- cell lymphomas. We defined the molecular mechanism for inhibiting CDK12 using THZ531 in these aggressive lym- phomas. Importantly, we investigated the molecular mech- anism conferring resistance to THZ531 and examined whether combined inhibitors of CDK12 and EZH2 cooper- atively reprogram transcriptional repression to overcome THZ531 resistance, and, ultimately, inhibit lymphoma growth and survival in these difficult-to-treat, aggressive B- cell malignancies.
Methods
Patients and tumor specimens
The primary samples from MCL patients were obtained from fresh biopsy-derived lymphoma tissues (lymph nodes) and from peripheral blood following informed consent from patients and approval by the Moffitt Cancer Center/University of South Florida InstitutionalReviewBoard.Forpreparationofviable,sterile,sin- gle-cell suspensions, the lymph node tissue was diced and forced through a cell strainer into RPMI-1640 tissue culture medium. Cells, obtained after low-speed centrifugation were re-suspended in medium. Lymphoma cells from peripheral blood were isolated by Ficoll-Plaque purification, and only lymphoma samples that had more than 80% tumor cells were used for experiments.
Image-based cell-viability assay
Cells were seeded in a 384-well plates of a reconstructed lym- phoma tumor microenvironemnt, including high physiological densities (1-10x106 cells/mL), extracellular matrix (collagen, Advanced BioMatrix, #5005-B), and lymphoma stromal cells. A panel of drugs at five serial diluted concentrations was added to the medium, and plates were continuously imaged every 30 min for 96 h (cell lines) or 144 h (primary samples). All images were analyzed using a digital imaging analysis algorithm to detect cell viability based on membrane motion (pseudo-colored in green), and changes in viability were quantified by the area under the curve (AUC) as described elsewhere.25-27
RNA-sequencing
All samples were prepared in biological triplicates. Cells (10x106) were treated with 100 nM THZ531 or dimethylsulfoxide (vehicle control) for 6 and 24 h. Total RNA was isolated using an RNA iso- lation kit, RNeasy Plus Mini (Qiagen Cat# 74134). Libraries were prepared using a TruSeq Stranded mRNA Library Prep Kit (Illumina Cat #RS-122-2101/2) according to the manufacturer’s instructions. RNA sequencing was performed on a HiSeq 2500v4 high output (50 bp, single-end reads). Tophat2 was used to align the Fastq files. Transcripts per kilobase million (TPM) values were calculated and normalized using Cuffnorm. Genes that had a P less than 0.05 and at least a 1.5-fold change were considered to be sig- nificantly altered between sensitive and resistant phenotypes. The cutoff value for expressed genes was a TPM value greater than or equal to 1.
Statistics
Unless otherwise stated, comparisons and statistical significance between two groups in this paper are based on a two-sided Student t-test. P values of less than 0.05 were considered statisti-
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