Efficacy of the novel CDK7 inhibitor QS1189 in mantle cell lymphoma

Mantle cell lymphoma (MCL) is typically an aggressive and rare form of non-Hodgkin lymphoma (NHL) with a poor prognosis despite recent advances in immunochemotherapy and targeted therapeutics against NHL. New therapeutic agents are needed for MCL. In this study, we generated a potent inhibitor of cyclin-dependent kinase 7 (CDK7), designated QS1189, and confirmed its anti-cancer effects towards MCL and other lymphomas. QS1189 was highly selective for CDK7 and showed potent anticancer effects in MCL compared to other targeted therapeutic agents, such as ibrutinib and venetoclax. Consistent with a conventional CDK7 inhibitor, QS1189 treatment significantly decreased phosphorylation of the carboxyl-terminal domain of RNA polymerase II and transcription-associated genes. QS1189 induced cell cycle arrest at the G2/M phase and apoptosis. Interestingly, QS1189 overcame the acquired resistance to venetoclax, which is mediated by Bcl-xL. Similarly, QS1189 showed potent tumour cell growth inhibition of various lymphomas. Thus, CDK7 might be a suitable therapeutic target for inhibiting lymphoma, and QS1189 is a promising therapeutic option for various lymphomas and cells with acquired resistance to targeted therapy.


Results
Development of novel CDK7 inhibitor. We generated a pharmaceutically active pyrazolo-triazine derivative as a selective inhibitor of CDK7. This compound was designated as QS1189. QS1189 potently inhibited CDK7 activity among a panel of 410 kinases in a multikinase inhibition assay in vitro ( Fig. 1a and Supplemental Table S1). Although QS1189 showed similar inhibition to other CDKs including CDK16/CycY, CDK5/p35NCK, CDK2/CycE1, and CDK5/p25NCK, QS1189 inhibited CDK7 activity in vitro with an IC 50 of 15 nM and showed IC 50 values that were 10-600-fold higher than those of other CDKs (Supplemental Table S2).
The BTK inhibitor ibrutinib and Bcl-2 inhibitor venetoclax show antitumor activity in the treatment of MCL [6][7][8] . We tested the antitumor activity of QS1189 compared to those of conventional drugs such as ibrutinib and venetoclax in MCL cells. Most MCL cells showed resistance to ibrutinib and venetoclax, although venetoclax showed growth inhibition with an IC 50 value in the nanomolar range (IC 50 value of 275.4 ± 62.9 and 50, 2.5 ± 0.4 nM for Mino and Maver-1 cells, respectively; Fig. 1b). However, QS1189 inhibited the growth of MCL cells with IC 50 values between 50 and 250 nM. Thus, QS1189 may be a more potent drug than other targeted agents for treating MCL.
Preclinical efficacy of QS1189 in MCL. CDK7 kinase activity has been implicated in phosphorylation of the CTD of RNAPII which plays a role in transcription initiation and RNAPII procession [29][30][31] . To evaluate the dose-dependent inhibition of CDK7 substrates by QS1189, we performed immunoblotting following treatment with QS1189. QS1189 treatment completely inhibited the phosphorylation of CDK7 substrate RNAPII CTD at Ser-2 and Ser-5 and slightly reduced RNAPII p-Ser7 CTD phosphorylation (Fig. 1c). Interestingly, QS1189 treatment led to reduced RNAPII expression. These results were also observed after 24 h of QS1189 treatment (Supplemental Fig. S1).
We examined the effect of CDK7 inhibition on gene expression in MCL cells following exposure to 1 µM QS1189 for 6 h by performing RNA sequencing. A total of 13,714 genes with detectable expression levels was retained, from which approximately 1,000 differentially expressed genes were downregulated in QS1189-treated MCL cells (Mino, 1793 genes; Maver-1, 1327 genes; Jeko-1, 1617 genes, Z-138, 1078 genes; log2-fold change ≤−2; Fig. 2a). Gene ontology analysis of downregulated genes revealed that genes involved in transcriptional regulation and cell cycle were enriched (Fig. 2b).
To better understand the mechanisms of the antitumor activity of QS1189 in MCL cells, we analysed apoptosis and the cell cycle. As shown in Fig. 2, QS1189 treatment induced G2/M arrest and apoptosis in all MCL cells (Fig. 3a,c). Consistent with these results, the expression of cdc2, cyclin B1, cyclin D1, and aurora-A/B, all of which participate in the G2/M phases, was significantly decreased (Supplemental Fig. S2), while QS1189 treatment induced apoptosis signalling, including PARP and caspase-3 activation (Fig. 3b,d). Taken together, these data suggest that QS1189 effectively inhibits the CDK7 substrate and induces G2/M arrest and apoptosis by suppressing transcription in MCL cells.
Anticancer efficacy of QS1189 in cells with acquired resistance to venetoclax. To evaluate whether the acquisition of resistance to venetoclax affects the anticancer efficacy of QS1189, we established 2 venetoclax-resistant cell lines (Mino and Maver-1) by long-term venetoclax exposure. The sublines with acquired resistance to venetoclax included two clones (Mino/VR-1 and Mino/VR-2) from Mino and two clones (Maver-1/ VR-1 and Maver-1/VR-2) from Maver-1 obtained by single-cell culture. As shown in Fig. 4a, all resistant cells showed an approximately 10-100-fold higher resistance to venetoclax than the parental cells, although Maver-1/ VRs cells acquired higher resistance than Mino/VRs cells. Consistent with the results of a previous study 32 , the induction of Bcl-xL was observed in both Maver-1/VR-1 and Maver-1/VR-2 cells (Fig. 4b). To determine whether the induction of Bcl-xL was associated with acquired resistance to venetoclax, we suppressed the Bcl-xL gene using two different Bcl-xL-specific shRNAs; suppression of the Bcl-xL protein was confirmed by immunoblotting (Fig. 4c). The responsiveness to venetoclax was restored in resistant cells through silencing of Bcl-xL (Fig. 4d). Next, we examined whether the acquisition of resistance to venetoclax affected the antitumor effects of QS1189. Interestingly, the acquisition of resistance to venetoclax did not affect the sensitivity to QS1189 (Fig. 4e). In addition, QS1189 treatment showed reduction of Bcl-xL and Mcl-1 which were associated with acquired resistance to venotoclax (Supplemental Fig. S3). As observed in parental cells, CDK7 substrate inhibition, G2/M arrest, and www.nature.com/scientificreports www.nature.com/scientificreports/ apoptosis by QS1189 treatment were observed in all resistant cells (Fig. 4f). Although we did not determine the mechanisms of acquired resistance to venetoclax in Mino/VRs cells, QS1189 treatment overcame the acquired resistance to venetoclax. www.nature.com/scientificreports www.nature.com/scientificreports/ Preclinical efficacy of QS1189 in various lymphomas. We further examined whether the anticancer effects of QS1189 extend to other subtypes of lymphoma including 4 Burkitt's lymphoma cell lines (Daudi, Namalwa, Raji, and Ramos) and 5 diffuse large B-cell lymphoma cell lines (OCI-Ly7, OCI-Ly10, SU-DHL-6, SU-DHL-8U2932 and U2932). The cells were treated with increasing concentrations of drugs for 72 h and growth inhibition was determined by the MTT assay (Fig. 5a). Although a few cell lines were sensitive to venetoclax (IC 50 values of 35.34, 66.93, and 121.2 nM for OCI-LY10, SU-DHL6, and U2932 cells, respectively), most cell lines were resistant to ibrutinib and venetoclax. However, QS1189 treatment inhibited the growth of Burkitt's lymphoma cells and diffuse large B-cell lymphoma cells with IC 50 values between 1 and 100 nM except for Raji cells. Additionally, QS1189 inhibited the CDK7 substrate, with concurrent induction of G2/M arrest and apoptosis in all cell lines, although there was a slight difference between cell lines (Fig. 5b). Taken together, these data suggest that QS1189 has potent anticancer effects towards MCL cells and various lymphoma cells.

Discussion
Treatment of mantle cell lymphoma is difficult because patients are typically diagnosed in stage IV, after the MCL has metastasised. Younger patients are typically treated with intensive chemotherapy or autologous stem cell transplantation 4,33,34 , whereas older patients are treated with tolerable regimens such as bendamustine and rituximab or R-CHOP with rituximab maintenance [35][36][37] . Although these regimens show a high response rate during initial therapy, most patients eventually relapse and succumb to their disease 3,4,33 . Thus, new effective drugs are needed. www.nature.com/scientificreports www.nature.com/scientificreports/ CDK7 is considered as an attractive target for treating human cancer, as it controls the activity of key enzymes involved in cell cycle progression, including other cyclin-dependent kinases such as CDK1, CDK2, CDK4, and CDK6. CDK7 function regulates the cell cycle of metazoans by controlling CDK activation through T-loop phosphorylation 19 . Moreover, CDK7 represents a component of the general transcription factor TFIIH and facilitates efficient transcriptional initiation, pause release, and elongation by phosphorylation of serine residues of RNAPII CTD (C-terminal domain). In our study, CDK7 inhibition by QS1189 diminished bulk Ser2, Ser5, and Ser7 phosphorylation of the RNAPII CTD in MCL cells as well as other lymphoma cells. Interestingly, we found that CDK7 inhibition led to reduced RNAPII transcription, although the effects of RNAPII transcription by CDK7 are controversial. Along with these results, QS1189 treatment induced G2/M arrest by decreasing the levels of cyclin B1, Aurora, and Cdc2 and then apoptosis in all lymphoma cells. Thus, the anticancer mechanisms of QS1189 in lymphoma included the induction of G2/M arrest and apoptosis by reducing RNAPII activity and transcription.
CDK7 inhibition resulted in the reduction of cellular mRNA levels because of the inhibition of RNAPII-dependent transcription by affecting the stability of preinitiation complexes 38 . Some studies demonstrated that CDK7 inhibitors are potential therapeutic agents that suppress master transcription-regulating oncogenes and disrupt their attendant super-enhancers [19][20][21]39 . Importantly, QS1189 treatment showed strong transcriptional inhibition of cyclin D1, Mcl-1, and c-Myc. Cyclin D1 is the genetic hallmark of MCL, and c-Myc and Mcl-1 are potent oncogenes in large B-cell lymphomas. Aberrant expression of these factors was associated with tumour proliferation and clinical outcomes [40][41][42] . Although QS1189 led to global mRNA downregulation, these results provide rationale for lymphoma treatment.   43 , the Akt/mTOR pathway 44 , B-cell receptor signaling 45 , anti-apoptotic pathway 8 , and cell-mediated immunity 46 . The impressive efficacy of some targeted agents including ibrutinib and venetoclax has changed the standard for care in specific subsets of patients. However, the use of these targeted agents is limited because of the development of acquired resistance to these drugs. For example, the mechanisms of acquired resistance to venetoclax are involved in mutations in Bcl-2 family proteins 47 and upregulation of Mcl-1 and Bcl-xL 48 . Consistent with the results of a previous study, we found that the upregulation of Bcl-xL was observed in cells with acquired resistance to venetoclax. Suppression of Bcl-xL led to the recovery of sensitivity to venentoclax in Maver-1/VR cells. However, the acquisition of resistance to venetoclax www.nature.com/scientificreports www.nature.com/scientificreports/ did not affect the sensitivity to QS1189. Although the mechanisms of acquired resistance to venetoclax in Mino/ VR cells were not examined in the current study, QS1189 treatment may be an attractive treatment option for overcoming acquired resistance to venetoclax.
In conclusion, we developed the novel CDK7 inhibitor QS1189 as an attractive option for efficiently suppressing MCL and other lymphoma cells. Additionally, QS1189 may be applicable for treating acquired resistance to venetoclax. Although the mechanisms of the anticancer effects of QS1189 included induction of G2/M arrest and apoptosis, further studies are needed before this QS1189 can be applied as an anticancer agent.

Generation of venetoclax-resistant cells.
Maver-1/VR cells were established by chronic, repeated exposure to increasing venetoclax doses up to 1 μM for more than 8 months. Maver-1/VR cells were cloned by single-cell culture, and these resistant sublines were designated as Maver-1/VR-1 and Maver-VR-2. In all studies, resistant cells were cultured in drug-fee medium for >1 week to eliminate the effects of venetoclax. The genetic identity of each subclone with the parental cells was verified by short tandem repeat (STR) analysis conducted at Cosmogenetech (Cosmogenetech co. Ltd., Seoul, Korea) according to established protocols. RNA sequencing. Cells were treated with 1 μM QS1189 or vehicle for 6 h. Cells were pelleted, and total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Genomic DNA was eliminated, and RNA integrity was verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA Sequencing was performed by a specialized company (Macrogen, Seoul, Korea) using the TruSeq RNA Sample Prep Kit v2 and HiSeq. 2500 platform (Illumina, San Diego, CA, USA). Sequenced reads were trimmed using TrimMomatic 0.32. The trimmed reads were mapped to the hg19 reference genomes using HISAT (version 2.0.5) and Bowtie2. Expression levels were measured as kilobase of transcript per million mapped reads using StringTie (version 1.3.3b). A |log2 fold-change| ≥2.0 was set as the threshold to screen out differentially expressed genes. Gene-enrichment and functional annotation analysis and pathway analysis for significant gene lists were performed using the DAVID Bioinformatics Resources 6.8 (http://david.ncifcrf.gov).

Flow cytometry analysis.
Each cell was treated with the indicated doses of QS1189 for 24 h. To validate the cell cycle, cells were fixed in 70% ethanol at −20 °C from 1 h to a few days, incubated with 5 μL RNase (10 mg/ mL), and finally stained with 10 μL propidium iodide (1 mg/mL). Cellular DNA content in the treated cells was analysed with a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Apoptosis was quantified using the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis kit (BD Biosciences) www.nature.com/scientificreports www.nature.com/scientificreports/ in accordance with the manufacturer's protocols. Cells were resuspended in annexin V-binding buffer (150 mM NaCl, 18 mM CaCl 2 , 10 nM HEPES, 5 mM KCl, and 1 mM MgCl 2 ). FITC-conjugated annexin V (1 μg/mL) and propidium iodide (50 μg/mL) were then added to the cells and incubated for 30 min at room temperature in the dark. Analyses were performed using an FACScan flow cytometer. Data were analysed with CellQuest software (BD Biosciences).
Statistics. Data are presented as the mean ± standard deviation. P values were determined using unpaired or paired t-tests between groups using the GraphPad Prism software.

Data Availability
All data generated or analyzed during this study are included in this published article and the Supplementary Information.