P-TEFb and CDK12 facilitate subsequent steps of transcriptional elongation by RNA polymerase II. Given the prominence of both kinases in cancer, gaining a better understanding of their interplay could inform the design of novel anti-cancer strategies. While down-regulation of DNA repair genes in CDK12-targeted cancer cells is being explored therapeutically, little was known about mechanisms and significance of transcriptional induction upon inhibition of CDK12.
In this work, we showed that selective targeting of CDK12 in colon cancer-derived cells activated P-TEFb through its release from the inhibitory 7SK snRNP complex. In turn, P-TEFb stimulated RNA Pol II pause-release at thousands of genes, most of which became newly dependent on P-TEFb. Amongst the induced genes, we identified those stimulated by hallmark pathways in cancer, including p53 and NF-κB. Consequently, CDK12-inhibited cancer cells exhibited hypersensitivity to inhibitors of P-TEFb and the NF-κB signalling pathway. Mechanistically, while blocking P-TEFb triggered apoptosis of CDK12-inhibited cells in a p53-dependent manner, it attenuated proliferation of these cells irrespective of p53 by preventing induction of genes downstream of the DNA damage-induced NF-κB signaling.
In summary, we demonstrate that stimulation of RNA Pol II pause-release at the signal-responsive genes underlies the functional dependence of CDK12-inhibited cancer cells on P-TEFb. Critically, our study establishes the mechanistic underpinning for combinatorial targeting of CDK12 with either P-TEFb or the induced oncogenic pathways in cancer.
Leading actors
Zhijia Wang, Samu V Himanen, Anniina Vihervaara, Matjaž Barborič
We published this study in Nucleic Acids Research, volume 51(20), Oxford University Press. You can read the entire manuscript here:
P-TEFb is the crucial player in RNA polymerase II pause-release that has emerged as a promising target in cancer. Because single-agent therapy is bound to fail in delivering durable response in the clinic, the targeting of P-TEFb shall benefit when deployed as a combination therapy. There, two drugs, if acting synergistically, could be used at lower doses, thus limiting a potential for toxicity and resistance linked to a particular compound.
In this study, we screened a comprehensive library of oncology compounds to uncover those that elicit cancer cell death in combination with a highly selective P-TEFb inhibitor NVP-2. This effort identified several clinically relevant groups of compounds, including top-performing antimetabolites and Mouse double minute 2 homolog (MDM2) inhibitors that activate tumor suppressor p53. By focusing on the pioneering MDM2 inhibitor Nutlin-3a, which dislodges the E3 ubiquitin ligase MDM2 from p53 and thus promises to be a powerful anti-cancer agent for patients with wild-type p53, we showed that selective perturbation of P-TEFb converts the fate of Nutlin-3a-treated cells from cell-cycle arrest to apoptosis. Mechanistically, we uncovered that the fate switching was enabled by the induction of p53-dependent pro-apoptotic genes and repression of P-TEFb-dependent pro-survival genes of the PI3K-AKT signaling cascade, which stimulated mitochondria-mediated intrinsic apoptosis pathway. Finally, we showed that a panel of identified combination treatments remained effective in a three-dimensional cell culture spheroid setting, suggesting a translational potential of our work.
Taken together, our work provides a foundation for combinatorial targeting of P-TEFb in cancer. Interestingly, our screen also identified SMAC mimetics amongst the top compounds that synergized with NVP-2 in cancer cell killing. By mimicking the action of SMAC that gets released into the cytosol during mitochondrial outer membrane permeabilization, SMAC mimetics target pro-survival IAP proteins to facilitate cell death by promoting Caspase 3, 7 and 9 activity. We therefore envision that targeting P-TEFb in combination with suppressors of intrinsic apoptosis pathway could become a viable therapeutic strategy for irreversible demise of cancer cells.
Leading actors
Zhijia Wang, Monika Mačáková, Andrii Bugai, Sergey G Kuznetsov, Caroline C Friedel, Matjaž Barborič
We published this study in Nucleic Acids Research, volume 51(4), Oxford University Press. You can read the entire manuscript here:
I had a chance to write a Preview, entitled 'The Interlocking Lives of LARP7: Fine-Tuning Transcription, RNA Modification, and Splicing through Multiple Non-coding RNAs' for Molecular Cell with Mikko J. Frilander of the Institute of Biotechnology (University of Helsinki), on two very exciting studies coming out of Meister, Fischer, and Liu laboratories published in Molecular Cell, issue 77 (5). The reports described a novel role of LARP7 in facilitating the 2′-O-methylation of the spliceosomal U6 snRNA via U6-specific snoRNAs. Previously, LARP7 has been known to regulate transcription by RNA polymerase II through enabling the assembly of 7SK snRNP, which sequesters CDK9 to inhibit its kinase activity (Figure 1, top; see also our NAR review article). Mehanistically, the authors show that LARP7 connects the U6-specific methylation guide snoRNAs with their target U6, facilitating the loading of U6 onto the snoRNPs, of which methyltransferase fibrillarin catalyzes the U6 2′-O-methylation (Figure 1, bottom). Functionally, the authors showed that this modification is required for fidelity of pre-mRNA splicing and development of male germ cells. These reinvigorating studies have widened our knowledge of LARP7, suggesting that this biomedically relevant protein is incorporated into distinct RNPs to exert a multifaceted regulatory role in gene expression in health and disease.
Actors
Mikko J. Frilander and Matjaž Barborič
The Preview was published in Molecular Cell, issue 78 (1), Cell press. You can read the entire manuscript here:
This is very good news for the lab! My Rivkin Center Pilot Study Award proposal entitled 'Targeting transcriptional kinases for novel ovarian cancer therapies' has been just announced to receive the funding. In our work we aim to lay a foundation for employing highly selective inhibitors of transcriptional CDKs in combinatorial therapies against ovarian cancer. For more information, follow this link to the Rivkin Center Pilot Study Awardees.
CONGRATULATIONS! Andrii has defended his doctoral thesis entitled 'REGULATION OF POL II TRANSCRIPTIONAL RESPONSE TO DNA DAMAGE BY RBM7 AND P-TEFb' at the public examination in the University of Helsinki Athena building, lecture hall 107, Siltavuorenpenger 3 A, on the January 22nd, 2020 at noon.
From left to right: Matjaz Barboric (Supervisor), Robert Fisher (Opponent), Andrii Bugai (Ph.D Candidate), Maciej Lalowski (Faculty representative), Dan Lindholm (Custos).
The Faculty of Medicine Council has in it’s meeting on the February 11th, 2020 approved Andrii's thesis with the grade passed with distinction.
The opponent Professor Robert Fisher, M.D., Ph.D. from the Icahn School of Medicine at Mt. Sinai, NY, USA, led a lively and inquisitive discussion. The custos Dan Lindholm, MD, and the Faculty of Medicine representative Maciej Lalowski also contributed to the great event.
In the evening, Andrii hosted the dissertation dinner party for his lab colleagues, friends, and family in the honor of his opponent. The dinner culminated the day's events in a relaxed and friendly atmosphere. Restaurant Spis with its delicious Nordic quisine and a wide array of excellent wines was a terrific choice.
Andrii moved on to start his postdoctoral studies with Professor Torben H. Jensen, Ph.D. at Aarhus University, Denmark. We wish him best of luck!
To detect and repair DNA lesions, cells have evolved DNA damage response. Activating this conserved response is critical for preserving integrity of genetic information, which prevents the onset of many human pathologies including hematological disorders, neurodegenerative diseases and cancer.
Previous work has documented that cells need to shut down gene transcription by Pol II to facilitate DNA repair and limit the production of unwanted transcripts. However, it has not been explored whether transcriptional activation is important for cells under genotoxic attack.
With the mix of biochemical, genome-wide and functional approaches, we have revealed that stimulating Pol II pause release at specific sets of genes is crucial for the survival of genotoxic-stressed cells (Figure 1). We found that this transcriptional activation was enforced by P-TEFb kinase, which became activated following DNA damage through its release from the inhibitory 7SK snRNP complex. This is achieved by RBM7, an RNA-binding protein that undergoes DNA-damage-induced phosphorylation, which enables its interaction with the core 7SK snRNP subunits 7SK, LARP7 and MePCE, triggering the release of P-TEFb. Importantly, interfering with the RBM7–P-TEFb axis prevented the transcriptional activation of critical pro-survival and DNA damage-response genes and caused hypersensitivity of many cell types to DNA damage-inducing agents, underscoring that activating Pol II pause release is critical for the cells to deal with genotoxic insult.
Figure 1. Model of P-TEFb activation by RBM7 during DNA damage response.
Collectively, our work places P-TEFb activation at the heart of cellular DNA damage reponse. It also raises the prospects for designing novel anti-cancer approaches. Highly specific CDK9 inhibitors are being developed for clinical use, and combining them with e.g. commonly applied chemoterapeutic agents might deliver a fatal blow to this mortal disease.
Leading actors
Andrii Bugai (above; holding the Molecular Cell issue) , Alexandre JC Quaresma, Caroline C Friedel, Tina Lenasi, Robert Düster, Matjaž Barborič
We published this study in Molecular Cell, issue 74(2), Cell Press. You can find press release of the work on University of Helsinki pages, an article highlighting this study on the national public radio and television web page (in Slovene), and read the entire manuscript here:
The editors highlighted our study in the issue and published a preview of our work written by Le May and Coin - you can read it here:
This is life-saving news for the lab! My 3-year Sigrid Juselius Senior Investigator grant proposal entitled 'Exploring CDK9-driven transcriptional dependency for novel anti-cancer interventions' has been selected for funding. In our work, we shall define parameters for dependency of cancer cells to CDK9-driven transcription elongation, opening up the prospects for novel therapies using highly selective small molecules targeting the dysregulated transcriptional landscape of cancer cells.
This is BIG news for the lab! My Academy of Finland 4-year research proposal entitled 'Significance of transcription elongation kinases in DNA damage response' has been just announced to receive the funding. We aim to dissect how CDK9 and CDK12 safeguard integrity of DNA to provide a rationale for developing novel anti-cancer therapies. For more information, follow this link to the Academy's decision page.
The classical work by Price laboratory on the identification of P-TEFb in late 1990’s, and Zhou and Bensaude laboratories on the inhibition of P-TEFb orchestrated by the non-coding 7SK snRNA in the fall of 2001 have laid the groundwork for our understanding of regulating Pol II pause release in multicellular organisms.
Since then, intensive research by many investigators has ensued, revealing the widespread control of gene transcription at the elongation step and critical importance for P-TEFb in promoting Pol II elongation. At the same time, the composition of 7SK snRNP as well as many fundamental principles governing the sequestration and inhibition of P-TEFb within the inhibitory complex have been worked out. Our recently reported review article provides a historical perspective on how the field has developed. Moreover, we have summarized recent progress describing fascinating mechanisms that tether 7SK snRNP to chromatin as well as those that release P-TEFb from 7SK snRNP (Table 1).
What emerges from recent developments is that 7SK snRNP is not an inert and isolated particle roaming the nucleoplasm and harboring inactive P-TEFb. Rather, it can be contacted by numerous auxiliary factors: while chromatin adaptor factors (Ch-AFs) deliver 7SK snRNP to gene promoters or enhancers, P-TEFb-release factors (P-REFs) stimulate P-TEFb activation at appropriate circumstances, for a controlled gene-specific transition of Pol II from pausing into productive elongation. In turn, we propose a unifying model of P-TEFb activation on chromatin (Figure 1).
We envision that each and every major step in our model, including the anchoring of 7SK snRNP to chromatin by Ch-AFs, release of P-TEFb from 7SK snRNP by P-REFs, assembly of P-TEFb with transcription factor and SECs, and sequestration of P-TEFb back into 7SK snRNP upon transcription shutdown, could be regulated. Many questions remain unanswered and interrogating the proposed framework shall yield exciting discoveries in the future.
Leading actors
Matjaž Barborič, Alexandre JC Quaresma, Andrii Bugai
This review was published in Nucleic Acids Research, Oxford University Press. You can read the entire manuscript here:
Ovarian cancer is the 5th most common cause of cancer death in women in United States (6th in Europe), and understanding molecular basis for its genesis is essential for removing the barriers to developing novel prognostic, diagnostic and therapeutic approaches. In high-grade serous ovarian carcinoma, the deadliest form of the disease, the work by The Cancer Genome Atlas (TCGA) consortium has identified CDK12 as one of only nine genes with statistically recurrent somatic mutations. The TCGA finding prompted us to explore whether the mutations in the kinase subunit could be detrimental to the activity and biological function of the novel Cdk12/CycK transcription elongation kinase.
Indeed, we found that most mutations prevented the assembly of the Cdk12/CycK complex and that all mutations except one disabled the kinase activity of Cdk12/CycK in vitro (Figure 1). When we examined positions of the mutations using the published Cdk12/CycK structure, we found that the mutations very likely provoke structural rearrangements detrimental to the Cdk12 activation process. Importantly, our analyses of mRNA expression in patient samples containing the CDK12 mutations revealed that genes essential to the homologous recombination (HR) DNA repair pathway were coordinately downregulated (Figure 1).
When we analyzed these genes in detail, we found that Cdk12/CycK was present at them to promote high Ser2-P levels on the CTD of Pol II, indicating that when active, the Cdk12/CycK complex directly facilitates their expression. Critically, we finally demonstrated that the mutant Cdk12 proteins containing the ovarian carcinoma CDK12 mutations failed to stimulate the error-free DNA double strand break repair by HR (Figure 3).
Together, our study provides the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. High-grade serous ovarian carcinoma has been known to possess a highly unstable genome, an enabling characteristic of any cancer. In fact, one half of the tumor cases display genetic and epigenetic defects in the components of the HR repair pathway. Our study demonstrates that mutating CDK12 equips the incipient cancer cells with an alternative source of defects in the HR repair pathway, which is achieved by the collective downregulation of critical HR genes. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations elicit defects HR-mediated and possibly other DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.
Leading actors
Kingsley M. Ekumi, Hana Paculova, Tina Lenasi, Vendula Pospichalova
We reported our work in Nucleic Acids Research, Oxford University Press. You can read the entire manuscript here: