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Multiple myeloma (MM) is the second most common hematological malignancy and remains incurable, thus demanding for new therapeutic targets. While the pathophysiology of MM is poorly understood, the substantial responsiveness of MM patients to proteasomal inhibitors (PIs) like bortezomib or carfilzomib hints towards a central role of the ubiquitin proteasome system (UPS). Deubiquitylases (DUBs) are therapeutically targetable components of the UPS, whose inhibition can destabilize oncoproteins. However, the identities of oncoprotein-regulating DUBs remain largely elusive. To identify new vulnerabilities in MM, a CRISPR/Cas9 screen targeting all human DUBs was performed. For validated candidates, phenotypical analysis regarding proliferation and cell cycle progression was performed, as well affinity and non-affinity mass spectrometry-based screens to identify substrates. We thereby identified OTUD6B as a novel oncogene that drives G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, was delineated as both a cell cycle-specific deubiquitylation substrate and activator of OTUD6B. RNA-Seq and qPCR analyses of OTUD6B and LIN28B depleted MM cells revealed that the stabilization of LIN28B drives MYC expression and activity at the G1/S transition, which in turn allows for rapid S-phase entry. Thus, silencing of OTUD6B as well as LIN28B inhibited MM outgrowth in xenograft experiments. Analyses of large MM patient cohorts revealed a progressive increase of OTUD6B expression along the transition from normal plasma cells to MGUS to MM and that high expression of OTUD6B was associated with a significantly adverse overall survival. Furthermore, OTUD6B expression was found to strongly correlate with MYC expression and significantly reduced progression-free survival in patients treated with the PI bortezomib. Knockout of OTUD6B in MM cells significantly enhance the anti-myeloma activity of the drug when using sub-lethal doses. Together, these results validate OTUD6B as a new therapeutically targetable oncogene, dependency, and prognostic factor in MM, that eventually serves as a master regulator of MYC activity to drive cell cycle progression. Citation Format: Ria Spallek, Carmen Paulmann, Oleksandra Karpiuk, Jana Zecha, Susan Klaeger, Isabell Schaeffer, Rupert Öllinger, Thomas Engleitner, Jan Krönke, Matthias Wirth, Ullrich Keller, Roland Rad, Bernahrd Kuster, Florian Bassermann. OTUD6B is a dependency in multiple myeloma that drives S-phase entry via MYC activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2298.

Sirtuin 2 (SIRT2) is a sirtuin family deacetylase that directs acetylome signaling, protects genome integrity, and is a murine tumor suppressor. We show that SIRT2 directs replication stress responses by regulating the activity of cyclin-dependent kinase 9 (CDK9), a protein required for recovery from replication arrest. SIRT2 deficiency results in replication stress sensitivity, impairment in recovery from replication arrest, spontaneous accumulation of replication protein A to foci and chromatin, and a G2/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 at lysine 48 in response to replication stress in a manner that is partially dependent on ataxia telangiectasia and Rad3 related (ATR) but not cyclin T or K, thereby stimulating CDK9 kinase activity and promoting recovery from replication arrest. Moreover, wild-type, but not acetylated CDK9, alleviates the replication stress response impairment of SIRT2 deficiency. Collectively, our results define a function for SIRT2 in regulating checkpoint pathways that respond to replication stress through deacetylation of CDK9, providing insight into how SIRT2 maintains genome integrity and a unique mechanism by which SIRT2 may function, at least in part, as a tumor suppressor protein.

Extensive changes in posttranslational histone modifications accompany the rewiring of the transcriptional program during stem cell differentiation. However, the mechanisms controlling the changes in specific chromatin modifications and their function during differentiation remain only poorly understood. We show that histone H2B monoubiquitination (H2Bub1) significantly increases during differentiation of human mesenchymal stem cells (hMSCs) and various lineage-committed precursor cells and in diverse organisms. Furthermore, the H2B ubiquitin ligase RNF40 is required for the induction of differentiation markers and transcriptional reprogramming of hMSCs. This function is dependent upon CDK9 and the WAC adaptor protein, which are required for H2B monoubiquitination. Finally, we show that RNF40 is required for the resolution of the H3K4me3/H3K27me3 bivalent poised state on lineage-specific genes during the transition from an inactive to an active chromatin conformation. Thus, these data indicate that H2Bub1 is required for maintaining multipotency of hMSCs and plays a central role in controlling stem cell differentiation.

The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53. MDM2 antagonizes the tumor suppressor p53. Wienken et al. report that MDM2 supports the Polycomb Repressor Complex 2 (PRC2), independent of p53. MDM2 thereby supports gene repression, stemness, and cancer cell survival, enhancing histone H2AK119 monoubiquitination and H3K27 trimethylation. MDM2 thus controls p53 and PRC2, each central decision-makers of cell fate.

Cyclin-dependent kinase-9 (CDK9) plays a central role in transcriptional elongation and controls multiple cotranscriptional histone modifications, including histone H2B monoubiquitination (H2Bub1). Like other CDK9-dependent histone modifications, the role of CDK9 in maintaining H2Bub1 was shown to be partially dependent upon the phosphorylation status of Ser2 of the RNA polymerase II (RNAPII) C-terminal domain (CTD). Since mutation of Ser2 within the RNAPII CTD resulted in a milder effect on H2Bub1 compared with CDK9 knockdown, we explored whether another CDK9 target may also influence H2Bub1. Based on its homology to yeast Bur1, we hypothesized that CDK9 may directly phosphorylate and activate the ubiquitin-conjugating enzyme utilized for H2B monoubiquitination. Indeed, we demonstrate that UBE2A specifically interacts with CDK9, but not CDK2. Furthermore, UBE2A is phosphorylated by CDK9 in vitro and increases UBE2A activity. Interestingly, CDK9 knockdown not only decreases UBE2A phosphorylation and H2Bub1, but also significantly impairs the induction of UBE2A-dependent monoubiquitination of proliferating cell nuclear antigen (PCNA). Thus, we provide the first evidence that CDK9 is required for the activity of UBE2A in humans, and that its activity is not only required for maintaining H2Bub1, but also for the monoubiquitination of PCNA. The common involvement of these two ubiquitinations in distinct DNA repair pathways may provide a mechanistic rationale for further exploring CDK9 as a combinatorial target for increasing the efficacy of existing cancer therapies based on the induction of DNA damage and are repaired by mechanisms which require H2Bub1 and/or PCNA ubiquitination.