NF-κB-Induced R-Loops and Genomic Instability in HTLV-1-Infected and Adult T-Cell Leukemia Cells.

Human T-cell leukemia virus type 1 (HTLV-1) is a human delta retrovirus that causes adult T-cell leukemia/lymphoma (ATL) in 3-5% of the infected population after decades of clinical latency. HTLV-1 Tax is a potent activator of IKK/NF-κB and a clastogen. While NF-κB activities are associated with cell survival and proliferation, constitutive NF-κB activation (NF-κB hyperactivation) by Tax leads to senescence and oncogenesis. Until recently, the mechanisms underlying the DNA damage and senescence induced by Tax and NF-κB were unknown. Current data indicate that NF-κB hyperactivation by Tax causes the accumulation of a nucleic acid structure known as an R-loop. R-loop excision by the transcription-coupled nucleotide excision repair (TC-NER) endonucleases, Xeroderma pigmentosum F (XPF), and XPG, in turn, promotes DNA double-strand breaks (DSBs). NF-κB blockade prevents Tax-induced R-loop accumulation, DNA damage, and senescence. In the same vein, the silencing of XPF and XPG mitigates Tax senescence, while deficiency in either or both frequently occurs in ATL of all types. ATL cells maintain constitutively active NF-κB, accumulate R-loops, and resist Tax-induced senescence. These results suggest that ATL cells must have acquired adaptive changes to prevent senescence and benefit from the survival and proliferation advantages conferred by Tax and NF-κB. In this review, the roles of R-loops in Tax- and NF-κB-induced DNA DSBs, senescence, and ATL development, and the epigenetic and genetic alterations that arise in ATL to reduce R-loop-associated DNA damage and avert senescence will be discussed.

NF-κB-induced R-loop accumulation and DNA damage select for nucleotide excision repair deficiencies in adult T cell leukemia.

Constitutive NF-κB activation (NF-κBCA) confers survival and proliferation advantages to cancer cells and frequently occurs in T/B cell malignancies including adult T cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1). Counterintuitively, NF-κBCA by the HTLV-1 transactivator/oncoprotein Tax induces a senescence response, and HTLV-1 infections in culture mostly result in senescence or cell-cycle arrest due to NF-κBCA How NF-κBCA induces senescence, and how ATL cells maintain NF-κBCA and avert senescence, remain unclear. Here we report that NF-κBCA by Tax increases R-loop accumulation and DNA double-strand breaks, leading to senescence. R-loop reduction via RNase H1 overexpression, and short hairpin RNA silencing of two transcription-coupled nucleotide excision repair (TC-NER) endonucleases that are critical for R-loop excision-Xeroderma pigmentosum F (XPF) and XPG-attenuate Tax senescence, enabling HTLV-1-infected cells to proliferate. Our data indicate that ATL cells are often deficient in XPF, XPG, or both and are hypersensitive to ultraviolet irradiation. This TC-NER deficiency is found in all ATL types. Finally, ATL cells accumulate R-loops in abundance. Thus, TC-NER deficits are positively selected during HTLV-1 infection because they facilitate the outgrowth of infected cells initially and aid the proliferation of ATL cells with NF-κBCA later. We suggest that TC-NER deficits and excess R-loop accumulation represent specific vulnerabilities that may be targeted for ATL treatment.

RNF8 Dysregulation and Down-regulation During HTLV-1 Infection Promote Genomic Instability in Adult T-Cell Leukemia.

The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.

OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination.

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48-linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1-E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1-/- knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin.

Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5∆ strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5∆ psh1∆ strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5∆ or psh1∆ strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.