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Purpose: TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 – all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC). Methods: TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data. TIPRL1-depleted HNSCC cells were generated by CRISPR/Cas9 editing, and effects on colony growth, micronuclei formation (microscopy), cell cycle (flow cytometry), DDR signaling (immunoblots) and proteome (mass spectrometry) following RT were assessed. Mass spectrometry was used for TIPRL1 phosphorylation and interactomics analysis in irradiated cells. Results: TIPRL1 expression was increased in tumor versus non-tumor tissue, with high tumoral TIPRL1 expression associating with lower locoregional control and decreased survival of RT-treated patients. TIPRL1 deletion in HNSCC cells resulted in increased RT sensitivity, a faster but prolonged cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Upon irradiation, ATM phosphorylates TIPRL1 at Ser265. A non-phospho Ser265Ala mutant could not rescue the increased radiosensitivity phenotype of TIPRL1-depleted cells. While binding to PP2A-like phosphatases was confirmed, DNA-dependent protein kinase (DNA-PKcs), RAD51 recombinase and nucleosomal histones were identified as novel TIPRL1 interactors. Histone binding, although stimulated by RT, was adversely affected by TIPRL1 Ser265 phosphorylation. Conclusions: Our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in RT resistance through modulation of the DDR, highlighting its potential as a new HNSCC predictive marker and therapeutic target. [ABSTRACT FROM AUTHOR]

Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98 fusion proteins. Abl1−/− murine hematopoietic cells transduced with AML1-ETO and NUP98-PMX1 gained proliferation advantage when compared to Abl1 + /+ counterparts. Conversely, overexpression and pharmacological stimulation of ABL1 kinase resulted in reduced proliferation. To pinpoint mechanisms facilitating the transformation of ABL1-deficient cells, Abl1 was knocked down in 32Dcl3-Abl1ko cells by CRISPR/Cas9 followed by the challenge of growth factor withdrawal. 32Dcl3-Abl1ko cells but not 32Dcl3-Abl1wt cells generated growth factor-independent clones. RNA-seq implicated PI3K signaling as one of the dominant mechanisms contributing to growth factor independence in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib exerted selective activity against Lin-cKit+ NUP98-PMX1;Abl1−/− cells when compared to the Abl1 + /+ counterparts. Since the role of ABL1 in DNA damage response (DDR) is well established, we also tested the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi). AML1-ETO;Abl1−/− and NUP98-PMX1;Abl1−/− cells were hypersensitive to DNA-PKi and ATRi, respectively, when compared to Abl1 + /+ counterparts. Moreover, ABL1 kinase inhibitor enhanced the sensitivity to PI3K, DNA-PKcs and ATR inhibitors. In conclusion, we showed that ABL1 kinase plays a tumor suppressor role in hematological malignancies induced by AML1-ETO and NUP98-PMX1 and modulates the response to PI3K and/or DDR inhibitors. [ABSTRACT FROM AUTHOR]

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation. Ubiquitin system factors are critical regulators of DNA repair pathways. Through a functional genetic screen, the authors identify a novel role of ubiquitin-conjugating enzyme UBE2D3 in promoting DNA repair at deprotected telomeres by limiting the accumulation of RNF168 and promoting ample ATM-dependent phosphorylation of KAP1. [ABSTRACT FROM AUTHOR]

Telomeres are repetitive nucleoprotein complexes at chromosomal termini essential for maintaining genome stability. Telomeric RNA, or TERRA, is a previously presumed long noncoding RNA of heterogeneous lengths that contributes to end-capping structure and function, and facilitates telomeric recombination in tumors that maintain telomere length via the telomerase-independent Alternative Lengthening of Telomeres (ALT) pathway. Here, we investigated TERRA in the radiation-induced DNA damage response (DDR) across astronauts, high-altitude climbers, healthy donors, and cellular models. Similar to astronauts in the space radiation environment and climbers of Mt. Everest, in vitro radiation exposure prompted increased transcription of TERRA, while simulated microgravity did not. Data suggest a specific TERRA DDR to telomeric double-strand breaks (DSBs), and provide direct demonstration of hybridized TERRA at telomere-specific DSB sites, indicative of protective TERRA:telomeric DNA hybrid formation. Targeted telomeric DSBs also resulted in accumulation of TERRA foci in G2-phase, supportive of TERRA's role in facilitating recombination-mediated telomere elongation. Results have important implications for scenarios involving persistent telomeric DNA damage, such as those associated with chronic oxidative stress (e.g., aging, systemic inflammation, environmental and occupational radiation exposures), which can trigger transient ALT in normal human cells, as well as for targeting TERRA as a therapeutic strategy against ALT-positive tumors. Transcriptomics (RNA-seq) data reveal significantly increased telomeric RNA, or TERRA, in response to spaceflight and radiation exposure (compared to baseline and ground control samples). [ABSTRACT FROM AUTHOR]

Trypanosoma cruzi, the etiological agent of Chagas disease, invades many cell types affecting numerous host-signalling pathways. During the T. cruzi infection, we demonstrated modulations in the host RNA polymerase II activity with the downregulation of ribonucleoproteins affecting host transcription and splicing machinery. These alterations could be a result of the initial damage to the host DNA caused by the presence of the parasite, however, the mechanisms are not well understood. Herein, we examined whether infection by T. cruzi coincided with enhanced DNA damage in the host cell. We studied the engagement of the DNA damage response (DDR) pathways at the different time points (0–24 h post-infection, hpi) by T. cruzi in LLC-MK2 cells. In response to double-strand breaks (DSB), maximum phosphorylation of the histone variant H2AX is observed at 2hpi and promotes recruitment of the DDR p53-binding protein (53BP1). During T. cruzi infection, Ataxia-telangiectasia mutated protein (ATM) and DNA-PK protein kinases remained active in a time-dependent manner and played roles in regulating the host response to DSB. The host DNA lesions caused by the infection are likely orchestrated by the non-homologous end joining (NHEJ) pathway to maintain the host genome integrity. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) are the most mutagenic form of DNA damage, and play a significant role in cancer biology, neurodegeneration and aging. However, studying DSB-induced mutagenesis is limited by our current approaches. Here, we describe iMUT-seq, a technique that profiles DSB-induced mutations at high-sensitivity and single-nucleotide resolution around endogenous DSBs. By depleting or inhibiting 20 DSB-repair factors we define their mutational signatures in detail, revealing insights into the mechanisms of DSB-induced mutagenesis. Notably, we find that homologous-recombination (HR) is more mutagenic than previously thought, inducing prevalent base substitutions and mononucleotide deletions at distance from the break due to DNA-polymerase errors. Simultaneously, HR reduces translocations, suggesting a primary role of HR is specifically the prevention of genomic rearrangements. The results presented here offer fundamental insights into DSB-induced mutagenesis and have significant implications for our understanding of cancer biology and the development of DDR-targeting chemotherapeutics. DNA double-strand breaks (DSBs) are highly mutagenic making them central to many pathologies. Here, the authors developed a highly sensitive sequencing approach to study DSB mutagenesis, yielding insights into mutagenic outcomes and characterising their underlying mechanisms. [ABSTRACT FROM AUTHOR]