Search

ATM kinase is a tumor suppressor and a master regulator of the DNA damage response. Most cancer-associated alterations to ATM are missense mutations at the PI3-kinase regulatory domain (PRD) or the kinase domain. Expression of kinase-dead (KD) ATM protein solely accelerates lymphomagenesis beyond ATM loss. To understand how PRD suppresses lymphomagenesis, we introduced the cancer-associated PRD mutation R3008H (R3016 in mouse) into mice. R3008H abrogated DNA damage- and oxidative stress-induced activation of ATM without consistently affecting ATM protein stability and recruitment. In contrast to the early embryonic lethality of AtmKD/KD mice, AtmR3016H (AtmR/R) mice were viable, immunodeficient, and displayed spontaneous craniofacial abnormalities and delayed lymphomagenesis compared with Atm−/− controls. Mechanistically, R3008H rescued the tardy exchange of ATM-KD at DNA damage foci, indicating that PRD coordinates ATM activation with its exchange at DNA-breaks. Taken together, our results reveal a unique tumorigenesis profile for PRD mutations that is distinct from null or KD mutations.Significant:This study functionally characterizes the most common ATM missense mutation R3008H in cancer and identifies a unique role of PI3-kinase regulatory domain in ATM activation.

Sup. Figures

Prevention of hypotension during the intra- and postoperative period is an important goal. Peripheral administration of low-concentration norepinephrine may be a safe and effective strategy to reduce the risk of hypotension.We conducted a 2-center, randomized pilot feasibility trial, with a target of 60 adult patients undergoing major noncardiac surgery. We randomized patients to receive a peripheral low-concentration (10 µg/mL) norepinephrine or placebo (saline 0.9%) infusion. The study drug infusion was titrated to achieve a minimum systolic blood pressure target, preselected within 10% of baseline value and within the range limit 100 to 120 mm Hg during surgery and for up to 4 or 24 hours postoperatively.We achieved a high consent rate (84%), successful study drug administration throughout surgery (98% of patients) and absence of unblinding. There were no important study drug-related adverse events. The average intraoperative systolic blood pressure was 120 ± 12.6 mm Hg in the norepinephrine group and 115 ± 14.9 mm Hg in the placebo group. The mean difference between the intraoperative systolic blood pressure achieved less the preselected minimum systolic blood pressure target was 10.0 ± 12.7 mm Hg in the norepinephrine group and 2.9 ± 14.7 mm Hg in the placebo group; difference in means, 7.1 (95% confidence interval, 0.2-14.0) mm Hg.A future large trial evaluating the effectiveness and safety of peripheral administration of low-concentration norepinephrine during the perioperative period is feasible, and likely to achieve a minimum systolic blood pressure threshold.

Discrimination is an urgent public health problem. A number of major cities and counties across the United States has declared racism a public health crisis. While there is a growing body of research on the discrimination-health connection, less is known regarding the social relational consequences of discrimination. The present study addresses this knowledge gap by investigating the relationship between discrimination, skin tone, and objective and subjective social isolation using a nationally representative sample of African Americans.This analysis was based upon the African American subsample (N = 3,570) of the National Survey of American Life. Discrimination was assessed with the Everyday Discrimination Scale. Objective and subjective isolation differentiated between respondents who were (a) socially isolated from both family and friends, (b) socially isolated from friends only, (c) socially isolated from family only, and (d) not socially isolated. Skin tone was self-reported. Multinomial logistic regression analyses were used to test the study hypotheses.The analyses indicated that more frequent discriminatory experiences were associated with increased risk for subjective and objective social isolation. Skin tone moderated the association between discrimination and subjective isolation; the discrimination-isolation relationship was stronger among participants with darker skin tones.These findings shed light on African Americans' nuanced experiences with discrimination and colorism. Further, the data demonstrate heterogeneity in the vulnerability to the adverse effects of discrimination within the African American population; the relationship between discrimination and subjective isolation was stratified by skin tone. This underscores the well-documented and persistent racialized social stratification system in the United States (PsycInfo Database Record (c) 2022 APA, all rights reserved).

ATM kinase is a tumor suppressor and a master regulator of the DNA damage response. Most cancer-associated alterations to ATM are missense mutations at the PI3-kinase regulatory domain (PRD) or the kinase domain. Expression of kinase-dead (KD) ATM protein solely accelerates lymphomagenesis beyond ATM loss. To understand how PRD suppresses lymphomagenesis, we introduced the cancer-associated PRD mutation R3008H (R3016 in mouse) into mice. R3008H abrogated DNA damage- and oxidative stress-induced activation of ATM without consistently affecting ATM protein stability and recruitment. In contrast to the early embryonic lethality of AtmKD/KD mice, AtmR3016H (AtmR/R) mice were viable, immunodeficient, and displayed spontaneous craniofacial abnormalities and delayed lymphomagenesis compared with Atm−/− controls. Mechanistically, R3008H rescued the tardy exchange of ATM-KD at DNA damage foci, indicating that PRD coordinates ATM activation with its exchange at DNA-breaks. Taken together, our results reveal a unique tumorigenesis profile for PRD mutations that is distinct from null or KD mutations. Significant: This study functionally characterizes the most common ATM missense mutation R3008H in cancer and identifies a unique role of PI3-kinase regulatory domain in ATM activation.

The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.

Fil: Reiter, R. Universidad de Texas. Departamento de Sistemas Celulares y Anatomía; Estados Unidos Fil: Sharma, Ramaswamy. Universidad de Texas. Departamento de Sistemas Celulares y Anatomía; Estados Unidos Fil: Simko, Fedor. Universidad Comenius. Facultad de Medicina. Instituto de Fisiopatología; República Eslovaca Fil: Domínguez Rodriguez, Alberto. Hospital Universitario de Canarias. Departamento de Cardiología; España Fil: Tesarik, Jan. Clínica MAR&Gen; España Fil: Neel, Richard L. Alcasian Care Enterprises; Estados Unidos Fil: Slominski, Andrzej T. Universidad de Alabama. Departamento de Dermatología; Estados Unidos Fil: Kleszczynsk, Konrad. Universidad de Münster. Departamento de Dermatología; Alemania Fil: Martin‑Gimenez, Verna M. Universidad Católica de Cuyo. Facultad de Cs. Químicas Y Tecnológicas. Instituto de Investigaciones en Ciencias Químicas; Argentina Fil: Manucha, Walter. Universidad Católica de Cuyo. Facultad de Cs. Químicas Y Tecnológicas. Instituto de Investigaciones en Ciencias Químicas; Argentina Fil: Cardinali, Daniel Pedro. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Abstract: Numerous pharmaceutical drugs have been repurposed for use as treatments for COVID-19 disease. These drugs have not consistently demonstrated high efcacy in preventing or treating this serious condition and all have side efects to difering degrees. We encourage the continued consideration of the use of the antioxidant and anti-infammatory agent, melatonin, as a countermeasure to a SARS-CoV-2 infection. More than 140 scientifc publications have identifed melatonin as a likely useful agent to treat this disease. Moreover, the publications cited provide the rationale for the use of melatonin as a prophylactic agent against this condition. Melatonin has pan-antiviral efects and it diminishes the severity of viral infections and reduces the death of animals infected with numerous diferent viruses, including three diferent coronaviruses. Network analyses, which compared drugs used to treat SARS-CoV-2 in humans, also predicted that melatonin would be the most efective agent for preventing/treating COVID-19. Finally, when seriously infected COVID-19 patients were treated with melatonin, either alone or in combination with other medications, these treatments reduced the severity of infection, lowered the death rate, and shortened the duration of hospitalization. Melatonin’s ability to arrest SARS-CoV-2 infections may reduce health care exhaustion by limiting the need for hospitalization. Importantly, melatonin has a high safety profle over a wide range of doses and lacks signifcant toxicity. Some molecular processes by which melatonin resists a SARS-CoV-2 infection are summarized. The authors believe that all available, potentially benefcial drugs, including melatonin, that lack toxicity should be used in pandemics such as that caused by SARS-CoV-2.

CtIP is a DNA end resection factor widely implicated in alternative end-joining (A-EJ)–mediated translocations in cell-based reporter systems. To address the physiological role of CtIP, an essential gene, in translocation-mediated lymphomagenesis, we introduced the T855A mutation at murine CtIP to nonhomologous end-joining and Tp53 double-deficient mice that routinely succumbed to lymphomas carrying A-EJ–mediated IgH-Myc translocations. T855 of CtIP is phosphorylated by ATM or ATR kinases upon DNA damage to promote end resection. Here, we reported that the T855A mutation of CtIP compromised the neonatal development of Xrcc4(−/−)Tp53(−/−) mice and the IgH-Myc translocation-driven lymphomagenesis in DNA-PKcs(−/−)Tp53(−/−) mice. Mechanistically, the T855A mutation limits DNA end resection length without affecting hairpin opening, translocation frequency, or fork stability. Meanwhile, after radiation, CtIP-T855A mutant cells showed a consistent decreased Chk1 phosphorylation and defects in the G2/M cell cycle checkpoint. Consistent with the role of T855A mutation in lymphomagenesis beyond translocation, the CtIP-T855A mutation also delays splenomegaly in λ-Myc mice. Collectively, our study revealed a role of CtIP-T855 phosphorylation in lymphomagenesis beyond A-EJ–mediated chromosomal translocation.

The classical nonhomologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70–Ku80 (KU) heterodimer and the large DNA-PK catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs−/−) abrogates end processing (e.g., hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD) abrogates end-ligation, suggesting a kinase-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs−/− and DNA-PKcsKD/KD mice because of the requirement for both hairpin opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056 cluster is the best-characterized autophosphorylation site in human DNA-PKcs. In this study, we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053. We also generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retained normal kinase activity and underwent efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053 cluster of murine DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.