JAK inhibition with tofacitinib rapidly increases contractile force in human skeletal muscle.

Reduction in muscle contractile force associated with many clinical conditions incurs serious morbidity and increased mortality. Here, we report the first evidence that JAK inhibition impacts contractile force in normal human muscle. Muscle biopsies were taken from patients who were randomized to receive tofacitinib (n = 16) or placebo (n = 17) for 48 h. Single-fiber contractile force and molecular studies were carried out. The contractile force of individual diaphragm myofibers pooled from the tofacitinib group (n = 248 fibers) was significantly higher than those from the placebo group (n = 238 fibers), with a 15.7% greater mean maximum specific force (P = 0.0016). Tofacitinib treatment similarly increased fiber force in the serratus anterior muscle. The increased force was associated with reduced muscle protein oxidation and FoxO-ubiquitination-proteasome signaling, and increased levels of smooth muscle MYLK. Inhibition of MYLK attenuated the tofacitinib-dependent increase in fiber force. These data demonstrate that tofacitinib increases the contractile force of skeletal muscle and offers several underlying mechanisms. Inhibition of the JAK-STAT pathway is thus a potential new therapy for the muscle dysfunction that occurs in many clinical conditions.

ATM and 53BP1 regulate alternative end joining-mediated V(D)J recombination.

G0-G1 phase alternative end joining (A-EJ) is a recently defined mutagenic pathway characterized by resected deletion and translocation joints that are predominantly direct and are distinguished from A-EJ in cycling cells that rely much more on microhomology-mediated end joining (MMEJ). Using chemical and genetic approaches, we systematically evaluate potential A-EJ factors and DNA damage response (DDR) genes to support this mechanism by mapping the repair fates of RAG1/2-initiated double-strand breaks in the context of Igκ locus V-J recombination and chromosome translocation. Our findings highlight a polymerase theta-independent Parp1-XRCC1/LigIII axis as central A-EJ components, supported by 53BP1 in the context of an Ataxia-telangiectasia mutated (ATM)-activated DDR. Mechanistically, we demonstrate varied changes in short-range resection, MMEJ, and translocation, imposed by compromising specific DDR activities, which include polymerase alpha, Ataxia-telangiectasia and Rad3-related (ATR), DNA2, and Mre11. This study advances our understanding of DNA damage repair within the 53BP1 regulatory domain and the RAG1/2 postcleavage complex.

DNA-PKcs suppresses illegitimate chromosome rearrangements.

Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in humans and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-exclusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.