Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

Abstract

Uterine fibroid (UF) driver mutations in Mediator complex subunit 12 (MED12) trigger genomic instability and tumor development through unknown mechanisms. Herein, we show that MED12 mutations trigger aberrant R-loop-induced replication stress, suggesting a possible route to genomic instability and a novel therapeutic vulnerability in this dominant UF subclass. Immunohistochemical analyses of patient-matched tissue samples revealed that MED12 mutation-positive UFs, compared to MED12 mutation-negative UFs and myometrium, exhibited significantly higher levels of R-loops and activated markers of Ataxia Telangiectasia and Rad3-related (ATR) kinase-dependent replication stress signaling in situ. Single molecule DNA fiber analysis revealed that primary cells from MED12 mutation-positive UFs, compared to those from patient-matched MED12 mutation-negative UFs and myometrium, exhibited defects in replication fork dynamics, including reduced fork speeds, increased and decreased numbers of stalled and restarted forks, respectively, and increased asymmetrical bidirectional forks. Notably, these phenotypes were recapitulated and functionally linked in cultured uterine smooth muscle cells following chemical inhibition of Mediator-associated CDK8/19 kinase activity that is known to be disrupted by UF driver mutations in MED12. Thus, Mediator kinase inhibition triggered enhanced R-loop formation and replication stress leading to an S-phase cell cycle delay, phenotypes that were rescued by overexpression of the R-loop resolving enzyme RNaseH. Altogether, these findings reveal MED12-mutant UFs to be uniquely characterized by aberrant R-loop induced replication stress, suggesting a possible basis for genomic instability and new avenues for therapeutic intervention that involve the replication stress phenotype in this dominant UF subtype.

Engineered MED12 mutations drive uterine fibroid-like transcriptional and metabolic programs by altering the 3D genome compartmentalization

“…To this end, we measured γ-H2AX as a proxy for DNA damage/repair activity. Notably, in line with recent ndings 34 , we did not see a signi cant increase in total γ-H2AX at the basal level by western blot (Fig. 4b).…”

“…9). The MED12 mutant Fibroid tumors are known to have higher abnormal progression of the replication fork and increased R-loop formation 34 . We, therefore, tested whether primary broid tumors also have higher DNA damage/repair activity compared to normal myometrium.…”

Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond

“…The targeting of R-loops with topoisomerase and poly ADP ribose polymerase inhibitors showed promising treatment strategies for this deadly disease [ 27 ]. Aberrant, unscheduled R-loop accumulation in Uterine fibroid (UF) patients (benign monoclonal neoplasms of the myometrium, which represent the most frequent non-cutaneous tumors) was demonstrated to specify potentially malignant tumor progression in the dominant UF subtype [ 124 ] ( Figure 2 A). In a study on Epidermal growth factor receptor variant III (EGFRvIII)-positive glioblastoma (GBM) patients, increased R-loop accumulation and genome instability caused by replication stress have been suggested to be associated with tumor heterogeneity and allow for the stratification of patients for individualized therapeutic approaches [ 125 ] ( Figure 2 B).…”

“…Along with an elevated expression of para-speckle genes, R-loops have been identified to correlate with MM’s progression [ 119 ]. Altogether, unscheduled R-loop accumulation could be used to identify distinct biological properties (heterogeneity) in tumors [ 119 , 124 , 125 ], can guide the monitoring of the severity of disorders [ 25 ], and has a strong prognostic potential.…”

“…Benign and Premalignant tumor Uterine fibroids are tissue-specifically characterized by aberrant R-loop accrual and its binding protein . Immunohistochemistry (IHC) staining with (upper panel) R-loop-specific S9.6 and (lower panel) pATR (Ser428)-specific antibodies indicating phosphorylated (activated) replication stress signaling correlative with R loop accumulation in different mutants (Re-adapted from Ref [ 124 ]). ( B ).…”