Abstract
The JAK2V617F mutation is present in a majority of patients with chronic myeloproliferative neoplasms (MPNs). Mutant JAK2 induces hyperactivation of multiple downstream signaling processes with the net effect of conferring cells with a pro-survival advantage. In particular, JAK2V617F-expressing cells tolerate increased DNA damage and higher levels of intracellular reactive oxygen species. However, the mechanisms by which increased genotoxic tolerance is mediated remain unclear.
Previously, we performed gene expression analysis on autologous wildtype and JAK2V617F-heterozygous erythroblasts from 36 MPN patients, and observed increased expression of the RECQL5 helicase in JAK2-mutant erythroblasts. Increased Recql5 transcript and protein levels were also validated in Hoxb8-immortalized, GMP-like cell lines derived from wildtype and Jak2V617F knock-in mice (WT-B8 and VF-B8 cells, respectively). Recql5 up-regulation was dependent on the Pi3k-Akt pathway, and was independent of Stat1/5 and Mapk/Erk activity. As the Recql family of helicases plays a critical role in replication fork stability, we tested whether Recql5 could modulate sensitivity of JAK2-expressing cells to agents which promote replication stress, such as hydroxyurea (HU) and aphidicolin (APH). Strikingly, VF-B8 cells transduced with two different Recql5 shRNAs were more susceptible to HU- and APH-induced apoptosis when compared to Recql5-deficient WT-B8 cells. Replication stress-induced cytotoxicity was accompanied by increased gamma-H2Ax-marked double stranded breaks (DSBs) and activation of DNA repair pathways. Importantly, re-introduction of an shRNA-resistant Recql5 cDNA successfully rescued Recql5-deficient VF-B8 cells from HU- and APH-cytotoxicity.
Molecularly, we show that Recql5 plays two roles to protect against DSB formation and cell death in Jak2-mutant cells. First, we visualized replication tracts on individual DNA fibers by chromosome combing and observed that Recql5-deficient VF-B8 cells treated with HU exhibit increased numbers of stalled replication forks. Moreover, Recql5 deficiency also led to an inability to restart forks stalled by HU treatment. This indicates that the absence of Recql5 leads to replication forks which are unstably arrested upon HU treatment, leading to fork collapse and the generation of DSBs. Second, we quantified the rate of single-stranded annealing (SSA) repair following Recql5 knockdown. Consistent with previous reports, we observed increased rates of SSA repair in VF-B8 cells compared to WT-B8 cells. However, this difference in the rate of SSA repair is abrogated upon Recql5 knockdown, suggesting that Recql5 functions as a mediator for the SSA DNA repair pathway. Cumulatively, these findings suggest that Recql5 up-regulation in Jak2V617F-expressing cells plays a role in protecting against DNA damage-induced cell death through (1) stabilization of stalled replication forks thus preventing their collapse, and (2) promoting rapid (albeit error prone) SSA DNA repair to ameliorate genomic instability.
Finally, we tested whether modulation of RECQL5 could also increase sensitivity of JAK2V617F-positive cells from primary MPN patients to HU. Following depletion with RECQL5, c-kit-enriched peripheral blood mononuclear cells from 2 essential thrombocythemia and 3 myelofibrosis patients were grown in semi-solid medium supplemented with HU for 14 days. Strikingly, we observed more specific eradication of JAK2V617F-positive erythroid progenitor colonies compared to autologous wildtype colonies from all patients examined. In contrast, no specific killing of JAK2V617F-positive erythroblasts was seen following transduction of control hairpins. This suggests that RECQL5 knockdown may potentially open a therapeutic window by sensitizing Jak2V617F-expressing cells to HU and other agents that induce replication stress.
Disclosures
No relevant conflicts of interest to declare.
The Nse5-Nse6 Dimer Mediates DNA Repair Roles of the Smc5-Smc6 Complex