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. 2015 Dec 22;13(11):2345-2352.
doi: 10.1016/j.celrep.2015.11.037. Epub 2015 Dec 10.

RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability

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Free PMC article

RECQL5 Suppresses Oncogenic JAK2-Induced Replication Stress and Genomic Instability

Edwin Chen et al. Cell Rep. .
Free PMC article

Abstract

JAK2V617F is the most common oncogenic lesion in patients with myeloproliferative neoplasms (MPNs). Despite the ability of JAK2V617F to instigate DNA damage in vitro, MPNs are nevertheless characterized by genomic stability. In this study, we address this paradox by identifying the DNA helicase RECQL5 as a suppressor of genomic instability in MPNs. We report increased RECQL5 expression in JAK2V617F-expressing cells and demonstrate that RECQL5 is required to counteract JAK2V617F-induced replication stress. Moreover, RECQL5 depletion sensitizes JAK2V617F mutant cells to hydroxyurea (HU), a pharmacological inducer of replication stress and the most common treatment for MPNs. Using single-fiber chromosome combing, we show that RECQL5 depletion in JAK2V617F mutant cells impairs replication dynamics following HU treatment, resulting in increased double-stranded breaks and apoptosis. Cumulatively, these findings identify RECQL5 as a critical regulator of genome stability in MPNs and demonstrate that replication stress-associated cytotoxicity can be amplified specifically in JAK2V617F mutant cells through RECQL5-targeted synthetic lethality.

Figures

Figure 1
Figure 1. JAK2V617F increases expression of RECQL5
(A) Gene expression profiles depicting expression of 25 DNA helicases in 40 MPN patients (20 ET, 16 PV and 4 MF). (B) qPCR validation of RECQL5 and RECQL1 expression in ET (blue) or PV (red) patients. (C) RECQL5 expression in SET-2 and HEL cells following treatment with 0–8 mM of the JAK2 inhibitor INCB18424. (D–E) Expression of Recq family members in WT-B8 and VF-B8 cells by qPCR (Panel D) and Western immunoblot (Panel E). (F) Recql5 expression in WT-B8 cells and in VF-B8 cells following knockdown of Jak2 (sh-Jak2), p85 subunit of Pi3-kinase (sh-Pi3k), Stat1 (sh-Stat1) or Stat5 (sh-Stat5). (G) RECQL5 expression in HEL cells following knockdown of JAK2 (sh-JAK2), STAT5 (sh-STAT5) or STAT1 (sh-STAT1). (H) RECQL5 expression in HEL cells following treatment for 16h with INCB018424 (1 µM), PI3K inhibitor PI103 (1 µM) or ERK1/2 inhibitor U0126 (5 µM). See also Figure S1.
Figure 2
Figure 2. Recql5 depletion sensitizes Jak2V617F-expressing cells to replication stress
(A–F) Viability of WT-B8 (black) and VF-B8 (red) cells transduced with empty vector (VA) or Recql5-targeting shRNAs, following serum deprivation in 0.5% FCS (SD) (Panel A), SD with 100 µM dNTPs (SD+dNTPs) (Panel B), HU (0–250 µM) (Panel C), CPT (0–10 nM) (Panel D), DOX (0–25 µM) (Panel E) or ETP (0–10 nM) (Panel F). For VA-transduced cultures, each point represents the mean of 3 independent cultures. For Recql5-knockdown cultures, each point represents the mean of 3 separate Recql5-targeting shRNAs. (G) Quantitation of annexin V-positivity in cells treated with 100 µM HU or 4 nM CPT. (H–J) VF-B8 cells were transduced with Recql5-targeting shRNA simultaneously with either a wildtype Recql5 cDNA (RQ5wt), shRNA-resistent Recql5 cDNA (RQ5res), or RQ5res cDNA harboring a K58R mutation (RQ5res-K58R). Immunoblotting for Recql5 protein levels was performed (Panel H) and cell viability was assessed (Panel I–J). Testing for statistical significance was performed using a student’s t-test (*: p<0.05; **: p<0.01; ***: p<0.001). See also Figure S2.
Figure 3
Figure 3. Recql5 protects against collapse of stalled replication forks in Jak2V617F-expressing cells exposed to replication stress
(A) Schematic of time course for chromosome combing experiments (top). Representative replication structures of a single combed DNA molecule labelled with IdU (red) and CldU (green) (bottom). (B) Fork rate in WT-B8 and VF-B8 cells transduced with empty vector alone (va) or with two different Recql5-targeting shRNAs (sh1 and sh2). Results represent the mean±S.D. for at least 50 fibres. (C) Quantification of HU-induced effects on fork dynamics. Relative fork progress is depicted as normalized ratio of the second (HU-treated) labelling step relative to the first (HU-free) labelling step. (D–F) Schematic of timeline for fork restart experiments (Panel D). Quantification of BrdU-positive cells (Panel E). Representative flow cytometric plots of BrdU staining (Panel F). (G–H) Quantification of γH2Ax-marked double-stranded breaks as assessed by immunofluorescent detection (Panel G) and Western immunoblot (Panel H). (D–H) Testing for statistical significance was performed using a student’s t-test (*: p<0.05; **: p<0.01). See also Figure S2.
Figure 4
Figure 4. RECQL5 depletion sensitizes JAK2V617F-positive cells from MPN patients to replication stressors
(A) Work flow for determining effect of RECQL5 knockdown on HU sensitivity of primary MPN samples. (B–C) CD34+ peripheral blood mononuclear cells from 3 myelofibrosis patients were transduced with a non-targeting shRNA (scr) or RECQL5-targeting shRNA (sh1 and sh2) and cultured on methylcellulose in the presence and absence of 2 µM HU. After 14 days, total BFU-Es were counted (Panel B) and genotyped for JAK2V617F (Panel C). (D) Model depicting interactions between JAK2V617F, RECQL5 depletion and exogenous pharmacological replication stress. See also Figure S4.

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