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. 2014 Oct 21;111(42):15190-5.
doi: 10.1073/pnas.1401873111. Epub 2014 Oct 6.

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response

Affiliations

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response

Edwin Chen et al. Proc Natl Acad Sci U S A. .

Abstract

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

Keywords: JAK2V617F; myeloproliferative neoplasm; replication stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
JAK2V617F induces replication fork stalling and activates the intra-S checkpoint in BJ human diploid fibroblasts. (A) Replication structures of single combed DNA molecules from BJWT and BJV617F cells labeled with IdU (red) and CldU (green), showing origins of replication (Orig), progressing forks (Prog), and stalled forks (Stall). (B) Scoring of fibers for different fork structures from BJWT and BJV617F cells. Fibers from each slide were scored by two independent observers. The data represent the mean ± SD of three independent experiments. (C) Quantitation of fork rate from BJWT and BJV617F cells. Results represent the mean ± SD for 110 fibers from BJWT cells and 65 fibers from BJV617F cells. (D) Origin symmetry in BJWT and BJV617F cells. Each point represents a single origin, with the length of one arm (arbitrarily designated as “left”) plotted relative to its opposite moving counterpart (designated as “right”). The dotted line represents a ratio of 0.7 between the two arms. (E) Analysis of pS345-CHK1 levels in BJWT and BJV617F cells by intracellular flow cytometry. The results are representative of four experiments. (F–H) Analysis of doubling time (F), cell death (G), and S-phase duration (H) of BJWT and BJV617F cells. (B–D) Testing for significance was performed using an unpaired t test (C and D) or a one-sample t test (E). *P < 0.05; **P < 0.01; ***P < 0.001. WT, wild-type.
Fig. 2.
Fig. 2.
JAK2V617F-induced fork stalling is PI3K-dependent. (A and B) pS345-CHK1 levels in BJWT and BJV617F cells after transfection with scrambled siRNA control (scr) or either pools of three to five siRNAs targeting STAT1, STAT5, or the p85 subunit of the PI3K (PIK3CA) (A) or individual siRNAs targeting STAT1, STAT5, or PIK3CA (B). (C and D) BJWT and BJV617F cells transfected with control siRNA or siRNAs targeting STAT1, STAT5, or PIK3CA were scored for frequency of origin (O), normal progressing forks (P), or stalled forks (S) (C) or fork rate (D). (AD) Data represent the mean ± SD of three independent experiments. Testing for significance was performed using an unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001. WT, wild-type.
Fig. 3.
Fig. 3.
JAK2V617F causes replication fork stalling in primary MPN patient samples. (A) Strategy for analysis of paired normal and JAK2V617F samples from MPN patients. (B) Scoring of fibers for structures resembling origins (Orig), progressing (Prog), or stalled (Stall) forks from erythroblasts from PV and ET patients. (C and D) Measurement of fork rate (C) and origin symmetry (D) in wild-type and JAK2-heterozygous erythroblasts from ET and PV patients. (E) Scoring of fork structures in control and PI103-treated wild-type and mutant erythroblasts. The results represent the mean ± SD for three MPN patients (one PV, two ET). (F) Fork rates of control and PI103-treated wild-type and mutant erythroblasts. The results represent the mean ± SD for three MPN patients (one PV, two ET). (B–F) The data are represented as mean ± SD. Each patient’s fibers were scored by two independent observers. Testing for statistical significance was performed using an unpaired t test or a one-sample t test. *P < 0.05; **P < 0.01; ***P < 0.001. WT, wild-type.
Fig. 4.
Fig. 4.
Disease-specific impairment of intra-S checkpoint activity. (A) Exemplary intracellular phospho-flow cytometry of pS345-CHK1 expression in autologous wild-type and V617F-heterozygous BFU-E colonies from ET and PV patients. (B) Dot plot depicting ratio of pCHK1 MFI in JAK2-mutant erythroblasts relative to autologous wild-type erythroblasts from seven ET and nine PV patients. (C) Immunofluorescent staining of cytospins from wild-type and V617F-heterozygous BFU-E pools from PV and ET patients for RPA (red), ATR (green), and DAPI (blue). The bar graphs depict median foci number per cell from three ET and four PV patients. (D) RPA, ATR, and DAPI staining of cytospins from wild-type and V617F-heterozygous BFU-E pools from PV patients grown in 4 nM camptothecin. Bar graph depicts data from four PV patients. (E–G) Effect of camptothecin treatment on cell death (E), cell proliferation (F), and colony size (G). For proliferation and cell death, the data are depicted as a normalized fold change relative to the untreated wild-type sample for each disease group. The results represent the mean ± SD for five ET and three PV patients. (B–G) Testing for statistical significance was performed using a Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. WT, wild-type.
Fig. 5.
Fig. 5.
Loss of p53 activity increases DNA damage in PV JAK2-mutant cells. (A) Representative immunocytochemical micrographs of γ-H2Ax (green) and DAPI (blue) staining of cytospins from wild-type and V617F-heterozygous BFU-E pools from PV and ET patients cultured in the presence of DMSO vehicle or 10 nM PFT-α. (B) Histogram depicts median γ-H2Ax foci per cell for three ET and three PV patients cultured with and without PFT-α. Testing for statistical significance was performed using a Student t test. *P < 0.05; **P < 0.01. (C–E) Effect of PFT-α treatment on cell death (C), cell proliferation (D), and colony size (E). For proliferation and cell death, the data are depicted as a normalized fold change relative to the untreated wild-type sample for each disease group. The results represent the mean ± SD for three ET and three PV patients. WT, wild-type.

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