DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

The cytoplasmic tyrosine kinase Jak2 plays a crucial role in cytokine receptor signaling in hematopoietic cells. The activated Jak2-V617F mutant is present in most cases of BCR/ABL-negative myeloproliferative neoplasms and constitutively activates downstream signals from homodimeric cytokine receptors, such as the erythropoietin receptor (EpoR). Here we examine the effects of DNA damage stress on Jak2 or Jak2-V617F and on induction of apoptosis in hematopoietic cells. Etoposide or doxorubicin dose-dependently decreased the expression level of Jak2 in UT7 or 32D cells expressing EpoR in the absence of Epo and that of exogenously expressed Jak2-V617F in UT7 cells when cotreated with the Jak2 inhibitor JakI-1 or AG490. Studies with pharmacological inhibitors and genetic manipulations further showed that downregulation of the PI3K/Akt pathway leading to the activation of GSK3β may be involved in downregulation of Jak2 or Jak2-V617F as well as in synergistic induction of Bax activation and apoptosis. The downregulation of Jak2 was inhibited by the proteasome inhibitor MG132 or by expression of both of loss-of-function mutants of c-Cbl and Cbl-b, E3 ubiquitin ligases which facilitated ubiquitination of Jak2-V617F when co-expressed in 293T cells. The pan-caspase inhibitor Boc-d-fmk also inhibited the Jak2 downregulation as well as appearance of a 100-kDa fragment that contained the N-terminal portion of Jak2 in response to DNA damage. Together, these data suggest that DNA damage stress with simultaneous inhibition of the kinase activity causes degradation of Jak2 or Jak2-V617F by caspase cleavage and proteasomal degradation through GSK3β activation, which is closely involved in synergistic induction of apoptosis in hematopoietic cells

Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL, Jak2-V617F, or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis

<div><p>Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.</p></div

Etoposide as well as doxorubicin downregulates Jak2 and Jak2-V617F when they are inactivated.

<p>(A) After cultured for 9 h in medium without Epo, UT7 cells were left untreated or treated with 5 µM etoposide (VP16) or 0.5 µM doxorubicin (DXR) for 4 hr in the absence or presence of 50 mU/ml Epo, as indicated. Cells were lysed and subjected to immunoblot analysis with anti-Jak2 antibody, followed by reprobing with anti-EpoR and anti-β-actin, as indicated. (B, C) After cultured for 3 h in medium without Epo, 32D/EpoR cells were treated for 5 h in the absence or presence of 100 mU/ml Epo, as indicated, with increasing concentrations of etoposide (C) or doxorubicin (D), as indicated. Cell lysates were analyzed by immunoblotting with antibodies against indicated proteins. (D) 32D/EpoR or parental 32Dcl3 cells, cultured in medium containing 10% WEHI conditioning medium as the source of IL-3, were washed out of cytokine for 1 h. Cells were further cultured with or without 5 µM etoposide (VP16) for 6 h, as indicated, and analyzed. (E, F) After cultured for 9 h in medium without Epo, UT7/Jak2-V617F cells were treated for 1 h with or without 2 µM JakI-1. Cells were subsequently treated with increasing concentrations of etoposide (E; 0, 1, 2, 5 µM) or doxorubicin (D; 0, 0.1, 0.2, 0.5 µM), as indicated, and analyzed. (G) UT7/Jak2-V617F cells starved from Epo were pretreated with indicated concentrations of AG490 for 1 h. Cells were then treated with 5 µM etoposide or 0.5 µM doxorubicin for 6 h, as indicated, and analyzed.</p

GSK3ß regulates etoposide-induced Chk1 activation and apoptosis in cytokine- or BCR/ABL-driven hematopoietic cells.

<p>(<b>A</b>) 32D/EpoR/pMXs-IG cells (Cont.) or the cells overexpressing the kinase inactive (KI) or S9A (SA) mutant as well as the wild-type (WT) GSK3ß, as indicated, were cultured for 16 h with 0.5 µM etoposide in the presence of 0.1 U/ml Epo and analyzed for the cellular DNA content. Percentages of apoptotic cells with sub-G1 DNA content and those of cells in the G2/M phase are plotted. Each data point represents the mean of three independent experiments, with error bars indicating standard deviations. The asterisks indicate statistically significant differences determined by Student’s <i>t</i>-test (*p<0.05, **p<0.01). (<b>B</b>) The cells indicated as in A were treated with indicated concentrations of etoposide for 7 h in the presence of 0.1 U/ml Epo and analyzed by Western blot analysis using indicated antibodies. Relative levels of Chk1-S345P, determined by densitometric analysis, are shown. (<b>C</b>) 32Dp210 cells were cultured for 16 h with 1 µM etoposide alone (Cont.) or also with 1 µM imatinib and 1 µM GSK3-I #5 (GSK3-I), as indicated, and analyzed for the cellular DNA content. (<b>D</b>) 32Dp210 cells were pretreated for 1 h with 1 µM imatinib, 1 µM GSK3-I #5, or 5 µM MG132, as indicated, or left untreated (Cont.). Cells were then treated with 1 µM etoposide for indicated times and analyzed by Western blot analysis.</p

Imatinib inhibits the G2/M arrest and prominently induces apoptosis in BCR/ABL-expressing cells treated with etoposide.

<p>(<b>A</b>) 32Dp210 cells were cultured for 24 h with 0.5 µM etoposide (Etop.), 0.6 µM imatinib, or 1 µM SB218078, as indicated, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content and those of cells in the G2/M phase are indicated. (<b>B</b>) K562 cells were cultured for 24 h with 1 µM etoposide, 1 µM imatinib, or 1 µM SB218078, as indicated, and analyzed. (<b>C</b>) 32Dp210 cells were cultured for 16 h with or without 50 ng/ml nocodazole in the presence of 1 µM etoposide and 1 µM imatinib, as indicated, and analyzed for the cellular DNA content by flow cytometry and for the mitotic index, as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079478#s2" target="_blank">Materials and methods</a>. Each data point represents the mean of three independent experiments, with error bars indicating standard deviations. The asterisk indicates a statistically significant difference determined by Student’s <i>t</i>-test (<i>p</i><0.01). (<b>D</b>) 32Dp210 cells were cultured for 16 h with 1 µM etoposide and 1 µM imatinib, as indicated, in the presence of 50 ng/ml nocodazole. Cells were analyzed for the DNA content and histone H3 phosphorylated on S10 (H3-S10-P) by flow cytometry. Percentages of cells in G2/M that are positive for H3-S10-P are indicated.</p

PI3K/Akt upstream of GSK3ß regulates etoposide-induced Chk1 activation and apoptosis in cytokine- or BCR/ABL-driven cells.

<p>(<b>A</b>) 32Dp210/Rev (Cont.) or 32Dp210/Rev-Akt1-myr (Akt-myr) cells were cultured with 0.5 µM etoposide (Etop.) or 2 µM imatinib, as indicated, for 24 h and analyzed for the cellular DNA content. (<b>B</b>) Ton.B210 cells cultured with DOX to induce BCR/ABL expression (BCR/ABL) in the absence of IL-3 or cultured without DOX in the presence of IL-3 (IL-3) were pretreated for 1 h with 1 µM GDC-0941 (GDC) or 5 µM MK-2206 (MK) or left untreated as control (Cont.). Cells were subsequently treated with 0.5 µM etoposide for 12 h or left untreated as control (Cont.), as indicated, and analyzed for the cellular DNA content. (<b>C</b>) Ton.B210 cells expressing BCR/ABL (BCR/ABL) or cultured with IL-3 (IL-3) were pretreated for 30 min with 2 µM GDC-0941, 3 µM MK-2206, or 20 µM SB216763, as indicated, or left untreated as control. Cells were subsequently treated with 1 µM etoposide for 8 h or left untreated, as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. A position of the caspase-cleaved fragment of PARP is indicated by an asterisk. (<b>D</b>) 32Dp210 cells were precultured for 1 h with 20 µM SB216763 or left untreated for control, as indicated. Cells were then treated for 24 h with 0.5 µM etoposide and 1 µM GDC-0941 or 5 µM MK-2206, as indicated, and analyzed.</p

Synergistic induction of apoptosis in UT7/Jak2-V617F by etoposide and JakI-1 and effects of Boc-d-fmk on Jak2 downregulation.

<p>(A) UT7/Jak2-V617F cells were cultured for 12 h with 0.5 µM etoposide (VP16), 0.5 µM JakI-1, 25 µM LY294002, and 1 µM GSK3I-5, as indicated in the absence of Epo, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (B) UT7/Jak2-V617F cells were cultured for 6 h with 5 µM etoposide (VP16), 1 µM JakI-1, and 100 µM Boc-d-fmk, as indicated, in the absence of Epo and analyzed for the cellular DNA content. (C, D) 32D/EpoR cells deprived of Epo for 2 h were pretreated for 1 h with 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated for 5 h with or without 5 µM etoposide (VP16) or 0.5 µM doxorubicin (DXR), as indicated. Cells were lysed and analyzed by immunoblotting. (E) UT7/Jak2-V617F cells, cultured without Epo for 12 h, were treated with 1 µM JakI-1 and 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated with or without 5 µM etoposide (VP16), as indicated, for 6 h and analyzed.</p

Involvement of the PI3K/Akt/GSK3β pathway in downregulation of Jak2 in response to DNA damage.

<p>(A) After cultured for 9 h in medium without Epo, UT7 cells were pretreated for 1 h with 50 µM LY294002 (PI3K-I) or 50 µM PD98059 (MEK-I), as indicated, or left untreated. Cells were subsequently treated with or without 10 µM etoposide (VP16) for 4 h, as indicated, in the presence of 20 mU/ml Epo or in its absence (Epo -). Cells were lysed and subjected to immunoblot analysis with anti-Jak2 antibody, followed by sequential reprobing with anti-phospho-GSK3α/β-S9/21 (GSK3β-P), anti-GSK3β, anti-β-actin, as indicated. (B) 32D/Akt-myr (Akt-myr) as well as control 32D/RevTRE (Cont.) cells were cultured for 24 h with 1 µg/ml doxycycline to induced the expression of Akt-myr in 32D/Akt-myr cells and subsequently washed out of WEHI conditioning medium for 12 h. Cells were then pretreated for 1 h with 1 µM JakI-1 or 10 µM LY294002 (PI3K-I), as indicated, or left untreated. Cells were finally treated with or without 10 µM etoposide (VP16), as indicated, for 4 h before analysis with indicated antibodies. (C) After cultured for 9 h in medium without Epo, UT7 cells were pretreated for 1 h with 10 µM SB216763 (SB216), 40 mM LiCl, or okadaic acid at 100 nM (OA100) or 200 nM (OA200), as indicated, or left untreated. Cells were subsequently treated with or without 10 µM etoposide (VP16) for 4 h, as indicated, and analyzed. (D) 32DE/STAT5A1*6 (STAT5A1*6) or control 32DE/pMX (Cont.) cells were pretreated for 1 h with 1 µM JakI-1 or 50 µM LY294002 (PI3K-I), as indicated, or left untreated in the absence of Epo. Cells were further treated with or without 5 µM etoposide (VP16) for 6 h, as indicated, before analysis. (E) 32DE/STAT5A1*6 (STAT5A1*6) or control 32DE/pMX (Cont.) cells were cultured overnight in the absence of Epo. Cells were lysed and subjected to immunoprecipitation of p85. Immunoprecipitates were analyzed by immunoblotting. (F) After cultured for 12 h in medium without Epo, UT7/Jak2-V617F cells were pretreated for 1 h with 50 µM LY294002 (PI3K-I), 2 µM JakI-1, 40 mM LiCl, or 10 µM MG132, as indicated, or left untreated as control (Cont.). Cells were subsequently treated with or without 5 µM etoposide (VP16), as indicated, for 6 h and analyzed.</p

Sorafenib or GDC-0941 inhibits etoposide-induced Chk1 activation and enhances apoptosis in cells expressing T315I-mutated BCR/ABL.

<p>(<b>A</b>) Ton.B210/T315I cells cultured with DOX to induce BCR/ABL with T315I (BCR/ABL-T315I) in the absence of IL-3 or cultured without DOX in the presence of IL-3 (IL-3) were left untreated as control (Cont.) or treated with 5 µM sorafenib and 1 µM etoposide (Etop.), as indicated, for 16 h, and analyzed for the cellular DNA content. (<b>B</b>) Ton.B210/T315I cells expressing BCR/ABL with T315I (T315I) or cultured with IL-3 (IL-3) were left untreated or treated with 10 µM sorafenib, as indicated, for 1 h. Cells were subsequently cultured with or without 1 µM etoposide for 6 h and subjected to Western blot analysis. A position of the caspase-cleaved fragment of PARP is indicated by an asterisk. (<b>C</b>) Ton.B210/T315I cells cultured with DOX were left untreated as control (Cont.) or treated with 5 µM imatinib, 50 nM dasatinib, 1 µM GDC-0941 (GDC) in the presence or absence of 0.5 µM etoposide, as indicated, for 16 h, and analyzed for the cellular DNA content. (<b>D</b>) Ton.B210/T315I cells cultured with DOX were left untreated or treated with 5 µM imatinib, 50 nM dasatinib, 1 µM GDC-0941, as indicated, for 1 h. Cells were subsequently cultured with or without 1 µM etoposide for 6 h and subjected to Western blot analysis.</p