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

Impact of genetic alterations on central nervous system progression of primary vitreoretinal lymphoma

Primary vitreoretinal lymphoma (PVRL) is a rare malignant lymphoma subtype with an unfavorable prognosis due to frequent central nervous system (CNS) progression. Thus, identifying factors associated with CNS progression is essential for improving the prognosis of PVRL patients. Accordingly, we conducted a comprehensive genetic analysis using archived vitreous humor samples of 36 PVRL patients diagnosed and treated at our institution and retrospectively examined the relationship between genetic alterations and CNS progression. Whole-exome sequencing (n = 2) and amplicon sequencing using a custom panel of 107 lymphomagenesis-related genes (n = 34) were performed to assess mutations and copy number alterations. The median number of pathogenic genetic alterations per case was 12 (range: 0– 22). Pathogenic genetic alterations of CDKN2A, MYD88, CDKN2B, PRDM1, PIM1, ETV6, CD79B, and IGLL5, as well as aberrant somatic hypermutations, were frequently detected. The frequency of ETV6 loss and PRDM1 alteration (mutation and loss) was 23% and 49%, respectively. Multivariate analysis revealed ETV6 loss (hazard ratio [HR]: 3.26, 95% confidence interval [CI]: 1.08–9.85) and PRDM1 alteration (HR: 2.52, 95% CI: 1.03–6.16) as candidate risk factors associated with CNS progression of PVRL. Moreover, these two genetic factors defined slow-, intermediate-, and rapid-progression groups (0, 1, and 2 factors, respectively), and the median period to CNS progression differed significantly among them (52 vs. 33 vs. 20 months, respectively). Our findings suggest that genetic factors predict the CNS progression of PVRL effectively, and the genetics-based CNS progression model might lead to stratification of treatment

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

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

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

Inhibition of Jak2-V617F downregulates etoposide-induced Chk1 activation as well as G2/M arrest and elicits apoptosis in a GSK3-dependent manner.

<p>(<b>A</b>) UT7/Jak2-V617F cells were cultured for 16 h with 0.5 µM etoposide (Etop.), 0.2 µM JakI-1, or 1 µM GSK3-I #5, as indicated, in the absence of Epo. Cells were then analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content (s-G1) and those of cells in the G2/M phase (G2/M) are indicated. (<b>B</b>) UT7-Jak2-V617F cells were pretreated for 60 min with 1 µM JakI-1 or 40 mM LiCl, as indicated, and subsequently treated with indicated concentrations of etoposide for 6 h. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Positions of GSK3ß are indicated by arrows, while the caspase-cleaved fragment of PARP by an asterisk.</p

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

Possible involvement of ubiquitin-proteasome pathway and Cbls in downregulation of Jak2.

<p>(A) 32D/EpoR cells deprived of Epo for 2 h were pretreated with 10 µM MG132 or left untreated as control, as indicated for 1 h in the absence of Epo. Cells were then treated for 6 h with or without 5 µM etoposide, as indicated. Cells were lysed and subjected to immunoblot analysis using indicated antibodies. (B) 293T cells were transfected on 6-well plate with 0.1 µg of pRK5-Ubiquitin-WT and 0.002 µg of pRK5-Jak2-Wt (Wt) or pRK5-Jak2-KE (KE) along with 0.1 µg of pXM-EpoR-Wt or empty plasmid, as indicated. Two days after transfection, cells were lysed, and Jak2 was immunoprecipitated. Immunoprecipitates were analyzed by immunoblotting using antibodies against polyubiquitin (poly-Ubi), Jak2, and Jak2 phosphorylated on Y1007 (Jak2-PY), as indicated. The vertical line indicates the smeary pattern characteristic of ubiquitination. (C) 293T cells were transfected on 6-well plate with 0.3 µg of pRevTRE-Jak2V617F and 0.2 µg of pTet-On along with 0.2 µg of pRK5-Ubiquitin-WT (Ubi), 0.5 µg of pRevTRE-c-Cbl (c-Cbl), or 0.5 µg of pRevTRE-Cbl-b (Cbl-b), as indicated. The amounts of plasmid DNA transfected were equalized by adding pRevTRE. Two days after transfection, cells were lysed, and lysates were analyzed by immunoblotting. The position of unmodified Jak2-V617F and those of ubiquitinated Jak2-V617F are indicated by asterisks and arrows, respectively. (D) Ton.32D/Flt3-Wt (Cont.) and Ton.32D/Flt3-Wt/c-Cbl-RQ/Cbl-b-CA (c-Cbl-RQ/Cbl-b-CA) cells, cultured in doxycycline-containing medium, were cultured for 3 h with or without 5 µM LY294002 (LY), as indicated, in the absence of IL-3. Cells were further treated for 6 h with 10 µM etoposide (VP16) or 1 µM doxorubicin (DXR), as indicated, and lysed. Cell lysates were analyzed by immunoblotting.</p

A schematic model for enhancement of apoptosis by inhibition of the aberrant tyrosine kinases in hematopoietic cells treated with chemotherapeutics.

<p>Chemotherapeutics induces Chk1-mediated G2/M cell cycle arrest to downregulate induction of apoptosis. Inhibition of BCR/ABL, FLT3-ITD, or Jak2-V617F by imatinib, sorafenib, of JakI-1, respectively, as well as inhibition of PI3K or Akt by GDC-0941 or MK-2206, respectively, drastically enhances apoptosis in hematopoietic cells treated with chemotherapeutics. The enhancing effect, prevented by inhibition of GSK3 by SB216763, may be at least partly due to the inhibition of PI3K/Akt pathway leading to activation of GSK3, which may prevent Chk1-mediated G2/M cell cycle arrest by chemotherapeutics. Inhibition of Chk1 by its inhibitor SB218078 also prominently enhances apoptosis induced by chemotherapeutics.</p