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JAK2V617F drives Mcl-1 expression and sensitizes hematologic cell lines to dual inhibition of JAK2 and Bcl-xL.

Guo J, Roberts L, Chen Z, Merta PJ, Glaser KB, Shah OJ - PLoS ONE (2015)

Bottom Line: We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling.Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression.Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation.

View Article: PubMed Central - PubMed

Affiliation: Oncology Division, Research & Development, AbbVie Inc., North Chicago, Illinois, United States of America.

ABSTRACT
Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating JAK2 mutations.

No MeSH data available.


Related in: MedlinePlus

Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in JAK2V617F-harboring AML cell lines.(A/B) HEL and K562 cells were treated for 6 hr with 1 μM JAKi-I followed by 3 hr with 0.15 μM ABT-263, then lysates or Bcl-XL immunoprecipitates were prepared and immunoblotted. (C) Cells were treated for 6 hr with 1 μM JAKi-I followed by 0.15 μM ABT-263 over a 3-hr time period. Caspase-3 activity was determined at each time point. Data are from duplicate samples and are representative of at least three independent experiments. (D-G) Cells were treated in combination as indicated, and cell viability was determined after 72 hr. Data are means of duplicate determinations, and are representative of at least three independent experiments. (H) Drug-drug interactions were determined using a matrix of pairwise combinations covering half-log dose responses from 0.03 to 1 μM for both JAKi-I and ABT-263. Drugs were added simultaneously, and cell viability was determined after 72 hr. The data were then analyzed using the drug-drug interaction model of Bliss additivity16 to define dose combinations that were synergistic (values >15; red), antagonistic (values <-15; blue), or without effect (-15<values<15; gray). (I) Model of JAK2/Bcl-2 family inhibitor synergy. JAK2V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional target, Mcl-1. Mcl-1 collaborates with Bcl-XL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon Bcl-XL. Neutralization of Bcl-XL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis.
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pone.0114363.g002: Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in JAK2V617F-harboring AML cell lines.(A/B) HEL and K562 cells were treated for 6 hr with 1 μM JAKi-I followed by 3 hr with 0.15 μM ABT-263, then lysates or Bcl-XL immunoprecipitates were prepared and immunoblotted. (C) Cells were treated for 6 hr with 1 μM JAKi-I followed by 0.15 μM ABT-263 over a 3-hr time period. Caspase-3 activity was determined at each time point. Data are from duplicate samples and are representative of at least three independent experiments. (D-G) Cells were treated in combination as indicated, and cell viability was determined after 72 hr. Data are means of duplicate determinations, and are representative of at least three independent experiments. (H) Drug-drug interactions were determined using a matrix of pairwise combinations covering half-log dose responses from 0.03 to 1 μM for both JAKi-I and ABT-263. Drugs were added simultaneously, and cell viability was determined after 72 hr. The data were then analyzed using the drug-drug interaction model of Bliss additivity16 to define dose combinations that were synergistic (values >15; red), antagonistic (values <-15; blue), or without effect (-15<values<15; gray). (I) Model of JAK2/Bcl-2 family inhibitor synergy. JAK2V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional target, Mcl-1. Mcl-1 collaborates with Bcl-XL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon Bcl-XL. Neutralization of Bcl-XL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis.

Mentions: Of the pro-apoptotic BH3-only proteins normally sequestered by anti-apoptotic members of the Bcl-2 family, Bim binds both Mcl-1 and Bcl-xL [17,18]. We therefore asked whether the loss of Mcl-1 induced by JAK inhibition resulted in increased binding of Bim to Bcl-xL. Although the abundance of total Bim protein was not altered following treatment with JAKi-I (Fig. 2A), Bim was enriched in Bcl-XL immunoprecipitates in the presence of the JAK2V617F mutation (Fig. 2B). In cells treated with ABT-263, Bim was displaced from Bcl-XL (Fig. 2B) irrespective of JAK2 mutational status. To assess whether suppression of Mcl-1 by treatment with JAKi-I would indeed potentiate apoptosis induced by Bcl-xL/-2 inhibition, we pretreated cell lines with JAKi-I for 6 hr (time sufficient for Mcl-1 levels to decline) followed by ABT-263 and monitored the activity of caspase-3. Whereas neither JAKi-I nor ABT-263 alone induced caspase-3 activity, a synergistic induction was evident within four hours specifically in cell lines harboring JAK2V617F (Fig. 2C).


JAK2V617F drives Mcl-1 expression and sensitizes hematologic cell lines to dual inhibition of JAK2 and Bcl-xL.

Guo J, Roberts L, Chen Z, Merta PJ, Glaser KB, Shah OJ - PLoS ONE (2015)

Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in JAK2V617F-harboring AML cell lines.(A/B) HEL and K562 cells were treated for 6 hr with 1 μM JAKi-I followed by 3 hr with 0.15 μM ABT-263, then lysates or Bcl-XL immunoprecipitates were prepared and immunoblotted. (C) Cells were treated for 6 hr with 1 μM JAKi-I followed by 0.15 μM ABT-263 over a 3-hr time period. Caspase-3 activity was determined at each time point. Data are from duplicate samples and are representative of at least three independent experiments. (D-G) Cells were treated in combination as indicated, and cell viability was determined after 72 hr. Data are means of duplicate determinations, and are representative of at least three independent experiments. (H) Drug-drug interactions were determined using a matrix of pairwise combinations covering half-log dose responses from 0.03 to 1 μM for both JAKi-I and ABT-263. Drugs were added simultaneously, and cell viability was determined after 72 hr. The data were then analyzed using the drug-drug interaction model of Bliss additivity16 to define dose combinations that were synergistic (values >15; red), antagonistic (values <-15; blue), or without effect (-15<values<15; gray). (I) Model of JAK2/Bcl-2 family inhibitor synergy. JAK2V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional target, Mcl-1. Mcl-1 collaborates with Bcl-XL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon Bcl-XL. Neutralization of Bcl-XL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362760&req=5

pone.0114363.g002: Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in JAK2V617F-harboring AML cell lines.(A/B) HEL and K562 cells were treated for 6 hr with 1 μM JAKi-I followed by 3 hr with 0.15 μM ABT-263, then lysates or Bcl-XL immunoprecipitates were prepared and immunoblotted. (C) Cells were treated for 6 hr with 1 μM JAKi-I followed by 0.15 μM ABT-263 over a 3-hr time period. Caspase-3 activity was determined at each time point. Data are from duplicate samples and are representative of at least three independent experiments. (D-G) Cells were treated in combination as indicated, and cell viability was determined after 72 hr. Data are means of duplicate determinations, and are representative of at least three independent experiments. (H) Drug-drug interactions were determined using a matrix of pairwise combinations covering half-log dose responses from 0.03 to 1 μM for both JAKi-I and ABT-263. Drugs were added simultaneously, and cell viability was determined after 72 hr. The data were then analyzed using the drug-drug interaction model of Bliss additivity16 to define dose combinations that were synergistic (values >15; red), antagonistic (values <-15; blue), or without effect (-15<values<15; gray). (I) Model of JAK2/Bcl-2 family inhibitor synergy. JAK2V617F constitutively phosphorylates and activates STAT3/5, thus enforcing expression of the transcriptional target, Mcl-1. Mcl-1 collaborates with Bcl-XL to oppose apoptosis and support viability. Inhibition of JAK2 in this context silences JAK/STAT-driven transcription of Mcl-1, leaving survival largely dependent upon Bcl-XL. Neutralization of Bcl-XL with ABT-263 is then achieved at a lower dose and is sufficient to induce apoptosis.
Mentions: Of the pro-apoptotic BH3-only proteins normally sequestered by anti-apoptotic members of the Bcl-2 family, Bim binds both Mcl-1 and Bcl-xL [17,18]. We therefore asked whether the loss of Mcl-1 induced by JAK inhibition resulted in increased binding of Bim to Bcl-xL. Although the abundance of total Bim protein was not altered following treatment with JAKi-I (Fig. 2A), Bim was enriched in Bcl-XL immunoprecipitates in the presence of the JAK2V617F mutation (Fig. 2B). In cells treated with ABT-263, Bim was displaced from Bcl-XL (Fig. 2B) irrespective of JAK2 mutational status. To assess whether suppression of Mcl-1 by treatment with JAKi-I would indeed potentiate apoptosis induced by Bcl-xL/-2 inhibition, we pretreated cell lines with JAKi-I for 6 hr (time sufficient for Mcl-1 levels to decline) followed by ABT-263 and monitored the activity of caspase-3. Whereas neither JAKi-I nor ABT-263 alone induced caspase-3 activity, a synergistic induction was evident within four hours specifically in cell lines harboring JAK2V617F (Fig. 2C).

Bottom Line: We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling.Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression.Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation.

View Article: PubMed Central - PubMed

Affiliation: Oncology Division, Research & Development, AbbVie Inc., North Chicago, Illinois, United States of America.

ABSTRACT
Constitutive activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) axis is fundamental to the molecular pathogenesis of a host of hematological disorders, including acute leukemias and myeloproliferative neoplasms (MPN). We demonstrate here that the major JAK2 mutation observed in these diseases (JAK2V617F) enforces Mcl-1 transcription via STAT3 signaling. Targeting this lesion with JAK inhibitor I (JAKi-I) attenuates STAT3 binding to the Mcl-1 promoter and suppresses Mcl-1 transcript and protein expression. The neutralization of Mcl-1 in JAK2V617F-harboring myelodyssplastic syndrome cell lines sensitizes them to apoptosis induced by the BH3-mimetic and Bcl-xL/Bcl-2 inhibitor, ABT-263. Moreover, simultaneously targeting JAK and Bcl-xL/-2 is synergistic in the presence of the JAK2V617F mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating JAK2 mutations.

No MeSH data available.


Related in: MedlinePlus