<i>JAK2^V617F</i> Drives Mcl-1 Expression and Sensitizes Hematologic Cell Lines to Dual Inhibition of JAK2 and Bcl-xL

<div><p>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 <i>JAK2</i> mutation observed in these diseases (<i>JAK2^V617F</i>) 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 <i>JAK2^V617F</i>-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 <i>JAK2^V617F</i> mutation. These findings suggest that JAK/Bcl-xL/-2 inhibitor combination therapy may have applicability in a range of hematological disorders characterized by activating <i>JAK2</i> mutations.</p></div

Regulation of Mcl-1 and Bcl-XL by <i>JAK2</i>^<i>V617F</i>.

<p>(A) JAKi-I was evaluated in a panel of 66 human protein kinases as detailed in the Methods section, and Ki values determined. Red, <0.01 μM; black, 0.01–1.67 μM, green, >1.67 μM. (B) UKE-1 (<i>JAK2</i>^<i>V617F</i>) AML cells were treated for 10 min with JAKi-I as indicated. Tyrosine phoshorylation of STAT3 and STAT5 was determined by immunoblotting. (C) The <i>JAK2</i>^<i>V617F</i>-positive AML cell lines, SET-2, UKE-1, and HEL, the chronic myelogenous leukemia line, K562 (<i>JAK2</i>^<i>V617F</i>-negative), and the AML cell line, MV4;11 (<i>JAK2</i>^<i>V617F</i>-negative), were cultured in the absence of serum for 2 hr, then treated with 1 μM JAKi-I for 1 hr. Constitutive tyrosine phosphorylation of STAT3 and STAT5 was determined by immunoblotting. (D and E) Cells were treated for 6 hr with JAKi-I, and the abundance of Mcl-1 and Bcl-XL mRNA was determined by qPCR. Data represent means +/- standard deviation for two independent determinations each performed in triplicate. (F) Cells were treated with JAKi-I or Ruxolitinib over a 24-hr time course, and Mcl-1 and Bcl-XL levels were determined by immunoblotting (similar results were observed for 2 separate immuoblots). (G) Quantification of the data shown in (F). Data are expressed as the ratio of intensity of Mcl-1/β-actin for each time point. (H and I) HEL or K562 cells were transfected with either non-targeting (siNT-1) or Mcl-1-specific (siMcl1–1–4) siRNAs, treated for 72 hr with ABT-263, then lysates were prepared, and cell viability was determined. Data are means of duplicate samples and are representative of two independent experiments. (J) Cells were treated for 6 hr with or without 1 μM JAKi-I then subjected chromatin immunoprecipitation assays using normal mouse IgG, anti-acetylated histone H3, or anti-STAT3. Mcl-1 promoter binding was determined by PCR on chromatin immunoprecipitates (for <u>immunoblots</u>, similar results were obtained twice).</p

Combination of JAK2 and Bcl-2 family inhibitors yields synergistic antiproliferative activity in <i>JAK2</i>^<i>V617F</i>-harboring AML cell lines.

<p>(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 (-15V617F 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.</p