Preclinical characterization of atiprimod, a novel JAK2 AND JAK3 inhibitor

Abstract

We herein report on the activity of the JAK2/JAK3 small molecule inhibitor atiprimod on mouse FDCP-EpoR cells carrying either wild-type (JAK2 (WT)) or mutant (JAK2 (V617F)) JAK2, human acute megakaryoblastic leukemia cells carrying JAK2 (V617F) (SET-2 cell line), and human acute megakaryocytic leukemia carrying mutated JAK3 (CMK cells). Atiprimod inhibited more efficaciously the proliferation of FDCP-EpoR JAK2 (V617F) (IC(50) 0.42 μM) and SET-2 cells (IC(50) 0.53 μM) than that of CMK (IC(50) 0.79 μM) or FDCP-EpoR JAK2 (WT) cells (IC(50) 0.69 μM). This activity was accompanied by inhibition of the phosphorylation of JAK2 and downstream signaling proteins STAT3, STAT5, and AKT in a dose- and time-dependent manner. Atiprimod-induced cell growth inhibition of JAK2 (V617F)-positive cells was coupled with induction of apoptosis, as evidenced by heightened mitochondrial membrane potential and caspase-3 activity, as well as PARP cleavage, increased turnover of the anti-apoptotic X-linked mammalian inhibitor of apoptosis (XIAP) protein, and inhibition of the pro-apoptotic protein BCL-2 in a time- and dose-dependent manner. Furthermore, atiprimod was more effective at inhibiting the proliferation of peripheral blood hematopoietic progenitors obtained from patients with JAK2 (V617F)-positive polycythemia vera than at inhibiting hematopoietic progenitors from normal individuals (p = 0.001). The effect on primary expanded erythroid progenitors was paralleled by a decrease in JAK2(V617F) mutant allele burden in single microaspirated BFU-E and CFU-GM colonies. Taken together, our data supports the clinical testing of atiprimod in patients with hematologic malignancies driven by constitutive activation of JAK2 or JAK3 kinases.

Fig. 1

Atiprimod inhibits the proliferation of cells harboring mutant JAK2^V617F. JAK2^wt- and JAK2^V617F-transduced FDCP-EpoR cells, human SET-2 and CMK cells were exposed to increasing concentrations of atiprimod for 72 h. (a) The MTS assay was used to evaluate cell proliferation. Data points are the mean ± SD from three independent determinations. (b) Non-linear regression model of the MTS results was used to estimate the inhibitory constants (IC) values for both cell lines

Fig. 2

Atiprimod inhibits the phosphorylation of JAK2, and JAK3. FDCP-EpoR cell lines carrying JAK2^WT or JAK2^V617F, human SET2 and CMK cells were treated with equipotent doses (IC₂₀, IC₅₀, and IC₈₀) of atiprimod for 4 h. Cells were lysed and whole lysates were immunoprecipitated with anti-JAK2 and AK3 antibodies, (a) Atiprimod inhibits the phosphorylation of JAK2 in FDCP-EpoR cell lines carrying constitutively active JAK2^V617F(a) and the phosphorylation of JAK2 in human SET2 cells and JAK3 in CMK cells (b)

Fig. 3

Atiprimod inhibits the phosphorylation of STAT3, STAT5, and AKT in FDCP-EpoR cells expressing mutant JAK2^V617F FDCP-EpoR cell lines carrying JAK2^WT or JAK2^V617F were treated with equipotent doses (IC₂₀, IC₅₀, and IC₈₀) of atiprimod for 24 h. Extracted cell lysates were separated on a 4–12% SDS-PAGE gel, and western blotting was performed using mouse monoclonal antibodies against the specified proteins. After stripping, membranes were reprobed with rabbit anti-total STAT3, STAT5 and AKT. β-actin served as loading control

Fig. 4

Desphosphorylation of STAT3, STAT5, AKT in human SET2 and CMK cells. Human SET-2 and CMK cell lines carrying JAK2^V617F or JAK2^wt respectively, were treated with equipotent doses (IC₂₀, IC₅₀, and IC₈₀) of atiprimod for 20 h. Whole cell lysates were separated on a 4–12% SDS-PAGE gel, and western blotting was performed using mouse monoclonal anti-p-STAT3, anti-p-STAT5, and anti-p-AKT. After stripping, the membrane was reprobed with rabbit anti-total STAT3, anti–total STAT5, and anti-total AKT. The quantity of protein was confirmed by probing the membranes with β-actin

Fig. 5

Atiprimod induces PARP and caspase-3 cleavage and decreases XIAP levels. (a) FDCP-EpoR cell lines carrying JAK2^WT or JAK2^V617F were treated with equipotent doses (IC₂₀, IC₅₀, and IC₈₀) of atiprimod for 24 h. Western blotting was performed using mouse anti-BCL2 and XIAP antibodies. β-actin served as loading control, (b) Human SET-2 and CMK cells were exposed to three different doses (IC₂₀, IC₅₀ and IC₈₀) of atiprimod for 24 h. Cell lysates were analyzed by western blot using mouse monoclonal anti-BCL2, XIAP, anti-PARP, and anti-caspase-3. β-actin served as loading control

Fig. 6

Effect of atiprimod on ex vivo expanded erythroid progenitors. In vitro expanded erythroid progenitor cells from three healthy controls and three patients with PV were harvested on day 5 of phase 2 of the expansion protocol and treated with increasing concentration of atiprimod for 48 h. The MTS assay was used to evaluate cell proliferation. Data points are the mean ± SD from three independent determinations, (b) Non-linear regression model of the MTS results was used to estimate the inhibitory concentration (IC) values for both cell lines

Fig. 7

Quantitation of the JAK2^V617F mutant allele and effect of atiprimod on JAK2^V617F allele burden. Peripheral blood low-density cells from 3 patients with PV were incubated in the presence of atripimod at doses ranging from 0.25 to 2.0 µM in clonogenic assays and evaluated after 14 days. Ten colonies at each dose were microaspirated and the JAK2^V617F allele burden was quantitated by qRT-PCR. After DNA extraction, gDNA was used for PCR amplification with JAK2-exonl4 primers. Quantitative allele-specific suppressive PCR (ASS-PCR) was performed using the purified PCR product on a sequence detection system 7,000 platform