Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms

Abstract

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.

Keywords: ABT-737; Bcl-xL; myeloproliferative neoplasms; therapeutic target.

FIGURE 1

A, Bcl‐xL mRNA expression in mature leucocytes from healthy donors (ctrl) and MPN patients (ET, essential thrombocythemia; PV, polycythemia vera; and PMF, myelofibrosis). Primary samples were divided into two groups based on high or low Bcl‐xL expression by dichotomizing for the average value. B, Bcl‐xL mRNA expression in leucocytes from ET, PV and PMF, classified as JAK2 wild‐type and mutated. C, Quantification of Bcl‐xL protein after immunofluorescence analysis in CD34 + cells (hematopoietic progenitor cells) from healthy donors and JAK2 wild‐type (wt) and mutated (mut) MPN patients. D, Quantification of Bcl‐xL expression after immunofluorescence analysis in CD235 + cells (erythroblasts) from healthy donors and JAK2 wt and mut MPN patients. Fluorescence intensity was measured in arbitrary units (a.u.)

FIGURE 2

Effects of JAK2 and Bcl‐xL inhibition in HEL cells. A, Left: Sigmoidal Dose‐Response curves in HEL cells treated with different concentrations (range: 0‐20 µmol\L) of ABT‐737, ruxolitinib, and ABT‐737 + ruxolitinib for 24 hrs. The experiment was performed in quadruplicate. Right: isobologram for IC50. B, Annexin V apoptosis assay was performed to estimate the apoptosis rate in HEL cells after treatment with 2.5 µmol\L ABT‐737, 5 µmol\L ruxolitinib and ABT‐737 + ruxolitinib for 24 hrs. The bar graph showed the percentage of apoptotic cells. C, Bcl‐xL mRNA expression was evaluated in untreated HEL cells and after treatment with 2.5 µmol\L ABT‐737, 5 µmol\L ruxolitinib and ABT‐737 + ruxolitinib for 24 hrs. D, Correlation between apoptosis rate and Bcl‐xL modulation. Linear regression analyses were performed plotting Bcl‐xL mRNA expression (average value for each condition, y‐axis) and the percentage of apoptotic cells (average value for each condition, x‐axis) in HEL cells treated with 2.5 µmol\L ABT‐737, 5 µmol\L ruxolitinib and drugs combination for 24 hrs. E, JAK2 phosphorylation status and Bcl‐xL protein expression were evaluated by SDS‐PAGE in untreated HEL cells and after treatment by single or combined administration of 2.5 µmol\L ABT‐737 and 5 µmol\L ruxolitinib for 24 hrs. The Western blot assays, shown here, represent an example of at least 4 independent experiments

FIGURE 3

Effects of JAK2 and Bcl‐xL inhibition in leucocytes from PV and PMF patients, both JAK2 mutated and wild‐type. A, Bcl‐xL mRNA expression was evaluated in untreated cells from PV and PMF patients and after treatment with 2.5 µmol\L ABT‐737, 5 µmol\L ruxolitinib and ABT‐737 + ruxolitinib for 24 hrs. In addition, Bcl‐xL levels in healthy patients were reported. B, JAK2 phosphorylation status and Bcl‐xL protein expression were evaluated by SDS‐PAGE in untreated cells from PV and PMF patients and after treatment with single or combined drugs at the concentration of 2.5 µmol\L for ABT‐737 and 5 µmol\L for ruxolitinib for 24 hrs. The Western blot assays shown here in PMF leucocytes is representative of at least 4 independent experiments. C, Annexin V apoptosis assay was performed to estimate the apoptosis rate in cells from PV and PMF patients after treatment with 2.5 µmol\L ABT‐737, 5 µmol\L ruxolitinib and ABT‐737 + ruxolitinib for 24 hrs. The bar graph showed the percentage of apoptotic cells. The Western blot assays, shown here, represent an example of at least 4 independent experiments