Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera

Abstract

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

Figure 1

Flow chart describing the three-steps to proteomic screening for differentially expressed proteins in polycythemia vera (PV) and essential thrombocythemia (ET). The training set includes two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS); the protein validation set includes immunohistochemistry (IHC); and the functional validation set includes burst formation unit-erythroid (BFU-E) and stem cells studies.

Figure 2

Two-dimensional difference gel electrophoresis (2D-DIGE) analysis and IHC validation. (A) Representative spots from three proteins from 2D-DIGE gels and intensity quantification with DeCyder software v7.0. three spots were identified as LTA4H, SERPINB1 and HSP70 (HSPA1A). These spots were over-expressed in polycythemia vera (PV) vs. essential thrombocythemia (ET) samples. (B) Representative images according the median of HSP70 staining of granulocytes from bone marrow of PV, and ET. (C) Box-plot of percentage of HSP70 positive granulocytes quantified from IHC.

Figure 3

KNK437 inhibition assay of cell cultures. (A) Viability test results of KNK437 (10, 50 and 100 μM) inhibition assay of mononuclear peripheral blood burst formation unit-erythroid (BFU-E) cultures, with and without EPO. (B) Viability test results of a KNK437 (50 and 100 μM) inhibition assay of CD34+ bone marrow BFU-E cultures, with and without EPO. (C) Viability test results of KNK437 treatment (50 μM) on HEL and Ba/F3 JAK2 V617F (BAF3MUT) cell lines. X axis: concentration (μM); Y axis: % of viability cells (viability test) or BFU-E (BFU-E cultures) (D) Molecular effects of KNK437 in HEL cell line Western blot of HSP90, HSP70, p-JAK2 (decreased with KNK437 treatment), p-ERK and p-P38. Actin was used as the housekeeping control. (E) Molecular effects of KNK437 in Ba/F3 cell line Western blot of JAK2, p-ERK, ERK, p-STAT5, STAT5 AND Actin, (F) Western blot of HSP90, HSP70 , JAK2, STAT5 and p-STAT5. Expression levels of HSP70, JAK2 and p-STAT5 decreased after HSP70 siRNA interference on HEL cell line. Actin was used as the housekeeping control.

Figure 4

Flow cytometry of KNK437 inhibition assay of CD34+ cell cultures. (A) FCM results of BFU-E cultured cells from CD34+ bone marrow of polycythemia vera (PV) and essential thrombocythemia (ET) samples, with and without KNK437 treatment (50 μM), and with the CD71 marker (y axis) and annexin V (x axis). (B) Box-plot of percentage of Annexin V positive CD34+ culture cells quantified by flow cytometry.