Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Abstract

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34+ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters.

Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34+ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34+ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly.

Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Figure 1

Gene expression in Control, ET and PMF CD34⁺ HSC. (A) anti-apoptotic gene A1 was increased in ET and PMF compared to control; (B) anti-apoptotic gene MCL1 was increased in ET compared to control; (C) pro-apoptotic gene BID was increased in ET compared to control; (D) pro-apoptotic gene BIK was increased in ET compared to control; (E) pro-apoptotic gene BAX was decreased in ET compared to control; (F) anti-apoptotic gene BCLW was increased in PMF compared to control; (G) pro-apoptotic gene BAK was increased in ET compared to control; (H) anti-apoptotic gene BCL2 was decreased in PMF compared to control. The significant "p values" are shown in the figure. Otherwise, p > 0.05. The horizontal bars show the median 2^-ΔΔCt for each group.

Figure 2

Gene expression in Control, ET and PMF leukocytes. (A) anti-apoptotic gene A1 was increased in ET compared to control; (B) anti-apoptotic gene BCL2 was increased in ET and PMF compared to control; (C) anti-apoptotic gene BCL-X_L was increased in ET and PMF compared to control and also significantly different between ET and PMF; (D) anti-apoptotic gene BCLW was increased in ET and PMF compared to control and also significantly different between ET and PMF; (E) pro-apoptotic gene BID was decreased in ET compared to control; (F) pro-apoptotic gene BIM_EL was decreased in ET compared to control. The significant "p values" are shown in the figure. Otherwise, p > 0.05. The horizontal bars show the median 2^-ΔΔCt for each group.

Figure 3

Protein expression in Control, ET and PMF leukocytes. (A) anti-apoptotic BCL-X_L showed higher expression in PMF leukocyte than in controls (fold change: 2.4); (B) pro-apoptotic BID showed lower expression in ET leukocyte than in controls (fold change: 0.64). Tubulin was probed as loading control and densitometry was used to confirm the fold change in the protein expression.

Figure 4

Apoptosis-related gene expression versus JAK2 V617F mutation. (A, B and C) pro-apoptotic genes BAX, BIK and BAD showed lower expression in ET JAK2 V617F positive patients compared to negative patients; (D, E and F) BAX, BIK and BAD expression in ET leukocytes also negatively correlated with the patients' JAK2 V617F allele burden. The significant "p values" and the "r value" for correlations are shown in the figure. The horizontal bars show the median 2^-ΔΔCt for each group.

Figure 5

PRV1 gene expression in Control, ET and PMF leukocytes and PRV1 expression versus gene expression. (A) PRV1 expression was significantly elevated in ET and PMF leukocytes; (B) anti-apoptotic gene A1 showed positive correlation with PRV1 expression in ET leukocytes; (C and D) pro-apoptotic genes BAX and BIK showed negative correlation with PRV1 expression in ET leukocytes; (E) anti-apoptotic gene BCLW showed positive correlation with PRV1 expression in PMF leukocytes. The significant "p values" are shown in the figure. Otherwise, p > 0.05. The horizontal bars show the median 2^-ΔΔCt for each group.

Figure 6

PRV1 expression versus JAK2 V617F mutation. (A) PRV1 expression was higher in ET JAK2 V617F positive patients than in JAK2 V617F negative patients; (B) a positive correlation between PRV1 expression in ET leukocytes and JAK2 V617F allele burden percentage was detected. The significant "p values" and the "r value" for correlations are shown in the figure.

Figure 7

Correlation of PRV1 gene expression with platelet count (PLT) and splenomegaly. PRV1 expression in ET (A) and PMF (B) leukocytes showed negative correlation with platelets (PLT); (C) PRV1 expression in PMF leukocytes showed positive correlation with the increase in centimeters of the spleen length (splenomegaly) determined by ultrasonography (reference value: 12 cm). The significant "p values" and the "r" value for correlations are shown in the figure.

Figure 8

Overview of the BCL2 family gene expression results: (A) in CD34⁺ HSC and (B) leukocytes from patients with ET (continuous arrows) and PMF (stippled arrows).