Primary myelofibrosis (PMF) is a Philadelphia-negative chronic myeloproliferative disorder characterized by splenomegaly, bone marrow fibrosis and a variable degree of myeloproliferation that can affect one or more hematopoietic lineages. The activation of the JAK/STAT pathway of the hematopoietic cells is a biological hallmark of the disease and is associated to acquired mutations that occur in JAK2, CALR or MPL genes. JAK/STAT pathway activation gives reason of the myeloproliferation; however, it does not completely explain how and why the pathological clone(s) emerge and expand in the bone marrow of PMF patients. The unfolded protein response (UPR) is an endoplasmic reticulum (ER)- specific pathway comprising a network of proteins that are activated in response to ER stress, due to accumulation of misfolded proteins. This can result from the exposure of the cell to stress stimuli, such as ROS overproduction, or DNA damage. UPR activation can result either in the recovery of the “stressed” cell through protein synthesis reduction and clearing of misfolded proteins or, if homeostasis is not restored, in its elimination through the activation of the apoptotic cascade. Recently, it has been reported that UPR activation maintains the integrity of hematopoietic stem/progenitor cells of healthy subjects by a balance between cell repair and induction of apoptosis (van Galen, P. et al.Nature 2014)
To assess the activation of the UPR pathway in CD34+ hematopoietic progenitor cells obtained from patients with PMF, by investigating the expression of the main components of the UPR, including those implicated in the apoptotic cascade.
RNA was obtained from CD34+ cells from the peripheral blood of 8 PMF patients (3 with a JAK2 p.V617F mutation, 2 with “Type 1” and 3 with “Type 2” CALR mutation) and 4 healthy subjects. Reverse transcription droplet digital PCR (RT-ddPCR) was used to analyze the expression of the main genes belonging to the three branches of the UPR: ATF4, DDIT3, DNAJB9 (as representative of PERK branch), XBP1 s, XBP1u, HSPA5 (IRE1-alpha branch) and ATF6 (ATF6 branch). The expression of BAX and BCL2 (involved in apoptosis) and of WT1 (as a measure of the myeloproliferative activity) was also analyzed.
As reported in Figure 1a, a significant downregulation of the genes belonging to the PERK and IRE1-alpha branches of the UPR pathway was observed in CD34+ cells from PMF patients compared to healthy subjects. In particular, CD34+ cells from CALR-mutated patients shows the lowest levels of expression in addition to downregulation of ATF6 (Figure 1b), whereas in JAK2V617F-mutated patients, the ratio between the spliced and unspliced form of XBP1, that measures the activity of the IRE1-alpha branch of UPR, is lower than in healthy subjects. This is accompanied by a significant upregulation of WT1 (Figure 1c). In CD34+ cell samples from all patients was also observed a reduced BAX/BCL2 ratio, mainly due to an increase of BCL2 expression.
Since UPR-dependent apoptosis activation can be induced both by ATF4-mediated BCL2 downregulation and by IRE1alpha-mediated BAX up-regulation, our data strongly suggest that UPR-mediated apoptosis is suppressed in hematopoietic progenitor cells from PMF patients. In keeping with this observation, the expression of WT1, a pro-proliferative gene, is increased in the same cells. We hypothesize that UPR downregulation, by abolishing a pre-differentiation checkpoint at stem/progenitor cell level, favors the emergence of the pathological clones.