Impaired apoptosis of megakaryocytes and bone marrow mononuclear cells in essential thrombocythemia: correlation with JAK2V617F mutational status and cytoreductive therapy

Abstract

Essential thrombocythemia (ET) is a clonal myeloproliferative disorder characterized by overproduction of megakaryocytes (MKCs) and platelets. The recent discovery of the JAK2 mutation has shed a new light on the development of ET but its pathogenesis still remains unknown. One of the possible mechanisms can be deregulation of apoptosis, resulting in accumulation of bone marrow MKCs. In this study, we investigated the apoptotic profile, as well as the expression of apoptosis-regulating protein in MKCs and bone marrow mononuclear cells (BMMCs) in 43 patients with ET. We found significantly lower percentages of apoptotic MKCs and BMMCs, as measured by the rate of annexin-V+ and caspase-3+ (Cas-3+) cells in relation to healthy volunteers. Additionally, the expression of Bax protein in ET patients naïve to cytoreductive treatment, as well as their Bax/Bcl-2 ratio, was significantly lower than in controls (p = <0.05 and p < 0.001, respectively). Patients positive for the JAK2V617F mutation had markedly higher activation of Cas-3, as well as higher Bax expression (p = 0.02 and p = 0.04, respectively) than JAK2V617F negative cases. There were no marked differences between patients already treated with anagrelide (ANA) or hydroxyurea (HU), although tendency toward the higher apoptosis rate was observed in the HU-treated group. In conclusion, these results demonstrate the inhibition of caspase-dependent apoptosis of both MKCs and BMMCs in untreated ET. This is associated with upregulation of Bcl-2 and downregulation of Bax proteins, predominantly in JAK2V617F negative cases. Patients treated with HU showed slightly higher pro-apoptotic Bax/Bcl-2 index than patients on ANA therapy, which may influence the better efficacy of HU therapy in ET.

Pro-Apoptotic Activity of Ruxolitinib Alone and in Combination with Hydroxyurea, Busulphan, and PI3K/mTOR Inhibitors in JAK2-Positive Human Cell Lines

“…We also found up-regulation of proapoptotic Bax protein in both HEL and SET-2 cells. In our previous study, we found that patients with essential thrombocythemia positive for the JAK2V617F mutation had markedly higher Bax expression than JAK2V617F negative cases [25]. As reported before, Mcl-1 protein has a crucial role in regulating the survival of hematopoietic stem cells [26].…”

Effects of polymorphic DNA genes involved in BER and caspase pathways on the clinical outcome of myeloproliferative neoplasms under treatment with hydroxyurea

“…In PV and ET patients it is very important to identify those whose risk of vascular and thrombotic complications is high enough to justify the use of risk-adapted treatment strategies, including aspirin, anticoagulants, hydroxyurea (HU), anagrelide, interferon alfa (IFN-alpha) or the recent JAK2 inhibitors (e.g., Ruxolitinib) (36). Patients are stratified and treatment is prescribed according to evaluation of the three major risk factors for thrombosis, namely previous history of arterial or venous thrombosis, possible presence of JAK2 mutation and age >60 years (36,38). The risk of thrombosis in older JAK2-unmutated patients without thrombosis history is low enough to occasionally consider, on an individual basis, skipping cytoreductive therapy (36).…”

“…HU is widely used as first-line cytoreductive therapy in 'high risk' patients, as result of consensus of randomized clinical trials (37,38). This drug is an antimetabolite, capable of inhibition of ribonucleoside diphosphate reductase necessary for synthesis and repair of DNA, with a subsequent myelosuppressive activity (38). Moreover, HU affects polymorphonuclear leukocytes function and interferes in their interactions with platelets, having an antithrombotic effect (38).…”

Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms

“…[6][7][8] The pathobiological basis underlying these numerical and morphological megakaryocytic abnormalities is thought to result from multiple molecular disruptions promoting proliferation and enhancing survival. [9][10][11][12][13] These megakaryocytes have impaired death mechanisms conferred by overexpression of antiapoptotic Bcl-XL and reductions in pro-death BNIP-3. 9 11-13 These changes are universal in the MPNs but there are differences between entities.…”

“…Limited studies have identified that apoptotic signalling processes are disrupted in megakaryocytes of the MPNs. [9][10][11][12][13] We intended to further delineate the biological basis of the apoptotic disturbances affecting megakaryocytes in the MPNs by assessing several biomarkers implicated in the intrinsic and extrinsic apoptotic pathways. We demonstrate through immunohistochemical analyses of human MPNs that the enhanced survival of myeloproliferative megakaryocytes occurs through inhibition of intrinsic death effectors despite concurrent extrinsic apoptotic activation.…”