Polycythemia vera (PV) and essential thrombocythemia (ET) are the two most common subtypes of Philadelphia chromosome-negative myeloproliferative neoplasm (MPN). PV results in erythrocytosis and ET ...in thrombocytosis. The discovery of JAK2 mutations in the majority of patients with MPN over the last 2 decades has led to the development of JAK inhibitors. Because PV and ET progress relatively slowly, the main treatment strategy for these two diseases is to prevent thrombotic complications. The first-line agent for both PV and ET is hydroxyurea, although some patients are intolerant or refractory to this compound and need other treatment options. Notably, hydroxyurea is contraindicated during pregnancy. In addition to JAK inhibitors, several new agents, such as HDAC inhibitors, LSD1 inhibitors, MDM2 inhibitors and hepcidin mimetics, have been developed as treatment options. Classical agents, such as busulfan and interferon, are still used to treat patients with PV or ET as well. Based on this context, treatment options and pregnancy management for patients with PV or ET are discussed in this review.
Deregulation of cytokine signaling is frequently associated with various pathological conditions, including malignancies. In patients with myeloproliferative neoplasms (MPNs), recurrent somatic ...mutations in the calreticulin (CALR) gene, which encodes a molecular chaperone that resides in the endoplasmic reticulum, have been reported. Studies have defined mutant CALR as an oncogene promoting the development of MPN, and deciphered a novel molecular mechanism by which mutant CALR constitutively activates thrombopoietin receptor MPL and its downstream molecules to induce cellular transformation. The mechanism of interaction and activation of MPL by mutant CALR is unique, not only due to the latter forming a homomultimeric complex through a novel mutant‐specific sequence generated by frameshift mutation, but also for its ability to interact with immature asparagine‐linked glycan for eventual engagement with immature MPL in the endoplasmic reticulum. The complex formed between mutant CALR and MPL is then transported to the cell surface, where it induces constitutive activation of downstream kinase JAK2 bound to MPL. Refined structural and cell biological studies can provide an in‐depth understanding of this unusual mechanism of receptor activation by a mutant molecular chaperone. Mutant CALR is also involved in modulation of the immune response, transcription, and intracellular homeostasis, which could contribute to the development of MPN. In the present article, we comprehensively review the current understanding of the underlying molecular mechanisms for mutant molecular chaperone‐induced cellular transformation.
In the present review, we would like to propose a novel and exciting model: mutant molecular chaperone serves as a ligand to constitutively activate a cytokine receptor on the cell surface for the induction of cellular transformation.
Recurrent somatic mutations of calreticulin (CALR) have been identified in patients harboring myeloproliferative neoplasms; however, their role in tumorigenesis remains elusive. Here, we found that ...the expression of mutant but not wild-type CALR induces the thrombopoietin (TPO)-independent growth of UT-7/TPO cells. We demonstrated that c-MPL, the TPO receptor, is required for this cytokine-independent growth of UT-7/TPO cells. Mutant CALR preferentially associates with c-MPL that is bound to Janus kinase 2 (JAK2) over the wild-type protein. Furthermore, we demonstrated that the mutant-specific carboxyl terminus portion of CALR interferes with the P-domain of CALR to allow the N-domain to interact with c-MPL, providing an explanation for the gain-of-function property of mutant CALR. We showed that mutant CALR induces the phosphorylation of JAK2 and its downstream signaling molecules in UT-7/TPO cells and that this induction was blocked by JAK2 inhibitor treatment. Finally, we demonstrated that c-MPL is required for TPO-independent megakaryopoiesis in induced pluripotent stem cell–derived hematopoietic stem cells harboring the CALR mutation. These findings imply that mutant CALR activates the JAK2 downstream pathway via its association with c-MPL. Considering these results, we propose that mutant CALR promotes myeloproliferative neoplasm development by activating c-MPL and its downstream pathway.
•Mutant CALR induces TPO-independent growth in the human megakaryocytic cell line UT-7/TPO.•Mutant CALR binds to the TPO receptor, inducing phosphorylation of JAK2 and activating downstream signaling.
Objective Thrombocytosis can occur as a primary event accompanying hematological diseases or as a secondary event. Since the publication of the World Health Organization classification in 2008, ...thrombocytosis is now generally defined as a platelet count above 450×109/L. Furthermore, the discovery of driver-gene mutations in myeloproliferative neoplasms (MPNs) has simplified the diagnostic approach for thrombocytosis. To identify the causes of thrombocytosis using this new definition, we conducted a retrospective study. Methods We identified outpatients and inpatients aged 20 years or older with platelet counts >450×109/L in a half-year period at a single institute and analyzed the causes of thrombocytosis and associated clinical characteristics. Results Among 1,202 patients with thrombocytosis, 150 (12.5%) had primary and 999 (83.1%) had secondary thrombocytosis. Of these patients with primary thrombocytosis, 129 (86%) had at least 1 molecular marker indicative of MPNs. The major causes of secondary thrombocytosis were tissue injury (32.2%), infection (17.1%), chronic inflammatory disorders (11.7%) and iron deficiency anemia (11.1%). The median platelet count and the incidence of thrombosis were significantly higher in patients with primary thrombocytosis than in those with secondary thrombocytosis. Conclusion Thrombocytosis mainly occurs as a secondary event; however, it is important to determine the cause of and prevent thrombosis, particularly in cases of primary thrombocytosis.
Pregnancy-associated primary red cell aplasia (pPRCA) is a rare disorder that may occur at various time points during pregnancy. Unlike pregnancy-associated aplastic anemia, pPRCA is usually ...reversible, and no maternal deaths attributable to pPRCA have been reported. Herein, we report a woman diagnosed with pPRCA in two consecutive pregnancies. Corticosteroids were found to be ineffective, and she required a large number of red blood cell transfusions during both pregnancies. Despite severe anemia developing in both pregnancies, two healthy babies were vaginally born and spontaneous remission of pPRCA was seen after delivery. Interestingly, in both events of pPRCA described here, a transient rise of reticulocytes was observed precedent to the authentic recovery phase of reticulocytes and remission of pPRCA, which is a novel finding that has not been reported. The significance of this phenomenon has yet to be elucidated. Along with this case report, we review all 15 cases with 21 events of pPRCA in the literature, including the present case.
Studies have previously shown that mutant calreticulin (CALR), found in a subset of patients with myeloproliferative neoplasms (MPNs), interacts with and subsequently promotes the activation of the ...thrombopoietin receptor (MPL). However, the molecular mechanism behind the activity of mutant CALR remains unknown. Here we show that mutant, but not wild-type, CALR interacts to form a homomultimeric complex. This intermolecular interaction among mutant CALR proteins depends on their carboxyl-terminal domain, which is generated by a unique frameshift mutation found in patients with MPN. With a competition assay, we demonstrated that the formation of mutant CALR homomultimers is required for the binding and activation of MPL. Since association with MPL is required for the oncogenicity of mutant CALR, we propose a model in which the constitutive activation of the MPL downstream pathway by mutant CALR multimers induces the development of MPN. This study provides a potential novel therapeutic strategy against mutant CALR-dependent tumorigenesis via targeting the intermolecular interaction among mutant CALR proteins.
Studies have shown that mutant calreticulin (CALR) constitutively activates the thrombopoietin (TPO) receptor MPL and thus plays a causal role in the development of myeloproliferative neoplasms ...(MPNs). To further elucidate the molecular mechanism by which mutant CALR promotes MPN development, we studied the subcellular localization of mutant CALR and its importance for the oncogenic properties of mutant CALR. Here, mutant CALR accumulated in the Golgi apparatus, and its entrance into the secretion pathway and capacity to interact with N-glycan were required for its oncogenic capacity via the constitutive activation of MPL. Mutant CALR-dependent MPL activation was resistant to blockade of intracellular protein trafficking, suggesting that MPL is activated before reaching the cell surface. However, removal of MPL from the cell surface with trypsin shut down downstream activation, implying that the surface localization of MPL is required for mutant CALR-dependent activation. Furthermore, we found that mutant CALR and MPL interact on the cell surface. Based on these findings, we propose a model in which mutant CALR induces MPL activation on the cell surface to promote MPN development.
Discrimination of Philadelphia‐negative myeloproliferative neoplasms (Ph‐MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and ...bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph‐MPNs from reactive hypercytosis and myelofibrosis by using RNA‐seq analysis utilizing platelet‐rich plasma (PRP)‐derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse‐transcription quantitative PCR and compared among patients with ET, other Ph‐MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation–positive Ph‐MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology‐affirmed triple‐negative ET (TN‐ET) patients were divided into a high– and low–CREB3L1‐expression group, and some patients in the low‐expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single‐handedly discriminate driver mutation–positive Ph‐MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN‐ET showing distinct clinical features including spontaneous remission.
CREB3L1 expression in platelet RNA can completely discriminate Philadelphia‐negative myeloproliferative neoplasms from other entities including reactive hypercytosis and myelofibrosis. The sensitivity and specificity of this testing are both 1.0000.
A subset of essential thrombocythemia (ET) cases are negative for disease-defining mutations on JAK2, MPL, and CALR and defined as triple negative (TN). The lack of recurrent mutations in TN-ET ...patients makes its pathogenesis ambiguous. Here, we screened 483 patients with suspected ET in a single institution, centrally reviewed bone marrow specimens, and identified 23 TN-ET patients. Analysis of clinical records revealed that TN-ET patients were mostly young female, without a history of thrombosis or progression to secondary myelofibrosis and leukemia. Sequencing analysis and human androgen receptor assays revealed that the majority of TN-ET patients exhibited polyclonal hematopoiesis, suggesting a possibility of reactive thrombocytosis in TN-ET. However, the serum levels of thrombopoietin (TPO) and interleukin-6 in TN-ET patients were not significantly different from those in ET patients with canonical mutations and healthy individuals. Rather, CD34-positive cells from TN-ET patients showed a capacity to form megakaryocytic colonies, even in the absence of TPO. No signs of thrombocytosis were observed before TN-ET development, denying the possibility of hereditary thrombocytosis in TN-ET. Overall, these findings indicate that TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and is paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis.
Patients with primary myelofibrosis (PMF) have a poorer prognosis than those with other subtypes of myeloproliferative neoplasms (MPNs). To investigate the relationship between gene mutations and the ...prognosis of Japanese PMF patients, we analyzed mutations in 72 regions located in 14 MPN-relevant genes (
CSF3R
,
MPL
,
JAK2
,
CALR
,
DNMT3A
,
TET2
,
EZH2
,
ASXL1
,
IDH1/2
,
SRSF2
,
SF3B1
,
U2AF1
, and
TP53
) utilizing a target resequencing platform. In our cohort,
ASXL1
mutations were more frequently detected in both overt and prefibrotic PMF patients than other mutations. The frequency of
ASXL1
mutations was slightly higher among overt PMF patients than among prefibrotic PMF patients (44.6% vs 25.0%, FDR = 0.472). Decision tree classification algorithms revealed that
ASXL1
,
EZH2,
and
SRSF2
mutations were associated with a poor prognosis for overt PMF. Overall survival was significantly shorter in patients harboring
ASXL1
,
EZH2
, or
SRSF2
mutations than in those without these mutations (
p
= 0.03). These results suggest that, as reported in Western countries, MIPSS70 is applicable to Japanese PMF patients and
ASXL1
,
EZH2
, and
SRSF2
mutations may be utilized as surrogate markers of a poor prognosis.
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