Mutant calreticulin (CALR) activates the thrombopoietin (TPO) receptor MPL, thereby inducing the development of essential thrombocythemia and primary myelofibrosis. Mutant CALR, due to a frameshift mutation, loses the endoplasmic reticulum (ER) retention signal, the KDEL sequence and is released extracellularly. To examine the significance of the loss of the KDEL sequence in mutant CALR-induced MPL activation, a series of constructs were prepared, including mutant CALR plus KDEL (mutant CALRKDEL), mutant CALR plus the Venus tag and KDEL (mutant CALRVenus-KDEL), and wild-type CALR minus KDEL (CALR WTΔKDEL). UT-7/TPO cells expressing mutant CALRKDEL exhibited autonomous cell growth in the absence of TPO, accompanied by the extracellular secretion of mutant CALRKDEL and subsequent activation of MPL. In contrast, UT-7/TPO cells expressing mutant CALRVenus-KDEL did not exhibit autonomous cell growth or MPL activation without TPO as well as the reduced extracellular secretion of mutant CALRVenus-KDEL. These results suggest that the loss of KDEL function in mutant CALR is closely linked to MPL activation and the extracellular secretion of mutant CALR. While UT-7/TPO cells expressing CALR WTΔKDEL did not exhibit autonomous cell growth, they were responsive to mutant CALR proteins added exogenously, as evidenced by STAT5 activity. Furthermore, CALR WTΔKDEL conferred mutant CALR sensitivity to MPL by recognizing the N-glycans of MPL while maintaining it in an immature form, which may bind to mutant CALR. In conclusion, deletion of the ER retention signal KDEL from CALR is a prerequisite for the expression of the immature form of MPL, which can interact with secreted mutant CALR.

CREB3L1, a gene encoding the endoplasmic reticulum stress transducer, is specifically overexpressed in platelet RNA from patients with myeloproliferative neoplasms (MPNs). However, the pathophysiological roles of CREB3L1 overexpression remain unclear. In the present study, we aimed to study CREB3L1 mRNA expression in the red blood cells (RBCs) of patients with MPN and its role in erythrocytosis. Elevated expression of CREB3L1 was exclusively observed in the RBCs of patients with polycythemia vera (PV) harboring JAK2 exon 12 mutations, but not in those harboring JAK2 V617F mutation or control subjects. In erythropoiesis, CREB3L1 expression was sharply induced in erythroblasts of bone marrow cells collected from patients with JAK2 exon 12 mutation. This was also evident when erythropoiesis was induced in vitro using hematopoietic stem and progenitor cells (HSPCs) with JAK2 exon 12 mutation. Interestingly, overexpression of CREB3L1 in RBCs was observed in patients with reactive erythrocytosis whose serum erythropoietin (EPO) levels exceeded 100 mIU/mL. Elevated CREB3L1 expression was also observed in the erythroblasts of a patient with acute erythroid leukemia. EPO-dependent induction of CREB3L1 was evident in erythroblasts differentiated from HSPCs in vitro, regardless of driver mutation status or MPN pathogenesis. These data strongly suggest that CREB3L1 overexpression in RBCs is associated with hyperactivation of the EPO receptor and its downstream molecule, JAK2. shRNA knockdown of CREB3L1 expression in HSPCs blocked erythroblast formation in vitro. These results suggest that CREB3L1 is required for erythropoiesis in the presence of JAK2 exon 12 mutation or high level of EPO, possibly by antagonizing cellular stress.

AbstractBackgroundAcquired erythrocytosis can be classified into polycythemia vera (PV) and non‐neoplastic erythrocytosis (NNE). The vast majority of PV patients harbor JAK2 mutations, but differentiating JAK2 mutation‐negative PV from NNE is challenging due to a lack of definitive molecular markers.MethodsWe studied the clinical features of 121 patients with erythrocytosis of which 47 (38.8%) were JAK2 mutation‐positive and also fulfilled the diagnostic criteria for PV, and 67 (55.4%) JAK2 mutation‐negative erythrocytosis patients who were diagnosed as NNE. Diagnosis was strictly based on driver mutation analysis and central pathology review.ResultsNo JAK2 mutation‐negative PV patients were found in our cohort. The NNE group showed significantly younger (p < 0.01) age with higher frequency of smoking (p < 0.001), alcohol consumption (p < 0.001), and diabetes mellitus (p < 0.05), whereas the PV group (n = 47) showed significantly higher white blood cell count, platelet count, and lactate dehydrogenase (p < 0.001). Although serum erythropoietin (EPO) levels were significantly higher in NNE compared to PV (p < 0.001), approximately 40% of the NNE patients had EPO levels below the lower range of normal, fulfilling a minor diagnostic criterion of PV and raising the possibility of PV misdiagnosis.ConclusionLow EPO levels in JAK2 mutation‐negative erythrocytosis may not be a reliable diagnostic criterion for distinguishing PV from NNE.

Identification and functional characterization of disease-associated genetic traits are crucial for understanding the pathogenesis of hematologic malignancies.Various in vitro and in vivo models, including cell lines, primary cells, and animal models, have been established to examine these genetic alterations.However, their nonphysiologic conditions, diverse genetic backgrounds, and species-specific differences often limit data interpretation.To evaluate somatic mutations in myeloproliferative neoplasms (MPNs), we used CRISPR/Cas9 combined with the piggyBac transposon system to establish isogenic induced pluripotent stem (iPS) cell lines with or without JAK2V617F mutation, a driver mutation of MPNs.We induced hematopoietic stem/progenitor cells (HSPCs) from these iPS cells and observed phenotypic differences during hematopoiesis using fluorescence-activated cell sorting analysis.HSPCs with pathogenic mutations exhibited cell-autonomous erythropoiesis and megakaryopoiesis, which are hallmarks in the bone marrow of patients with MPNs.Furthermore, we used these HSPCs as a model to validate therapeutic compounds and showed that interferon alpha selectively inhibited erythropoiesis and megakaryopoiesis in mutant HSPCs.These results demonstrate that genome editing is feasible for establishing isogenic iPS cells, studying genetic elements to understand the pathogenesis of MPNs, and evaluating therapeutic compounds against MPNs.

Cyclic AMP-response element-binding protein 3-like 1 (CREB3L1) is a gene involved in the unfolded protein response (UPR). Recently, we demonstrated that CREB3L1 is specifically overexpressed in the platelets of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). In this study, we aimed to show the clinical and biological relevance of CREB3L1 in these hematological diseases. Overexpression of CREB3L1 was specific to platelets in MPNs and associated with a higher risk of thrombosis and fibrotic transformation in essential thrombocythemia (ET) and polycythemia vera (PV) cases, respectively. Furthermore, we found that UPR genes were downregulated in platelets of patients with ET and PV, which were more pronounced in patients harboring the JAK2 V617F mutation. However, CREB3L1 overexpression does not alter UPR gene expression or cell proliferation in UT-7/TPO/CALRm cells exogenously expressing mutated calreticulin and HEL cells harboring endogenous JAK2 V617F. Furthermore, CREB3L1 overexpression did not modulate sensitivity to endoplasmic reticulum stress in these cell lines. Taken together, our data show 1) a potential role of CREB3L1 expression in platelets as a new marker of high-risk MPNs and 2) an association between CREB3L1 overexpression and UPR gene downregulation in these patients' platelets, with CREB3L1 not altering UPR in our in vitro models and possibly further in vivo mechanisms being involved.

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that traffics to the cell surface and constitutively activates the receptor. However, the molecular basis for this mechanism is elusive because oncogenic activation occurs only in the cell-intrinsic complex and is thus cannot be replicated with external agonists. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. In-depth characterization of lectin-induced activation showed that, similar to oncogenic activation, sugar chain-mediated activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. Overall, the existence of sugar-mediated MPL activation, in which the mode of activation is different from the original ligand, suggests that receptor activation is unpredictably diverse in living organisms.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G 0 marker (G 0 M), we narrow down CML LSCs as G 0 M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive 'imaging' data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.

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.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis is imperative because treatment strategies differ greatly, and an exhaustive search for the underlying cause becomes mandatory in reactive cases. However, discrimination is not necessarily easy in the real-world setting, and a simple and universally utilizable method that can efficiently differentiate Ph-MPNs from reactive cases is awaited. We extracted platelet rich plasma (PRP) derived RNAs from 9 essential thrombocythemia (ET) patients (3 patients with JAK2V617F, 3 patients with MPLW515L/K, and 3 patients with CALR exon 9 frameshift mutation) and 6 patients with reactive thrombocytosis (3 cases due to chronic inflammation, and 3 cases due to rebound thrombocytosis) and performed RNA-seq to identify a gene expressed specifically in ET. RNA-seq analysis followed by differential expression and principal component analysis revealed that CREB3L1 had the highest impact in discriminating ET from reactive cases. Subsequently, expression levels of CREB3L1 in PRP were quantified by reverse transcription quantitative PCR and compared within patients with various Ph-MPNs harboring either JAK2, MPL, or CALR mutations, chronic myeloid leukemia (CML), and reactive cases, and found that CREB3L1 expression levels were significantly higher in 66 ET compared to 33 reactive thrombocytosis (p < 0.0001), 26 polycythemia vera (PV) compared to 23 reactive erythrocytosis (p < 0.0001), 22 primary myelofibrosis and 15 post-PV/ET myelofibrosis (MF) compared to 3 reactive MF (p < 0.001, and p < 0.01, respectively), and the entire cohort of 129 Ph-MPN compared to 5 CML patients (p < 0.001). A clear cut-off value discriminating Ph-MPNs and non-Ph-MPNs was determined, and sensitivity and specificity were both 1.0000. Furthermore, when we tested CREB3L1 expression levels of triple-negative cases with thrombocytosis, all patients with CREB3L1 overexpression were pathologically diagnosed as ET by bone marrow biopsy. We demonstrate that CREB3L1 overexpression can single-handedly and reliably discriminate Ph-MPNs from reactive hypercytosis, reactive myelofibrosis, and CML. Early utilization of this method in the diagnostic process can guide patients to an efficient diagnosis and free many patients from unnecessary testing. Disclosures Komatsu: Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

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.

Ruxolitinib (RUX), a JAK1/2-inhibitor, is effective for myeloproliferative neoplasm (MPN) with both JAK2V617 F and calreticulin (CALR) mutations. However, many MPN patients develop resistance to RUX. Although mechanisms of RUX-resistance in cells with JAK2V617 F have already been characterized, those in cells with CALR mutations remain to be elucidated. In this study, we established RUX-resistant human cell lines with CALR mutations and characterized mechanisms of RUX-resistance. Here, we found that RUX-resistant cells had high levels of MPL transcripts, overexpression of both MPL and JAK2, and increased phosphorylation of JAK2 and STAT5. We also found that mature MPL proteins were more stable in RUX-resistant cells. Knockdown of MPL in RUX-resistant cells by shRNAs decreased JAK/STAT signaling. Immunoprecipitation assays showed that binding of mutant CALR to MPL was increased in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells. When resistant cells were cultured in the absence of RUX, the RUX-resistance was reversed, with reduction of the mutant-CALR/MPL complex. In conclusion, MPL overexpression induces higher levels of a mutant-CALR/MPL complex, which may cause RUX-resistance in cells with CALR mutations. This mechanism may be a new therapeutic target to overcome RUX-resistance.

Mutant calreticulin (CALR) has been shown to play a causal role in the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF) via activation of the thrombopoietin receptor MPL. The oncogenic property of mutant CALR originates from a +1 frameshift mutation in its carboxyl-terminal domain, which is found in approximately 30% of patients with ET and PMF. Because the domain is uniquely found in mutant CALR, it has been recognized as a neoantigen and can therefore be used to target CALR-mutant cells using immunotherapy. In the present study, we found that a large portion of the domain generated by the frameshift in the mutant CALR was cleaved by an endoprotease belonging to the subtilisin family in multiple cell lines and primary cells. The cleaved form of mutant CALR was detected in the cell lysate; however, it was more abundant in the culture supernatant, implying that the cleavage occurred on the cell surface and/or outside the cells. Using mass spectrometric analysis, we determined the cleavage site of mutant CALR. To examine whether the cleavage was required for the oncogenic properties of mutant CALR, we introduced point mutations at the cleavage site. The mutant CALR construct that was resistant to protease cleavage exhibited full oncogenic capacity when expressed in UT-7/TPO cells. Consistent with this observation, chemical inhibition of the protease, which blocked the cleavage of mutant CALR, did not interfere with mutant CALR-dependent cell growth in UT-7/TPO cells. Next, we generated B3, a rat monoclonal antibody that recognized the mutant-specific sequence, even after cleavage. B3 recognized both the uncleaved and cleaved forms of mutant CALR by immunoblot in cell lysates prepared from the platelets and peripheral blood cells of CALR-mutant ET and PMF patients. B3 also recognized mutant CALR expressed on the cell surfaces of monocytes and granulocytes from CALR-mutant ET and PMF patients. Based on these results, we developed B3-chimera, a mouse chimeric antibody of B3, and evaluated its therapeutic potential for ET in vivo. We used an ET mouse model created by transplantation of LSK (lin-sca1+c-kit+) cells transduced with CALR del52 into the bone marrow. Intravenous injection of B3-chimera markedly suppressed the thrombocytosis induced by CALR del52, which was associated with a significant reduction in the megakaryocyte count in the bone marrow (Figure 1). In conclusion, we demonstrated that targeting the cleaved form of mutant CALR on the cell surface was a promising strategy for the treatment of CALR-mutant myeloproliferative neoplasms. Disclosures Akazawa: ITOCHU CHEMICAL FRONTIER Corporation: Membership on an entity's Board of Directors or advisory committees. Komatsu:Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

Somatic mutations in JAK2, MPL, and CALR are found in approximately 80% of patients with essential thrombocythemia (ET), whereas the remaining patients are negative for disease-defining mutations and are defined as triple-negative (TN). Studies have shown that some patients with TN-ET harbor non-canonical mutations in JAK2 and MPL; however, the failure to identify recurrent mutations in most patients has made the pathogenesis of TN-ET ambiguous (Milosevic Feenstra et al. Blood 2016, Cabagnols et al. Blood 2016). In this study, we screened 483 patients suspected as having ET in a single center, performed mutation analysis for JAK2 V617F, CALR exon 9, and MPL exon 10, and centrally reviewed bone marrow specimens. We identified 23 patients with TN-ET based on the WHO 2016 criteria. Sequencing analysis of these patients revealed non-canonical mutations in JAK2 and MPL in 4 cases. Whole exome-sequencing analysis of genomic DNA from peripheral blood and CD3-positive cells from 9 patients revealed that 2 patients harbored somatic mutations in other genes; 7 patients showed no detectable somatic mutation. A STAT5 reporter assay revealed that unlike JAK2 V617F and MPL W515L, all non-canonical mutants of JAK2 or MPL activated STAT5 similar to wild-type proteins, suggesting that these mutations did not drive the disease. Statistical analysis of clinical records revealed that patients with TN-ET were mostly young (median age of 36.0 years), female (18/23, 78.3%), and had neither a history of thrombosis nor progression to secondary myelofibrosis and leukemia, demonstrating the unique characteristics of TN-ET. The presence of clonal hematopoiesis, analyzed using genomic DNA purified from granulocytes of peripheral blood from female patients in a human androgen receptor assay, revealed that only 1 out of 15 patients was clonal. Hypothesizing that TN-ET was reactive thrombocytosis, the concentrations of cytokines promoting platelet production such as thrombopoietin (TPO) and interleukin-6 (IL-6) in the serum were analyzed. However, no significant differences in concentrations were observed among ET with driver mutations, TN-ET, and healthy individuals. We next examined the capacity of hematopoietic stem cells from patients with TN-ET to form megakaryocytic colonies. CD34-positive cells purified from cryopreserved bone marrow cells were cultured in the absence or presence of TPO. CD34-positive cells derived from patients with TN-ET exhibited an equivalent capacity to form megakaryocytic colonies compared to those from patients with ET harboring a driver mutation, even in the absence of TPO (Figure 1). Thus, in TN-ET, megakaryopoiesis may have been induced in a cell-autonomous manner. In 10 patients with TN-ET with available blood count data, no sign of thrombocytosis was observed before ET development, indicating that thrombocytosis was not hereditary but rather occurred via an alternate mechanism, such as aberrations in epigenomic regulation that induced cellular transformation (Ohnishi et al, Cell 2015). Taken together, TN-ET is a distinctive disease entity associated with polyclonal hematopoiesis and paradoxically caused by hematopoietic stem cells harboring a capacity for cell-autonomous megakaryopoiesis. Figure 1 Disclosures Komatsu: Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy.

Objective Prefibrotic/early primary myelofibrosis (pre-PMF) and essential thrombocythemia (ET) exhibited different features of bone marrow; however, this is not always easy to judge objectively, making pathologists' distinction often suboptimal. In the WHO 2008 criteria, pre-PMF was not defined as a subgroup of PMF; therefore, affected patients were at a higher risk of misdiagnosis with ET. In this study, we examined the prevalence of pre-PMF patients among those previously diagnosed with ET in Japan. Method We reviewed bone marrow specimens and clinical and molecular parameters of patients who were previously diagnosed with ET by the WHO 2008 criteria. Results Among 107 ET patients, 13 patients were redefined as having pre-PMF. Pre-PMF patients exhibited a higher frequency of MPL mutation and increased platelet counts compared to true ET patients. Molecular analysis revealed the frequencies of high-risk molecular mutations, such as ASXL1, EZH2, and SRSF2, were significantly increased in pre-PMF patients than those in true ET patients. Conclusion These results demonstrated the value of reexamining clinical records for patients diagnosed with ET by the WHO 2008 criteria and emphasized that adequate examinations of patients' bone marrow are crucial for an accurate diagnosis of pre-PMF and ET.

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 CALRdependent 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.

In myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), somatic mutation in the Splicing Factor 3B subunit 1 gene (SF3B1) at the rate of approximately 85% was concurrently found with one of the MPN driver mutations, such as JAK2 V617F (50%), calreticulin (CALR) exon 9 (1–3%) and MPL W515 (0–3%) mutations (Patnaik and Tefferi 2017). Impaired erythropoiesis induced by mutant SF3B1 (Mupo et al, 2017, Obeng et al, 2016), and an increase in one or more lineages of blood cells induced by MPN driver mutations (Vainchenker and Kralovics 2017) have led to the notion that the overlapping phenotypes of MDS/MPN are at least partly, if not totally, due to overlapping disease-defining mutations (Cazzola et al, 2013). The MPN driver mutation, JAK2 exon 12, is rare in MPN and has been exclusively found in polycythaemia vera (PV) (Scott 2011), with no reports of this mutation in MDS/MPN-RS-T to date. A bone marrow biopsy of a patient, who was originally followed for Raynaud disease in our hospital and presented with anaemia associated with persistent leucocytosis and thrombocytosis (Fig S1 and Table SI), displayed MDS/MPN features (Fig S2) and lacked any chromosomal abnormality, including Philadelphia chromosome; the patient was thus suspected to have MDS/MPN-RS-T. Sequence analysis (see Data S1) of genomic DNA obtained from a peripheral blood sample revealed the allelic frequencies of SF3B1 E622D, JAK2 H538_K539delinsL (hereinafter called JAK2 exon 12, one of the typical mutations of JAK2 exon 12; Scott 2011), and TET2 I1873T (Fig S3) to be 44%, 75% and 14%, respectively. Based on this evidence, the patient was diagnosed with MDS/MPN-RS-T. As no report on the identification of JAK2 exon 12 mutation in MDS/MPN-RS-T was available, and only limited assessment has been performed for the capacity of haematopoietic cell differentiation in MDS/MPN-RS-T harbouring SF3B1 and MPN driver mutations, we examined the colony-forming ability of the patient's bone marrow mononuclear cells (BM-MNCs). Unlike BM-MNCs obtained from a patient with PV harbouring JAK2 exon 12 mutation and a patient with malignant lymphoma without bone marrow infiltration (hereinafter called control), those from the patient with MDS/MPN-RS-T formed smaller colonies of burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte (CFU-G), CFU-macrophage (CFU-M), and CFU–granulocyte, macrophage (CFU-GM) (Fig 1A). Significantly lower number of BFU-Es was formed from BM-MNCs of the MDS/MPN-RS-T samples than from the PV and control samples, even in the presence of erythropoietin (EPO) (Fig 1B). EPO-independent BFU-Es were formed only in those with PV but not in those with MDS/MPN-RS-T harbouring JAK2 exon 12 mutation (Fig 1B), confirming that the patient had neither PV nor post-PV myelofibrosis. Based on the mutation status in individual colonies (Fig 1C and S3) determined by Sanger sequencing (see Data S1), diagrams were created to represent a hypothetical clonal evolution of haematopoietic progenitor cells (Fig 2A,2B). As expected from the patient's anaemic phenotype, an acquisition of SF3B1 mutation occurred earlier than any other mutation. JAK2 exon 12 mutation was always concurrently detected with the SF3B1 mutation, implying that BFU-E formation was blocked by mutant SF3B1, leading to anaemia in the patient, regardless of JAK2 exon 12 mutation. In contrast, in MPN, acquisition of SF3B1 mutation seems to be a later event (Boiocchi et al, 2019). Therefore, we hypothesized that the order of acquisition of SF3B1 mutation and an MPN driver mutation is likely to define the disease type (Fig 2C). Seventeen out of 28 BFU-Es (60.7%) were defined as wild-type (Fig 2A), which is contradictory to the anaemic phenotype of our patient. This discrepancy is presumably owing to the weakened capacity of erythroid cell differentiation in progenitor cells, considering the smaller size of BFU-Es, regardless of SF3B1 and JAK2 exon 12 mutations (Fig 1A). In contrast to the BFU-E formation, the ratio of wild-type (n = 4, 16.0%) versus SF3B1 mutant (n = 21, 84.0%) in CFU-G/M/GMs was significantly reduced compared to that in BFU-Es (P < 0.001, Table SII). This suggests skewing of the progenitor cells harbouring the SF3B1 mutation toward the myeloid lineage. Although the leucocytosis-like phenotype was not recapitulated in the colony assay, these genetic analyses implied that SF3B1 mutation, in combination with JAK2 exon 12 mutation, contributed to the leucocytosis in the patient by skewing the haematopoietic lineage towards myeloid (Fig 1C). Although this needs to be confirmed with bone marrow cells from multiple patients, it definitely suggests the possibility of cell extrinsic factor(s), such as cytokines and the bone marrow niche, playing a key role in the induction of leucocytosis in the patient with MDS/MPN-RS-T. Screening for JAK2 exon 12 mutations may not be routinely performed in daily clinical practice. The identification of concurrent SF3B1 mutation and MPN driver mutation supports the diagnosis of MDS/MPN-RS-T, the prognosis of which is different and treatment options vary across MDS or MPN. We would like to emphasize the importance of analysing JAK2 exon 12 mutation in patients with suspected MDS/MPN-RS-T, but who are negative for JAK2 V617F, CALR exon 9 and MPL W515 mutations. Furthermore, we would like to note that MDS/MPN-RS-T might often be misdiagnosed as essential thrombocythaemia (ET) or prefibrotic/early primary myelofibrosis (pre-PMF), especially when the bone marrow assessment, such as iron staining, is not properly performed. Detection of SF3B1 mutation does not support diagnosis, because this occurs in 5% of ET and 10% of PMF cases (Tefferi et al, 2016a, Tefferi et al, 2016b). A mild anaemia and megakaryocytic dysplasia are associated with pre-PMF. Therefore, we would like to emphasize that the absence or small number, if any, of ring sideroblasts and that of erythroid dysplasia should be confirmed in patients suspected with ET and pre-PMF, especially when they harbour SF3B1 mutation. In summary, we identified a patient with MDS/MPN-RS-T harboring SF3B1 and JAK2 exon 12 mutations. Considering the possibility that the order of acquisition of SF3B1 mutation and an MPN driver mutation defines the disease type, this study highlighted the importance of a comprehensive assessment of gene alterations, as well as pathology, for the diagnosis of MPN and MDS/MPN-RS-T. We thank the members of the Department of Haematology, Juntendo University Graduate School of Medicine for encouraging this study. TI designed the study, carried out the experiment, and wrote the initial draft of the manuscript. MA, YH and NK contributed to manuscript preparation, analysis and interpretation of data. MI performed histological diagnosis of clinical samples. SJ, HN, TO, KM, YF contributed to data collection. MI, YE, AO, NK supervised the study. This work was funded in part by the MEXT's Promotion Plan for the Platform of Human Resource Development for Cancer Project; the JSPS KAKENHI Grant (#17K16195); and Grant for Cross-disciplinary Collaboration in Juntendo University (#30-8). The funders had no role in manuscript preparation. Data S1. Patients and methods. Figure S1. Progressive leukocytosis, thrombocytosis, and anemia observed in the patient. Figure S2. Bone marrow morphologies of the patient harboring JAK2 exon 12 mutation, representing features associated with MDS/MPN-RS-T. Figure S3. Detection of SF3B1, JAK2 exon 12, and TET2 mutations in peripheral blood and colonies by Sanger sequencing. Table SI. Hematological and biochemical data for the patient with MDS/MPN-RS-T harboring JAK2 exon 12 mutation at initial diagnosis. Table SII. Enrichment of mutant cells in myeloid lineages. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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Myeloproliferative neoplasms (MPN), such as polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are characterized by the expansion of myeloid lineage cells caused by an acquired driver mutation, such as JAK2 V617F, CALR exon 9, or MPL W515K/L.[1][1] Hematopoietic