Calreticulin Mutations Research Articles

Prevalence of Calreticulin exon 9 Mutation in Iranian Cardiovascular Patients

Background: Calreticulin (CALR) is a 46 kDa protein in the endoplasmic reticulum and is one of the major proteins in ca2+ binding; it has a key role in oxidative stress, transcription factor activation, and as a chaperone in newly synthesized protein and glycoprotein folding. The high expression of CALR is pivotal for cardiac development in the embryonic period. It has been showed that mutation in exon 9 of CALR causes loss of C-terminal function and contributes to cardiovascular disease (CVD) development. Objective: It could conceivably be hypothesized that in addition to the general risk factors, the specific gene defects which are less considered can contribute to CVD development. In this regard in this study the possible existence of CALR mutations in CVD development is determine in patients younger than 40. Method: Thirty patients younger than 40 were recruited for this study, 86.7% (26) were male, and just 13.3% (4) were female. The amplification refractory mutation system-PCR was used to identified mutation in exon 9. The CVD risk factors, including blood pressure, type 2 diabetes, dyslipidemia, history of smoking, alcohol drinking, and familial CVD development were evaluated. Result: In none of the patients, CALR mutations were detected. Since CALR defect causes accumulation of glycogen in the heart's cells and contributes to CVD development, our results confirm this, so that 76.7% of patients did not have diabetes. Conclusion: The findings of the current study show there is no significant differences between exon 9 CALR mutation and CVD development.

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Hematoxylin binds to mutant calreticulin and disrupts its abnormal interaction with thrombopoietin receptor

Somatic mutations of calreticulin (CALR) have been identified as a main disease driver of myeloproliferative neoplasms, suggesting that development of drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD) abolishes the ability of mutant CALR to oncogenically activate the thrombopoietin receptor (MPL). We therefore hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing a selective growth inhibitory effect on cells that depend on oncogenic CALR for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR-mutated cells than in CALR wild-type cells. Additionally, knockout or C-terminal truncation of CALR eliminated drug hypersensitivity in CALR-mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, disruption of the mutant CALR-MPL interaction, inhibition of the JAK2-STAT5 pathway, and reduction at the intracellular level of mutant CALR upon drug treatment. Our data indicate that small molecules targeting the GBD of CALR can selectively kill CALR-mutated cells by disrupting the CALR-MPL interaction and inhibiting oncogenic signaling.

Zinc-dependent multimerization of mutant calreticulin is required for MPL binding and MPN pathogenesis

Calreticulin (CALR) is mutated in the majority of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs). Mutant CALR (CALRdel52) exerts its effect by binding to the thrombopoietin receptor MPL to cause constitutive activation of JAK-STAT signaling. In this study, we performed an extensive mutagenesis screen of the CALR globular N-domain and revealed 2 motifs critical for CALRdel52 oncogenic activity: (1) the glycan-binding lectin motif and (2) the zinc-binding domain. Further analysis demonstrated that the zinc-binding domain was essential for formation of CALRdel52 multimers, which was a co-requisite for MPL binding. CALRdel52 variants incapable of binding zinc were unable to homomultimerize, form CALRdel52-MPL heteromeric complexes, or stimulate JAK-STAT signaling. Finally, treatment with zinc chelation disrupted CALRdel52-MPL complexes in hematopoietic cells in conjunction with preferential eradication of cells expressing CALRdel52 relative to cells expressing other MPN oncogenes. In addition, zinc chelators exhibited a therapeutic effect in preferentially impairing growth of CALRdel52-mutant erythroblasts relative to unmutated erythroblasts in primary cultures of MPN patients. Together, our data implicate zinc as an essential cofactor for CALRdel52 oncogenic activity by enabling CALRdel52 multimerization and interaction with MPL, and suggests that perturbation of intracellular zinc levels may represent a new approach to abrogate the oncogenic activity of CALRdel52 in the treatment of MPNs.

Mutation profile in BCR-ABL1-negative myeloproliferative neoplasms: A single-center experience from India

Objective/BackgroundRecurrent somatic mutations in the JAK2, calreticulin (CALR), and the MPL genes are described as drivers of BCR-ABL1-negative myeloproliferative neoplasms (MPN) that includes polycythemia vera (PV), essential thrombocytosis (ET), primary myelofibrosis (PMF), and MPN unclassified (MPN-U). MethodsWe describe the mutation profile and clinical features of MPN cases diagnosed at a tertiary care center. JAK2V617F and MPL (S505/W515) mutations were screened by allele-specific polymerase chain reaction, while CALR exon 9 and JAK2 exon 12 mutations were screened by fragment analysis/Sanger sequencing. Among the 1,570 patients tested for these mutations during the study period, 407 were classified as MPN with a diagnosis of PV, ET, PMF, and MPN-U seen in 30%, 17%, 36%, and 17%, respectively, screened. ResultsSimilar to previous reports from Asian countries, the incidence of PMF was the highest among the classic MPN. JAK2V617F mutation was detected in 90% of PV, 38% of ET, 48% of PMF, and 65% of MPN-U. JAK2 exon 12 mutations were seen in 5.7% of PV and 1.4% of PMF. CALR exon 9 mutations were seen in 33% of ET, 33% of PMF, and 12% of MPN-U. MPL mutations were detected in 2.8%, 2.7%, and 2.9% of ET, PMF, and MPN-U, respectively. Fifteen % of PMF, 26% of ET, and 22% of MPN-U were triple negative. ConclusionThere was a significantly higher incidence of CALR mutation in PMF and ET cases. Our study highlights the challenges in the diagnosis of JAK2-negative PV and the need for harmonization of criteria for the same.

Therapeutic Cancer Vaccination With a Peptide Derived From the Calreticulin Exon 9 Mutations Induces Strong Cellular Immune Responses in Patients With CALR-Mutant Chronic Myeloproliferative Neoplasms.

BackgroundThe calreticulin (CALR) exon 9 mutations that are identified in 20% of patients with Philadelphia chromosome negative chronic myeloproliferative neoplasms (MPN) generate immunogenic antigens. Thus, therapeutic cancer vaccination against mutant CALR could be a new treatment modality in CALR-mutant MPN.MethodsThe safety and efficacy of vaccination with the peptide CALRLong36 derived from the CALR exon 9 mutations was tested in a phase I clinical vaccination trial with montanide as adjuvant. Ten patients with CALRmut MPN were included in the trial and received 15 vaccines over the course of one year. The primary end point was evaluation of safety and toxicity of the vaccine. Secondary endpoint was assessment of the immune response to the vaccination epitope (www.clinicaltrials.gov identifier NCT03566446).ResultsPatients had a median age of 59.5 years and a median disease duration of 6.5 years. All patients received the intended 15 vaccines, and the vaccines were deemed safe and tolerable as only two grade three AE were detected, and none of these were considered to be related to the vaccine. A decline in platelet counts relative to the platelets counts at baseline was detected during the first 100 days, however this did not translate into neither a clinical nor a molecular response in any of the patients. Immunomonitoring revealed that four of 10 patients had an in vitro interferon (IFN)-γ ELISPOT response to the CALRLong36 peptide at baseline, and four additional patients displayed a response in ELISPOT upon receiving three or more vaccines. The amplitude of the immune response increased during the entire vaccination schedule for patients with essential thrombocythemia. In contrast, the immune response in patients with primary myelofibrosis did not increase after three vaccines.ConclusionTherapeutic cancer vaccination with peptide vaccines derived from mutant CALR with montanide as an adjuvant, is safe and tolerable. The vaccines did not induce any clinical responses. However, the majority of patients displayed a marked T-cell response to the vaccine upon completion of the trial. This suggests that vaccines directed against mutant CALR may be used with other cancer therapeutic modalities to enhance the anti-tumor immune response.

Elevated levels of oxidized nucleosides in individuals with the JAK2V617F mutation from a general population study

It is unknown if the somatic mutations in chronic myeloproliferative neoplasms (MPNs), JAK2V617F and Calreticulin, are associated with oxidative stress, or impaired mitochondrial defense against reactive oxygen species. In the Danish General Suburban Population Study (GESUS), including 116 JAK2V617F-mutated, 8 CALR-mutated, and 3310 mutation-negative participants without overt MPN, and in a study of 39 patients with myelofibrosis, the most advances type of MPNs, and 179 matched controls, we compared the urinary concentration of oxidized nucleosides – 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) – as markers of oxidative stress. In GESUS, we performed Mendelian randomization analyses, using the Ala16Val single nucleotide polymorphism in the superoxide dismutase2 (SOD2) gene. In the multivariate analyses in GESUS, the 8-oxodG and 8-oxoGuo concentration were 13% (95%CI: 6–21%, p < 0.001) and 6% (95%CI: 0.4–11%, p = 0.035) higher in mutation-positive than in mutation-negative participants, respectively. Each SOD2 T allele was associated with an odds ratio of being mutation-positive of 1.69 (95%CI: 1.12–2.55, p = 0.013) through 8-oxodG. The 8-oxodG and 8-oxoGuo concentrations were 77% (95%CI: 49–110%, p < 0.001) and 105% (95%CI: 80–133%, p < 0.001) higher in myelofibrosis patients than in controls, respectively. In conclusion, an impaired mitochondrial antioxidative defense, that is causatively associated with markers of oxidative stress, may contribute to the development of mutations associated with MPNs.

Calreticulin Immunohistochemistry in Myeloproliferative Neoplasms - Evolution of a New Cost-Effective Diagnostic Tool: A Retrospective Study with Histological and Molecular Correlation.

Recent WHO 2017 guidelines mandates mutational analysis for the diagnosis of myeloproliferative disorders (MPN). JAK2V617F has been found in only 50-60% of Primary myelofibrosis (PMF) and Essential thrombocythaemia (ET). A recently discovered somatic Calreticulin (CALR) mutation has been linked to MPN. This mutation leads to a common 36 amino acid C-terminus that can be detected accurately by immunohistochemistry (IHC). Limited published literature exists on the utility of CAL2IHC as a diagnostic tool. The study aimed to validate the sensitivity and specificity of CAL2IHC for its use as a cost effective and rapid diagnostic tool. Subjects included 23 patients of MPN (15 PMF, 6 ET, 2 PV (Polycythaemia Vera)), diagnosed between January 2014 to November 2016 with adequate available tissue for histopathological and mutational analysis. Mutational analysis had been performed with Bidirectional Sanger sequencing. CAL2IHC was performed in all cases and the sensitivity and specificity of CAL2 IHC to identify the Calreticulin mutation was evaluated with respect to comparison with the gold standard mutation analysis. In the 23 MPN patients, CAL2 IHC detected CALR mutation with a sensitivity of 95% and a specificity of 100%. Both cases of PV were negative for CAL2IHC. CAL2IHC showed cytoplasmic positivity in ET (2-3+) and PMF (1-3+) with (62-69%) positive megakaryocyte staining. All 6 ET cases and all 14/15 PMF cases were CAL2IHC positive, and these results were concordant with CALR mutational analysis. Anti-CAL2 immunohistochemistry is a specific and a sensitive marker to detect CALR mutation. Its' cost effectiveness and fast results are quite advantageous as compared to molecular analysis.

Impact of Calreticulin and Its Mutants on EndoplasmicReticulum Function in Health and Disease.

The endoplasmic reticulum (ER) performs key cellular functions including protein synthesis, lipid metabolism and signaling. While these functions are spatially isolated in structurally distinct regions of the ER, there is cross-talk between the pathways. One vital player that is involved in ER function is the ER-resident protein calreticulin (CALR). It is a calcium ion-dependent lectin chaperone that primarily assists in glycoprotein synthesis in the ER as part of the protein quality control machinery. CALR also buffers calcium ion release and mediates other glycan-independent protein interactions. Mutations in CALR have been reported in a subset of chronic blood tumors called myeloproliferative neoplasms. The mutations consist of insertions or deletions in the CALR gene that all cause a+1bp shift in the reading frame and lead to a dramatic alteration of the amino acid sequence of the C-terminal domain of CALR. This alters CALR function and affects cell homeostasis. This chapter will discuss how CALR and mutant CALR affect ER health and disease.

Calreticulin mutations in myeloproliferative neoplasms.

Calreticulin (CALR) is a chaperone present in the endoplasmic reticulum, which is involved in the quality control of N-glycosylated proteins and storage of calcium ions. In 2013, the C-terminal mutation in CALR was identified in half of the patients with essential thrombocythemia and primary myelofibrosis who did not have a JAK2 or MPL mutation. The results of 8 years of intensive research are changing the clinical practice associated with treating myeloproliferative neoplasms (MPNs). The presence or absence of CALR mutations and their mutation types already provide important information for diagnosis and treatment decision making. In addition, the interaction with the thrombopoietin receptor MPL, which is the main mechanism of transformation by CALR mutation, and the expression of the mutant protein on the cell surface have a great potential as targets for molecular-targeted drugs and immunotherapy. This chapter presents recent findings on the clinical significance of the CALR mutation and the molecular basis by which this mutation drives MPNs.

Roles of Calreticulin in Protein Folding, Immunity, Calcium Signaling and Cell Transformation.

The endoplasmic reticulum (ER) is an organelle that mediates the proper folding and assembly of proteins destined for the cell surface, the extracellular space and subcellular compartments such as the lysosomes. The ER contains a wide range of molecular chaperones to handle the folding requirements of a diverse set of proteins that traffic through this compartment. The lectin-like chaperones calreticulin and calnexin are an important class of structurally-related chaperones relevant for the folding and assembly of many N-linked glycoproteins. Despite the conserved mechanism of action of these two chaperones in nascent protein recognition and folding, calreticulin has unique functions in cellular calcium signaling and in the immune response. The ER-related functions of calreticulin in the assembly of major histocompatibility complex (MHC) class I molecules are well-studied and provide many insights into the modes of substrate and co-chaperone recognition by calreticulin. Calreticulin is also detectable on the cell surface under some conditions, where it induces the phagocytosis of apoptotic cells. Furthermore, mutations of calreticulin induce cell transformation in myeloproliferative neoplasms (MPN). Studies of the functions of the mutant calreticulin in cell transformation and immunity have provided many insights into the normal biology of calreticulin, which are discussed.

Calreticulin haploinsufficiency augments stem cell activity and is required for onset of myeloproliferative neoplasms in mice

AbstractMutations in JAK2, myeloproliferative leukemia virus (MPL), or calreticulin (CALR) occur in hematopoietic stem cells (HSCs) and are detected in more than 80% of patients with myeloproliferative neoplasms (MPNs). They are thought to play a driver role in MPN pathogenesis via autosomal activation of the JAK-STAT signaling cascade. Mutant CALR binds to MPL, activates downstream MPL signaling cascades, and induces essential thrombocythemia in mice. However, embryonic lethality of Calr-deficient mice precludes determination of a role for CALR in hematopoiesis. To clarify the role of CALR in normal hematopoiesis and MPN pathogenesis, we generated hematopoietic cell-specific Calr-deficient mice. CALR deficiency had little effect on the leukocyte count, hemoglobin levels, or platelet count in peripheral blood. However, Calr-deficient mice showed some hematopoietic properties of MPN, including decreased erythropoiesis and increased myeloid progenitor cells in the bone marrow and extramedullary hematopoiesis in the spleen. Transplantation experiments revealed that Calr haploinsufficiency promoted the self-renewal capacity of HSCs. We generated CALRdel52 mutant transgenic mice with Calr haploinsufficiency as a model that mimics human MPN patients and found that Calr haploinsufficiency restored the self-renewal capacity of HSCs damaged by CALR mutations. Only recipient mice transplanted with Lineage−Sca1+c-kit+ cells harboring both CALR mutation and Calr haploinsufficiency developed MPN in competitive conditions, showing that CALR haploinsufficiency was necessary for the onset of CALR-mutated MPNs.

JAK2V617F, CALR and MPL515L/K mutations and plateletcrit in essential thrombocythemia: A single centre's experience.

JAK2V617F (JAK2), calreticulin (CALR) and MPL515L/K (MPL) mutations are important in essential thrombocythemia (ET) and may be associated with various clinical consequences of the disease. This study aimed to compare the clinical and haematological parameters of ET patients regarding the mentioned mutations and the role of plateletcrit (PCT). Seventy patients who were diagnosed with ET between 2005 and 2017 in a single centre were included in this descriptive study. The initial symptoms and clinical findings were retrieved from the electronic database. JAK2 gene V617F mutations, MPL gene exon 10 mutations and CALR gene exon 9 DNA sequence analyses were performed. Forty-one healthy volunteers were included to perform ROC curve analysis for interpreting PCT value. The distributions of patients according to the mutations were as follows: Thirty-seven (52.9%) patients were JAK2-positive, 15 (21.4%) were CALR-positive, 2 (2.8%) patients were positive for both CALR and JAK2, and 1 (1.4%) was only MPL-positive. Fifteen (21.4%) patients were triple-negative. The ET patients with JAK2 mutation showed a higher level of haemoglobin at the time of diagnosis. The ET patients with CALR mutation presented with higher platelet and LDH levels (P=.002 and P=.001, respectively). The PCT level was higher in the CALR-positive group when compared to the others (P=.026). A sensitivity value of 97.6% and specificity value of 98.6% were determined regarding PCT% at a cut-off value of 0.37 in ET patients. In CALR-positive patients, the sensitivity and specificity values were 100% for the PCT at a cut-off value of 0.42%. We determined that the platelet count and blood LDH level was high in the ET patient group with CALR mutation. Besides, we found that the blood haemoglobin level was higher in the ET patient group with JAK2 mutation. Additionally, the PCT level was higher in the CALR group when compared to the other patient groups.

The stem/progenitor landscape is reshaped in a mouse model of essential thrombocythemia and causes excess megakaryocyte production.

Frameshift mutations in CALR (calreticulin) are associated with essential thrombocythemia (ET), but the stages at and mechanisms by which mutant CALR drives transformation remain incompletely defined. Here, we use single-cell approaches to examine the hematopoietic stem/progenitor cell landscape in a mouse model of mutant CALR-driven ET. We identify a trajectory linking hematopoietic stem cells (HSCs) with megakaryocytes and prospectively identify a previously unknown intermediate population that is overrepresented in the disease state. We also show that mutant CALR drives transformation primarily from the earliest stem cell compartment, with some contribution from megakaryocyte progenitors. Last, relative to wild-type HSCs, mutant CALR HSCs show increases in JAK-STAT signaling, the unfolded protein response, cell cycle, and a previously undescribed up-regulation of cholesterol biosynthesis. Overall, we have identified a novel megakaryocyte-biased cell population that is increased in a mouse model of ET and described transcriptomic changes linking CALR mutations to increased HSC proliferation and megakaryopoiesis.

Therapeutic Potential of an Antibody Targeting the Cleaved Form of Mutant Calreticulin in Myeloproliferative Neoplasms

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 &amp; Royalties: PCT/JP2020/008434, Research Funding.

Co-Expression of JAK2-V617F and Calr-del52 In Vivo Enhances Myeloproliferative Phenotype in Mice and Does Not Influence Competitive Fitness of Hematopoietic Stem Cells

Myeloproliferative neoplasms (MPNs) are a group of hematopoietic stem cell disorders driven by mutations that constitutively activate physiologic signal transduction pathways essential for hematopoiesis. The majority of patients with classical MPNs harbor mutations within the Janus activated kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor (MPL) genes. The occurrence of driver mutations among patients is mutually exclusive but rare double positive cases have been reported. Employment of targeted sequencing methods for diagnostics revealed more double positive cases and reviewing published studies we estimate the CALR and JAK2 double positive MPN frequency to be about 0.5% in all MPNs and 2% in essential thrombocythemia. However, the mutual exclusivity of CALR and JAK2 mutations in double positive cases was confirmed at single cell level in few studies where clonogenic assays were performed with subsequent genotyping of colonies. In our MPN biobank of over 800 samples, we identified one case diagnosed with PMF, carrying both in JAK2 and CALR, with allelic burdens of 8% and 41%, respectively. Using a clonogenic assay, we confirmed mutual exclusivity of the mutations at CFU level confirming previous findings. Mutations can be mutually exclusive due to their synthetic lethal interaction. Such synthetic lethal interaction has been recently described in splicing factor mutated MDS, showing that SF3B1 and SRSF2 double mutant hematopoietic cells (HSC) have reduced fitness in vivo providing explanation why such patients are never observed. In this study, we tested the hypothesis that JAK2-V617F and CALR-del52 mutations are synthetic lethal if they occur in the same HSC. We have generated mice that co-expresses both JAK2-V617F and CALR-del52 mutations in hematopoietic lineages and analyzed their phenotype. First, we co-expressed JAK2-V617F and CALR-del52 on the Vav1-Cre backgound in which Cre recombinase activates the floxed transgenes in embryonic HSC. Double positive offspring were born at expected Mendelian frequency compared to single positive littermates, suggesting no signs of synthetic lethality in utero. The phenotype of the JAK2-V617F and CALR-del52 double positive mice was significantly more severe compared to single mutant mice. More specifically, double positive mice showed more pronounced splenomegaly, higher white blood cell, lymphocyte, granulocyte, monocyte, and platelet counts in peripheral blood. In the bone marrow, double positive mice had more prominent megakaryocyte dyspoiesis and altered myeloid to erythroid ratios, without evident myelofibrosis as observed in histological sections. This increase in megakaryocyte numbers was also confirmed by FACS. In addition, double positive mice had more obscured follicular architecture and more signs of enhanced extramedullary hematopoiesis in the spleen, and more pronounced megakaryocytic sequestration in the lungs when compared to the JAK2-V617F histology findings. These mice also had lower overall survival compared to the JAK2-V617F and CALR-del52 mice. Next, we performed competitive bone marrow transplantation (BMT) to examine HSC fitness in primary and secondary transplants. Wild type bone marrow (BM) derived from F1 hybrid CD45.1/CD45.2 mice was mixed with BM form either mice bearing single mutation or double mutations (CD45.2), and ingrafted into CD45.1 recipients. The changes in chimerism were followed in peripheral blood by FACS. Double positive BM engrafted recipients equally well as JAK2-V617F or CALR-del52 cells suggesting no functional defect at HSC level. Same results were seen also in secondary BMT. In summary, double positive mice have an enhanced MPN phenotype with lower overall survival compared to single positive JAK2-V617F and CALR-del52 animals. Our results suggest that the mutual exclusivity of MPN driver mutations JAK2-V617F and CALR-del52 is not due to synthetic lethality or loss of HSC fitness. It is possible that once the second mutation is acquired, JAK2-V617F and CALR-del52 double positive cells do not gain additional competitive advantage over single positive HSCs, and therefore, do not grow out into a significant population. Another reason why we do not observe JAK2-V617F and CALR-del52 double positive colonies in patients is the very low likelihood of such HSC arising. Our data shows that such MPN patients may be found and very likely will have more severe MPN. Disclosures No relevant conflicts of interest to declare.

The frequency of calreticulin and mpl gene mutations in bcr-abl and jak2 unmutated chronic myeloproliferative neoplasms and its effect on the outcome

The frequency of calreticulin and mpl gene mutations in bcr-abl and jak2 unmutated chronic myeloproliferative neoplasms and its effect on the outcome

Genetic analysis of BCR-ABL negative chronic myeloproliferative diseases at initial diagnosis and their clinical effects

Purpose: The aim of this study to discuss frequency and clinical significance of JAK2-V617F, Calreticulin (CALR type 1 and type-2) and MPL-W515K/L mutations in patients at initial diagnosis of bcr-abl negative chronic myeloproliferative diseases (CMPD).Materials and Methods: In this study, the demographic characteristics, subtype, risk status and mutation analysis were investigated between July 2017 and March 2019 in patients diagnosed with bcr-abl negative CMPD.Results: JAK2 V617F mutation was detected in sum of 27 patients, 18 of them (85,7%) diagnosed with polycythemia vera (PV) and rest of them (N=9, 56,2%) diagnosed with essential thrombocytosis (ET). Calreticulin mutation was positive in 4 (57,1%) patients, who were also JAK2 V617F negative, diagnosed with ET. CALR-type 1 mutation was detected in three patients and CALR-type 2 was in one. MPL-W515K/L was not detected in any of patients diagnosed with ET. Thrombotic event was accompanied 12,6% of patients with PV and 6,25% patients with ET. Splenomegaly was noted in 14 (37,8%) of patients. Conclusion: Pathogenesis, classification, and risk groups of CMPD have been well characterized with the identification of some genetic mutations in recent years. JAK2 V617F, CALR and MPL are the most common somatic mutations in the pathogenesis of CMPD, which are important in the diagnosis, risk classification and follow-up of the disease and gain importance in personalized medicine.

Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN

Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.

Frequency of coexistence and kinetics of the BCR-ABL1 transcript level and allele burden of JAK2V617F and CALR Type 1, 2 gene mutations in patients with chronic myeloid leukemia

Introduction. The pathogenesis of myeloproliferative neoplasms is associated with the chimeric gene BCR-ABL1 or with one of the driver mutations in the genes JAK2, MPL and CALR (Calreticulin). However, the classifi cation of the World Health Organization lists no myeloid neoplasms with more than one driver genetic abnormality. Aim. To search for mutations in the genes JAK2, MPL and CALR in patients with BCR-ABL1-positive chronic myeloid leukemia (CML), as well as to evaluate the kinetics of the discovered mutations during tyrosine kinase inhibitor (TKI) therapy. Materials and methods. mRNA and DNA samples isolated from blood and bone marrow cells of 567 CML patients, who underwent periodic monitoring of the BCR-ABL1 transcript level over the 2012–2019 period were included in the study The BCR-ABL1 transcript level was determined using a highly sensitive quantitative real-time polymerase chain reaction. The mutations JAK2V617F and MPLW515L/K were detected using real-time quantitative allele-specifi c polymerase chain reaction. Mutations in the CALR gene were investigated using fragment analysis followed by Sanger sequencing. Results. The combination of the BCR-ABL1, JAK2 and CALR gene mutations among CML patients receiving TKIs was 1.23 % (7/567). Out of these, the combination of BCR-ABL1 with JAK2V617F and the combination of BCR-ABL1 with CALR gene mutations were detected in 0.88 % (5/567) and 0.35 % (2/567) of cases, respectively. During TKI therapy, in 5 out of 7 patients, the level of BCR-ABL1 reached major molecular response (MR). In 4 of these patients, the therapy was discontinued. These patients are currently in molecular remission. In the remaining 2 patients, major MR was not achieved, despite the use of second-generation TKI preparations. Conclusions. The combination of the BCR-ABL1 chimeric gene with gene mutations Jak2 or CALR was a rare event and amounted to 0.88 and 0.35 % of cases, respectively. The combination of BCR-ABL1 with Jak2V617F and CALR mutations does not always impede the achievement of major MR.