A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5215-5215
Author(s):  
Munazza Rashid ◽  
Rifat Zubair Ahmed ◽  
Shariq Ahmed ◽  
Muhammad Nadeem ◽  
Nuzhat Ahmed ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Diagnostic Algorithm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Common Mutation ◽  
Hypochondriac Region ◽  
Exon 9 ◽  
Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

JAK2 V617F-Negative and MPL W515K/L-Negative Essential Thrombocythemia: A High Resolution SNP Array Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5258-5258
Author(s):  
Carla AL Assaf ◽  
Els Lierman ◽  
Timothy Devos ◽  
Carlos Graux ◽  
Johan Billiet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Essential Thrombocythemia ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Snp Array ◽  
Jak2 V617f ◽  
Common Mutation ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Array Data

Abstract Background JAK2 V617F is the most common mutation in essential thrombocythemia (ET), occurring in approximately 50 % of cases. Second to JAK2 V617F is MPL W515K/L, accounting for about 10 % of cases. The molecular cause of the remaining ET cases is still largely unknown. Aims We sought to investigate JAK2 V617F-negative and MPL W515K/L-negative ET for regions of copy number variations (CNV) and loss of heterozygosity (LOH). Methods We studied blood or bone marrow samples from a series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET cases. They were subjected to 2.7M SNP array by Affymetrix, which has 2,761,979 copy number markers including 400,103 SNP markers. The array data were analyzed for recurrent CNVs with Array Studio (OmicSoft), and for individual CNVs or recurrent LOHs (≥3 Mbs) with the Chromosome analysis suite (ChAS, Affymetrix). Results Only 8 recurrent gains were identified, in 5/64 patients. Interestingly, the most common gain, occurring in 5 cases was a gain of chr7 q22.3, including the gene encoding Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is known to be overexpressed in several cancers such as multiple myeloma. It catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway. It is also required for cell growth and survival. We checked in the 5 patients for NAMPT amplification by quantitative PCR (qPCR) on genomic DNA in comparison to controls and by normalizing to ALB and RPPH1. We were able to validate the gain in 2/5 patients. The gain in these 2 patients was demonstrated to be acquired by qPCR of NAMPT in buccal swab DNA. Other recurrent gains involved regions of chromosomes 2, 5, 7, 12, 13, and 22. These gains included, amongst others, LCP1 on chr13 q14.3 and CYTIP on chr2 q24.1, occurring in 2/64 and in 3/64 respectively. We also checked for non-recurrent gains and losses in our cohort. This analysis generated a total of 8 CNVs in 6 different patients, comprising 5 regional gains in chromosomes 2, 8, 12, and 15 and 3 regional losses in chromosomes 5, 8 and 11. The array data were also analyzed for recurrent LOHs on ChAS, yielding 17 recurrent copy neutral LOHs (CN-LOH) in 35 patients (circos plot). The most common CN-LOH region was on chromosome 3 appearing in 8 patients. Other CN-LOH regions involved chromosomes 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, and 17 and they occurred in 2-5/64 patients. However, as small regions of CN-LOH can be constitutional, we suspect that most of these CN-LOH regions are not acquired. The largest region of CN-LOH observed was 12 Mbs in size. Conclusions Previous studies in unselected series of BCR-ABL1-negative myeloproliferative neoplasms have shown that copy number alterations are rare in ET as well as in polycythemia vera. In this series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET patients we found recurrent gains not reported previously in the database of genomic variants in only 8% of patients, and small areas of CN-LOH in ∼55% of cases. However, most of the latter probably are constitutional. Our SNP array study provides further evidence that gains, losses or CN-LOH of small genomic regions do not play an essential role in the pathogenesis of the majority of JAK2 V617F-negative and MPL W515K/L-negative ET. However, the low frequency of megakaryocytes and unknown level of clonal involvement of the myeloid compartment in JAK2 V617F-negative and MPL W515K/L-negative ET bone marrow remain a caveat. Next generation sequencing technology is expected to bring new insights on the molecular pathogenesis of this elusive ET subset. Circos plot showing the recurrent CN-LOHs Left half represents a total of 35 patients carrying recurrent CN-LOHs and the right half represents the chromosomes and their associated properties. Right outermost layer depicts 1+log-gene density (min, 1; max 42) where cancer, OMIM and other genes are colored in red, blue and green respectively. Right middle and innermost layers designates SNP density (blue, values < 0,02; gray values, <0,06; red values >0,06; max scale 0,013) and absolute SNP numbers (min, 1; max, 12074) per windows of 50kb. Each 5Mb distance is marked with a tick underneath the innermost layer. Links from each chromosome are colored differently. Regions that are more confined on the same chromosome are least transparent and regions that are shared by more patients are drawn on top of lesser links. Disclosures: No relevant conflicts of interest to declare.

Calr Is up-Regulated in Patients with Essential Thrombocythemia Independently from Calr and JAK2 Mutations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5581-5581
Author(s):  
Lilia Brown ◽  
Ciaren Graham ◽  
Ciro Rinaldi
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Over Expression ◽  
Molecular Abnormalities ◽  
Mutational Status ◽  
Significant Difference ◽  
Exon 9 ◽  
Calr Mutations

Abstract Somatic mutations in exon 9 of calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations, and absent in patient with polycythemia vera (PV). Among patients with ET or PMF with un-mutated JAK2 or MPL, CALR mutations were detected in 67% of those with ET and 88% of those with primary PMF. Several types of insertions or deletions were identified and all resulted in a frameshift in exon 9 generating a novel C-terminal peptide in the mutant CALR protein. Over expression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with myeloproliferative neoplasms carrying CALR mutations present with higher platelet counts and lower haemoglobin levels than patients with mutated JAK2. Studies suggest these patients have a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. We analysed by Real Time PCR, CALR expression in peripheral blood (PB) of 38 patients affected by ET, 17 JAK2 mutated (45%), 4 CALR (10.5%) mutated, 1 MPL mutated (3%) and 14 with no apparent molecular abnormalities. These were compared with a cohort of healthy volunteers. We found a significant over expression of CALR (median 5.15; range 1.13-270.08) comparing with controls (median 0.38, range 0.18-1). CALR mRNA expression is independent from the CALR mutational status. No significant difference was found comparing CALR expression in CALR mutated (median 4.9, range 1.51-37.14) and CALR/JAK2 un-mutated patients (4.68, range 1.51-28.71). CALR up-regulation is not mutually exclusive with JAK2 mutations; no difference was seen in CALR mRNA between JAK2 mutated (median 5.09, range 1.13-270) and wild type ET patients (median 5.08, range 1.51-37). There was no significant difference when we correlated CALR expression with PLT counts, spleen size or type of cytoreductive therapy. A larger cohort of patients is required to confirm these preliminary findings. Disclosures No relevant conflicts of interest to declare.

Frequency and Molecular Characteristics of Calreticulin Gene (CALR) Mutations in Patients with JAK2-Negative Myeloproliferative Neoplasms

2014 ◽  
Vol 133 (2) ◽  
pp. 193-198 ◽  
Author(s):  
Marzena Wojtaszewska ◽  
Małgorzata Iwoła ◽  
Krzysztof Lewandowski
Keyword(s):  
Data Analysis ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Molecular Characteristics ◽  
New Mutation ◽  
Clinical Data Analysis ◽  
Leukocyte Counts ◽  
Exon 9 ◽  
Calr Mutations

In 2013, Nangalia et al. and Klampfl et al. found a recurrent and abundant mutation in the calreticulin gene (CALR), mutually exclusive with JAK2 and MPL alterations. At present, the data concerning the new mutation, i.e. its prevalence, allele burden and clinical significance, are scarce. We report the incidence and molecular characteristics of CALR mutations in a group of 184 Polish patients with myeloproliferative neoplasms (MPNs). Clinical data analysis revealed significant differences between JAK2 V617F-mutated and CALR-mutated groups. In essential thrombocythemia patients, hemoglobin levels and leukocyte counts were significantly higher in JAK2-positive than in CALR-positive patients (p = 0.023 and p = 0.017, respectively), but the CALR-positive patients had significantly higher platelet counts (p = 0.022). Patients harboring CALR mutations were also younger at the time of diagnosis (p = 0.039). In primary myelofibrosis patients, the degree of anemia was less severe in those who were CALR exon 9 mutation-positive than in those who were JAK2 V617F-positive (p = 0.048). © 2014 S. Karger AG, Basel

Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5569-5569
Author(s):  
Amin Ben Lassoued ◽  
Nathalie Beaufils ◽  
Caroline Bonnefoy ◽  
Jean Gabert
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Gene Mutations ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Molecular Diagnostic ◽  
Platelet Counts ◽  
Study Population ◽  
Calr Mutations

Abstract Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region. The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations. The study was approved by the local ethics committee. Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included. Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test. A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants. Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients. Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L). Disclosures No relevant conflicts of interest to declare.

scholarly journals JAK2 V617F, MPL, and CALR Mutations in Korean Patients with Essential Thrombocythemia and Primary Myelofibrosis

2015 ◽  
Vol 30 (7) ◽  
pp. 882 ◽  
Author(s):  
Bo Hyun Kim ◽  
Young-Uk Cho ◽  
Mi-Hyun Bae ◽  
Seongsoo Jang ◽  
Eul-Ju Seo ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Korean Patients ◽  
Calr Mutations

scholarly journals STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1021 ◽  
Author(s):  
Emir Hadzijusufovic ◽  
Alexandra Keller ◽  
Daniela Berger ◽  
Georg Greiner ◽  
Bettina Wingelhofer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Janus Kinase ◽  
Signaling Cascades ◽  
Nuclear Compartment ◽  
Downstream Signaling ◽  
Direct Targeting ◽  
Calr Mutations

Janus kinase 2 (JAK2) and signal transducer and activator of transcription-5 (STAT5) play a key role in the pathogenesis of myeloproliferative neoplasms (MPN). In most patients, JAK2 V617F or CALR mutations are found and lead to activation of various downstream signaling cascades and molecules, including STAT5. We examined the presence and distribution of phosphorylated (p) STAT5 in neoplastic cells in patients with MPN, including polycythemia vera (PV, n = 10), essential thrombocythemia (ET, n = 15) and primary myelofibrosis (PMF, n = 9), and in the JAK2 V617F-positive cell lines HEL and SET-2. As assessed by immunohistochemistry, MPN cells displayed pSTAT5 in all patients examined. Phosphorylated STAT5 was also detected in putative CD34+/CD38− MPN stem cells (MPN-SC) by flow cytometry. Immunostaining experiments and Western blotting demonstrated pSTAT5 expression in both the cytoplasmic and nuclear compartment of MPN cells. Confirming previous studies, we also found that JAK2-targeting drugs counteract the expression of pSTAT5 and growth in HEL and SET-2 cells. Growth-inhibition of MPN cells was also induced by the STAT5-targeting drugs piceatannol, pimozide, AC-3-019 and AC-4-130. Together, we show that CD34+/CD38− MPN-SC express pSTAT5 and that pSTAT5 is expressed in the nuclear and cytoplasmic compartment of MPN cells. Whether direct targeting of pSTAT5 in MPN-SC is efficacious in MPN patients remains unknown.

Evaluation of the JAK2 V617F gene mutation in myeloproliferative neoplasms cases: a one-center study from Eastern Anatolia

2019 ◽  
Vol 44 (4) ◽  
pp. 492-498
Author(s):  
Gonca Gulbay ◽  
Elif Yesilada ◽  
Mehmet Ali Erkurt ◽  
Harika Gozukara Bag ◽  
Irfan Kuku ◽  
...  
Keyword(s):  
Blood Cell ◽  
Essential Thrombocythemia ◽  
Myeloproliferative Neoplasms ◽  
Hematological Parameters ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Myeloproliferative Disorders ◽  
Jak2 V617f Mutation ◽  
V617f Mutation ◽  
The Difference

AbstractObjectiveDetection ofJAK2V617F in myeloproliferative neoplasms (MPNs) is very important in both diagnosis and disease progression. In our study, we investigated the frequency ofJAK2V617F mutation in patients with myeloproliferative disorders.MethodsWe retrospectively reviewed the records of 720 patients (174 females and 546 males) who were tested for JAK2 V617F mutation from January 2007 to December 2017.ResultsIn our patients were determined 22.6%JAK2V617F mutation. 33.3% in women, 19.2% in men have been positive forJAK2V617F mutation. In our studyJAK2V617F present in 48.6% of essential thrombocythemia, 80.5% of polycythemia rubra vera (PV), 47.5% of primary myelofibrosis, 10% of MPNs, unclassifiable, 0.8% of others. We also investigated the difference in hematological parameters [white blood cell, hemoglobin (Hb), hematocrit (HCT), red blood cell distribution widths (RDW) and platelets count (PLT)] betweenJAK2V617F positive andJAK2V617F negative patients.ConclusionsInvestigation of the JAK2 V617F mutation is very important in cases of MPNs. In our study JAK2 V617F mutation was higher in PV, essential thrombocythemia, and primary myelofibrosis patients. However, there were significant differences in Hb, HCT, RDW and PLT levels in mutation-positive patients.

Analysis of JAK2 V617F Mutation and Its Clinical Significance in Patients with Thrombocythemia and Other Myeloproliferative Neoplasms in Chinese

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4687-4687
Author(s):  
Yue Xu ◽  
Changxin Yin ◽  
Han He ◽  
Lingling Shu ◽  
Fuqun Wu ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Jak2 Mutation ◽  
Western Countries ◽  
Arms Pcr ◽  
V617f Mutation

Abstract Abstract 4687 JAK2 mutation is commonly found in Philadelphia-negative myeloproliferative neoplasms (MPNs). In Western countries, this mutation is found in approximately 96 percent of people with polycythemia vera, half of individuals with essential thrombocythemia or primary myelofibrosis. We used the method of amplification refractory mutation PCR (ARMS-PCR) to investigate MPN patients in China. We focused our study on patients with essential thrombocythemia (ET). ARMS-PCR was used to detect JAK2 V617F mutation in the bone barrow (BM) or peripheral blood of 37 MPN patients, which consisting of 7 ET, 5 polycythemia vera (PV), 5 chronic myeloid leukemia (CML), 5 chronic idiopathic myelofibrosis (CIMF), as well as 15 suspected MPNs. 17 cases of JAK2 V617F mutation (45.9%) were found in 37 patients, including 4 ET (57.1%), 4 PV (80.0%), 3 CIMF (60.0%), 6 suspected MPNs (40.0%). We did not find JAK2 V617F in the patients with CML. Our results indicated that the frequency of JAK2 V617F mutation in bcr/abl-negative MPNs in Chinese is similar to that in MPN patients in Western countries. At the same time, ARMS-PCR can distinguish the mutation is heterozygous or homozygous. Most patients were heterozygous for JAK2 but only a few were homozygous. In conclusion, our study showed that JAK2 V617F mutation frequency in Chinese MPN patients is similar to that in patients with this disorder in the West. It is the major molecular genetic abnormality in bcr-abl negative MPN and it can be used for diagnosis of MPN in China. Disclosures: No relevant conflicts of interest to declare.

Calreticulin Mutations in JAK2-Negative Myeloproliferative Neoplasms. Experience in Our Center

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5586-5586
Author(s):  
Maria Jose Penalva Moreno ◽  
Carolina Martinez-Laperche ◽  
Santiago Osorio Prendes ◽  
Elena Buces Gonzalez ◽  
Jose Luis Diez-Martin ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Statistical Correlation ◽  
Type I ◽  
Type Ii ◽  
Multifunctional Protein ◽  
High Clinical Suspicion ◽  
Fluorescent Pcr ◽  
Calr Mutations

Abstract Introduction: Calreticulin (CALR) is a multifunctional protein regulated by calcium that is located in the endoplasmic reticulum. Recently, mutations in the calreticulin gene have been described in patients with the diagnosis of essential thrombocytemia (ET) and primary myelofibrosis (PMF), mainly in JAK2-negative cases. CALR mutations are localized to exon 9 and generate deletions or insertions that lead to a frameshift change resulting in a mutant protein. The detection of these mutations helps in the actual diagnosis of JAK2 mieloproliferative syndromes (MPN). Our aim is to assess the utility of the determination of these mutations in the management of patients with diagnosis of MPN in our center. Patients and methods: This study includes 94 patients with diagnosis of JAK2-negative MPN retrospectively selected following clinical and analytical criteria between 2008 and 2014 in our center (Table 1, 2). CALR mutations were performed with the use of fluorescent PCR following the methods described by Klampf et al. (NEJM, 2013). Results: 94 patients were analyzed, 77 of them had the diagnosis of TE, 8 of PMF and 9 of others disorders of myelodisplastic/mieloproliferative. 22% of the cases of ET had mutations in CALR (Table 1). In these mutations, a total of 53% were type I mutations (52-bp deletion) and 47% were type II mutations (5-bp insertion). Only one mutation was infrequent, a 46-pb deletion. We have found statistical correlation in the number of platelets depending on the presence of the mutation and in the largest number of platelets in type II mutations. 33% of the cases of PMF had mutations in CALR, all of them type I. Among other diseases not included in MPN, one of them had a type I mutation (data not shown). Conclusions: Our results are close to recently published results regarding the frequency of mutation and as the largest number of platelets in type II mutations with respect to mutation type I. This study confirms the importance of CALR mutations determination in the diagnosis of JAK2-negative ET and PMF with high clinical suspicion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Physical Interaction Between Mutant Calreticulin and the Thrombopoietin Receptor Is Required for Hematopoietic Transformation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. LBA-4-LBA-4 ◽  
Author(s):  
Shannon Elf ◽  
Nouran Abdelfattah ◽  
Edwin Chen ◽  
Javier Perales-Patón ◽  
Emily Rosen ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Gene Set Enrichment Analysis ◽  
Common Mutation ◽  
Physical Interaction ◽  
C Terminus ◽  
Thrombopoietin Receptor ◽  
Arginine Residues ◽  
Calr Mutations

Abstract Somatic mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, are found in up to 40% of patients with myeloproliferative neoplasms (MPN). All pathologic CALR mutations are out-of-frame insertion and/or deletions (indels) in exon 9, generating a 1 base-pair (bp) frame shift and a common mutant-specific C-terminus, with the most common mutation being a 52 bp deletion (del52). The observation that CALR mutations are mutually exclusive with other MPN-initiating mutations such as JAK2V617F suggests a key pathogenic role for mutant CALR. To determine if mutant CALR alone is sufficient to induce MPN we began by over-expressing CALR-del52 in a retroviral bone marrow transplant (BMT) mouse model. We found that CALR-del52-expressing mice develop thrombocytosis and megakaryocytic hyperplasia, recapitulating the megakaryocyte-specific phenotype of CALR-mutant MPN patients. These findings suggest that the thrombopoietin receptor, MPL plays a key role in the pathogenesis of mutant CALR-driven MPN. To evaluate the role of MPL in mutant CALR driven oncogenesis, we over-expressed CALR-del52 in interleukin-3 (IL-3)-dependent Ba/F3 hematopoietic cells. We found that CALR-del52 over-expression results in transformation to IL3-independent growth only in Ba/F3 cells co-expressing MPL, but not in parental Ba/F3 cells or Ba/F3 cells co-expressing the EPO receptor (EPOR) or the G-CSF receptor (GCSFR). We found similar results in human cytokine-dependent UT-7 cells. We also introduced +1 frameshift mutations into the endogenous Calr locus in Ba/F3-MPL cells using CRISPR/Cas9 gene editing and successfully engendered IL-3 independent growth, indicating that endogenous levels of mutant Calr expression are sufficient for transformation. Together, these data indicate that MPL is specifically required for the transforming capacity of mutant CALR. Using RNA-sequencing followed by gene set enrichment analysis (GSEA), we confirmed that mutant CALR transformed Ba/F3-MPL cells display strong enrichment of Stat5 and Stat3 gene expression signatures. Concordantly, we also saw differential phosphorylation of Stat5 and Stat3 in these cells. Furthermore, we found that the IL-3 independent proliferation of mutant CALR expressing Ba/F3-MPL cells is decreased upon shRNA-mediated knockdown of Jak2, and that differential activation of Stat5 and Stat3 is abrogated by the JAK2 inhibitor, ruxolitinib. Together, these data demonstrate that mutant CALR signals through the JAK/STAT axis downstream of MPL. We next sought to define the specific domains within mutant CALR required for oncogenic transformation. We found that neither expression of the mutant C-terminus alone nor expression of CALR lacking the C-terminus leads to cytokine-independent growth, suggesting that the novel C-terminus is necessary (but not sufficient) for transformation. We therefore generated an extensive series of truncation, domain deletion and point mutations within the C-terminus and assessed their respective transforming capabilities. Surprisingly, we found that the oncogenic activity of mutant CALR is not encoded within a specific sequence or domain of the mutant C-terminus. Rather, we found that the positive electrostatic charge of the mutant C-terminus is critical for its transforming capacity. Mutagenizing all 18 lysine/arginine residues (positively charged) within the C-terminus to a neutral glycine residue abrogates CALR-del52 transformation activity. In contrast, mutagenizing the 18 non-lysine/arginine residues within the C-terminus to glycine does not affect transforming activity, a remarkable finding considering that, in this mutant, 50% of the amino acids have been modified. Finally, using co-immunoprecipitation assays we found that mutant CALR, but not wild-type CALR, physically interacts with MPL, and that neither the mutant C-terminus alone nor mutant CALR lacking the C-terminus can bind to MPL. This suggests that the tertiary structure of mutant CALR is required for binding to MPL. Moreover, we found that the ability of our engineered CALR mutants to bind MPL perfectly correlates with their ability to mediate transformation, suggesting that the interaction with MPL is critical for mutant CALR-mediated transformation. Together, our findings elucidate a novel mechanism of pathogenesis in MPN and provide insights into how CALR mutations drive the development of MPN. Disclosures: No relevant conflicts of interest to declare.

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