Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

Abstract First and second authors contributed equally During female embryogenesis, most of the genes in either the maternal or paternal X-chromosome are randomly inactivated in each cell, a process that remains remarkably constant in their progeny. X-chromosome inactivation has been used to define clonality in myeloproliferative neoplasms (MPNs) such polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET). One such method to determine clonality uses a quantitative, transcriptional clonality assay based on conservative exonic polymorphisms in five X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD). Females who are heterozygous for any of these polymorphisms are considered “informative” and can be studied for clonality by interrogating their platelets’ and granulocytes’ RNA allelic usage ratio. JAK2 mutations occur in >95% of PV and 50-60% of ET and PMF; cMPL mutations are found in another 5-10% of ET and MF. Somatic calreticulin (CALR) mutations have been identified in a majority of patients with ET and MF who lack JAK2 and cMPL mutations. CALR mutations are reported to be associated with a more favorable prognosis and are believed to be acquired early in the disease course. More than 30 CALR mutations have been described, but type 1 (52-bp deletion; c.1092_1143del) and type 2 (5-bp insertion; c.1154_1155insTTGTC) mutations are the most frequent. We analyzed 61 females informative for a transcriptional clonality assay and 44 males with unexplained thrombocytosis or marrow fibrosis and no detectable JAK2 or cMPL mutations for CALR mutations in their granulocytes. With the exception of an absence of a clonal marker, these patients met WHO criteria for ET or PMF. A CALR mutation (20 type 1 and 17 type 2) was present in 37 of these 105 patients (22 females and 15 males). One of the CALR mutated females had a paternal grandmother with JAK2V617F –positive PV, confirming a previous report that, in familial clustering of MPNs, affected individuals may carry different disease-defining somatic mutations. In those CALR positive patients who had available T cells, no detectable CALR mutations were found in their T cells. In one of these subjects, CD34+ cells were available and had a similar mutation level as in the granulocytes. Of the 22 females with a CALR mutation, 19 had clonal hematopoiesis, but 3 had polyclonal hematopoiesis; all 3 had previously unexplained thrombocytosis. None of these patients had any prior treatment for thrombocytosis. Clonal hematopoiesis was present in 26 of the 39 females without a CALR mutation. All female patients with myelofibrosis had clonal hematopoiesis, regardless of CALR mutation status. In contrast to the polyclonal hematopoiesis seen in some CALR positive ET patients, 166 informative PV and JAK2V617F-positive ET or PMF females all had clonal hematopoiesis. We report that CALR mutations are associated with polyclonal hematopoiesis in some ET patients. This finding differs from JAK2V617F-positive ET and PMF and PV females, where clonal hematopoiesis was always seen. This indicates that CALR mutated clones have a weaker suppressive effect on residual normal hematopoietic stem cells than JAK2 mutated clones and may contribute to the possibly more benign course of CALR mutated ET. The CALR mutation was not detected in T cells, which also differs from JAK2V617F mutated MPNs, where a small level of the JAK2 mutation is often detected in T cells. Similar to other reports, we found a lower prevalence of the CALR mutation in JAK2 or cMPL non-mutated ET and PMF than initially described. Disclosures No relevant conflicts of interest to declare.

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Clinicopathologic Analysis of Calr Mutation Subtypes in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.2822.2822 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2822-2822 ◽

Cited By ~ 1

Author(s):

Yin Xu ◽

Brian Kwok ◽

Aine Yung ◽

Rachel Flamholz ◽

Zhao Wu ◽

...

Keyword(s):

Platelet Count ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Disease Evolution ◽

Cytogenetic Abnormalities ◽

Calr Mutations ◽

Marrow Cellularity ◽

Calr Mutation

Introduction: The discovery of JAK2, MPL, and CALR mutations has significantly improved the diagnostic approach to BCR-ABL1-negative myeloproliferative neoplasms (MPN). Approximately 60% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) harbor a JAK2 or MPL mutation. CALR mutations account for the majority of the remaining cases, and are found in 50-70% of ET and 60-90% of PMF cases that are negative for JAK2 and MPL mutations. Most CALR mutations cause a 52-bp deletion (type 1) or a 5-bp insertion (type 2). These mutations are acquired early during disease evolution and activate JAK/STAT signaling. Prior studies have shown that CALR type 1 mutations are associated with a favorable impact on survival of PMF patients, but not those with ET. Some data also suggested that CALR type 2 mutations may be associated with unfavorable prognosis in PMF. To assess the clinicopathologic impacts of CALR mutation subtypes in ET and PMF, we evaluated a series of CALR-mutated cases and correlated subtypes of mutations with several clinical, laboratory, and genetic parameters. Methods: MPN cases positive for CALR mutations were retrieved from our database over a period of 14 months. CALR, JAK2, and MPL mutation analyses were performed by either fragment analysis with Sanger sequencing confirmation or Next-Generation sequencing. Chromosome analysis and FISH with probes for 5p15/5q31, 7p11/7q31, 8cen, 20q, and t(9;22) were performed in all cases. Other parameters obtained included age, gender, hemoglobin, WBC, platelet count, bone marrow blasts and histology, and JAK2/MPL mutation status. The data were analyzed with independent sample t-tests and a 2-tailed chi-square test. Results: A total of 100 consecutive cases of CALR mutated MPNs were identified, 86 of which had available marrow specimens for morphologic subclassification. We further studied the cohort of 86 cases, including 37 ET and 49 PMF patients. 49 were male and 37 female with a median age of 67 (range 31-88) years. 49 (57%) patients had type 1, 28 (33%) had type 2, and 9 (10%) exhibited other types of mutations. No JAK or MPL mutation was found in any cases. Among patients with type 1 mutations, 22 (46%) were ET and 27 (54%) were PMF. Type 2 mutations were seen in 9 (33%) ET and 19 (67%) PMF patients. Notably, 5 cases of ET with type 2 mutations displayed atypical megakaryocytic hyperplasia with variable size and tight aggregates. In contrast, ET with type 1 mutations generally exhibited large megakaryocytes with hyperlobated nuclei. Two cases of PMF with type 2 mutations had a remote history of ET and may represent myelofibrotic transformation. ET patients with type 2 mutations had lower marrow cellularity (mean: 40% vs. 57%; p=0.014) than those with type 1 mutations. There were no statistically significant differences in age, gender, average hemoglobin, WBC, platelet count, marrow blasts, or reticulin fibrosis between the two ET subgroups. While no significant differences in various parameters were observed between PMF patients with type 1 and type 2 mutations, type 2 mutations showed a trend toward a higher platelet count (mean: 714 K/uL vs. 513 K/uL; p=0.086). Chromosome abnormalities were seen in 12 cases (23%), including 11 cases of PMF and 1 case of ET. Among PMF cases, cytogenetic abnormalities were less frequently associated with type 1 mutation (3/27) than type 2 and other types of mutations (8/22) (6% vs. 36%; p=0.035). The number of cases with other types of CALR mutations was small (3 ET and 6 PMF); therefore, comparison of those cases with cases from type 1 or type 2 mutated groups was precluded. Conclusions: ET patients with type 2 mutations showed less marrow cellularity and more megakaryocytic abnormalities associated with PMF compared to those with type 1 mutations. Our observations may raise the question whether ET patients with type 2 CALR mutations are more likely to progress to post-ET myelofibrosis. Type 2 mutations were also associated with a higher platelet count and higher frequency of cytogenetic abnormalities in PMF. Thus, CALR type 2 mutations may have a greater impact on megakaryocytic hyperplasia and platelet count production. We hypothesize that CALR type 1 and type 2 mutations represent different disease subgroups with pathogenic and prognostic implications. Disclosures No relevant conflicts of interest to declare.

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Differential Association of Calreticulin Type 1 and Type 2 Mutations with Myelofibrosis and Essential Thrombocytemia: Relevance for Disease Evolution

Blood ◽

10.1182/blood.v124.21.1823.1823 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1823-1823 ◽

Cited By ~ 1

Author(s):

Xenia Cabagnols ◽

Jean-Philippe Defour ◽

Valérie Ugo ◽

Jean Christophe Ianotto ◽

Pascal Mossuz ◽

...

Keyword(s):

Platelet Count ◽

Triple Negative ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hemoglobin Concentration ◽

Disease Evolution ◽

Calr Mutations ◽

Calr Mutation

Abstract Recent advances in myeloproliferative neoplasms (MPN) have highlighted the prevalence of mutations in the calreticulin gene (CALR), bringing a major new actor in these disorders. CALR mutations were reported in 25% of ET and in 35% of MF patients, which were non-mutated for JAK2 and MPL. CALR mutations lead to a frame-shift generating a common 36 amino acids C-terminal end and loss of the KDEL motif. Two variants account for 85% of the CALR mutations in ET and PMF: type 1, a 52-bp deletion and type2, a 5-bp insertion. 572 MPN patients negative for JAK2 and MPL mutations were collected from several French and Belgian hospitals. In our series, 396 patients were diagnosed as ET, 108 as MF and 68 as mixed MDS/MPN. We identified mutations of CALR in 368 patients (63.3%). The remaining 204 patients were designated as triple negative. In MF there was an over representation of type 1 mutation (70%) and an under representation of type 2 mutation (13%) as compared to patients with ET. This bias was associated with a higher allelic burden of CALR mutation in MF. MF patients represent a quite homogeneous group, mostly composed of men diagnosed at a median age of 62.5 with a low hemoglobin concentration (10.1 g/dl) and a low platelet count (median at 237 x 109/l). In ET patients the clinical presentation was more heterogeneous. They were mostly women (more than 61%) at a median age of diagnosis of 57 with a median platelet count of 724 x 109/l. In CALR mutated patients there were no sex prevalence and a more important thrombocytosis (785 x 109/l). The type of CALR mutation impacted also age and platelet count. We report the caracterisation of triple negative patients. In ETs they were mostly women (76.9%), particularly for ET patients under 50 years old that were almost exclusively women (27/28). In MF, triple negative patients presented a low hemoglobin concentration (8.85 g/dl) and a low leukocyte count (1.995 x 109/l). A striking characteristic is their platelet count, which was significantly lower than their group mates either in ET or in MF. This lower platelet count may suggest that in the general population, putative asymptomatic triple negative ET male patients could be retrieved, which would only be diagnosed at more advanced age with a symptomatic MF. Taken together, our results underline the differences between the two most frequent types of CALR mutation and show that CALR mutated patients should not be considered as a single entity. Disclosures No relevant conflicts of interest to declare.

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Mutation Type As a Major Determinant of Clinical Phenotype in Myeloproliferative Neoplasms Associated with Mutant Calreticulin

Blood ◽

10.1182/blood.v124.21.3215.3215 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3215-3215 ◽

Cited By ~ 1

Author(s):

Daniela Pietra ◽

Elisa Rumi ◽

Chiara Milanesi ◽

Christian A Di Buduo ◽

Marta Bellini ◽

...

Keyword(s):

Amino Acids ◽

Clinical Phenotype ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Charged Amino Acids ◽

Calr Mutations ◽

Mutation Type ◽

Calr Mutation

Abstract About 25% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) carry a somatic mutation of CALR, the calreticulin gene [N Engl J Med. 2013;369:2379-90]. So far, more than 50 different indels in CALR exon 9 have been found, but a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2) are the most common lesions. All indels generate a novel C-terminus of the mutant protein, in which the endoplasmic reticulum retention signal KDEL is lost and the negatively charged amino acids are replaced by neutral and positively charged amino acids, disrupting the Ca-binding site. This suggests that both cellular dislocation and impaired Ca-binding activity may be involved in the abnormal proliferation of cells expressing a mutant calreticulin. It is still unclear, however, why the same mutant gene is associated with 2 different disease phenotypes (ET and PMF). In particular, little in known about the effect of the mutant protein on megakaryocyte biology and bone marrow collagen deposition. We studied the relationships between CALR mutation type, megakaryocyte biology, and clinical phenotype in patients with myeloproliferative neoplasms. According to the 2008 WHO criteria, 716 out of 892 patients had ET and 176 had PMF. Overall, 578 (65%) patients carried JAK2 (V617F), 230 (26%) had a CALR indel, and 84 (9%) had nonmutated JAK2 and CALR. Patients with MPL mutations were excluded. Twenty-six different types of CALR lesions were identified: 120 (52%) patients had type 1 mutation, 75 (33%) had type 2, and 35 (15%) carried other indels. The frequency of type 1 mutation was significantly higher in PMF than in ET (71% vs 46%, P=.004). All these variants involved 3 different stretches of negatively charged amino acids, with an increase in the isoelectric points (pI) of the mutant protein. As type 1 and type 2 mutations affected stretch I and III, respectively, the 26 indels were categorized into 3 groups on the basis of the stretch they affected: i) type 1-like (61%), affecting stretch I; ii) type 2-like (36%), stretch III; iii) and other types (3%), stretch II. The pI values were significantly different in the 3 groups (P<.001). The frequency of type-1 like mutations was significantly higher in PMF than in ET (82% vs 55%, P=.001). In vitro differentiated megakaryocytes from CALR-mutant patients displayed a significant increase in the extent of both intracellular Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry inside the cytoplasm, as compared with healthy controls. Megakaryocytes carrying type 1-like CALR mutations exhibited the highest amplitude of Ca2+ flows regardless of the type of disease. In ET, impaired Ca2+ homeostasis was accompanied by atypical proplatelet architecture (ie, more branches and bifurcations). With respect to clinical phenotype at diagnosis, ET patients with type 2-like CALR mutation showed a trend towards higher PLT count (P=.063) and lower age (P=.053), and significantly lower LDH values (P=.021) than those with type 1-like mutation. In a hierarchical cluster analysis including demographic, clinical and molecular data, CALR mutation type (1 vs 2) identified the 2 clusters with the highest dissimilarity. Considering all patients, those with type 2-like CALR lesions had a better survival than those with JAK2 (V617F) (96.1% vs 84.4% at 10 years, P=.039), while no difference was found between the 2 CALR mutation types. ET patients with type 2-like CALR mutations showed a lower risk of thrombosis than those with JAK2 (V617F) (P=.010). By contrast, ET patients with type 1-like CALR mutations had a higher risk of myelofibrotic transformation that those with type 2-like CALR mutations (P=.029) and especially those with JAK2 (V617F) (P=.011). Finally, PMF patients with type 1-like CALR variants had a better survival than those with JAK2 (V617F) (80.1% vs 48% at 10 years, P=.008). In summary, abnormalities in megakaryocyte calcium metabolism and proplatelet architecture are found in patients with CALR-mutant myeloproliferative neoplasms, and their extent is related to mutation type. Type 2-like CALR mutations are more likely to be associated with isolated thrombocytosis without bone marrow fibrosis, ie, with an ET phenotype. By contrast, type 1-like CALR mutations are generally associated with bone marrow fibrosis, ie, with a PMF phenotype. Thus, in CALR-mutant myeloproliferative neoplasms, the mutation type is a major determinant of the clinical phenotype. Disclosures No relevant conflicts of interest to declare.

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Knock-Ins of Type-2 Calr Mutants Cause Myeloproliferative Neoplasm (MPN)-like Hematopoiesis in Mice

Blood ◽

10.1182/blood-2019-124575 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2964-2964

Author(s):

Keiji Minakawa ◽

Koki Ueda ◽

Osamu Nakajima ◽

Tetsuro Yokokawa ◽

Yusuke Kinishima ◽

...

Keyword(s):

Research Funding ◽

Gene Set Enrichment Analysis ◽

Hematopoietic Stem ◽

Cell Counts ◽

Calr Mutations ◽

Calr Mutation ◽

Mouse Lines ◽

Stat Pathway

MPNs, including polycythemia vera, essential thrombocythemia (ET) and myelofibrosis (MF), are characterized by proliferation of mature myeloid cells. A somatic mutation in a hematopoietic stem cell (HSC) that activates JAK/STAT pathway drives MPN. Following the JAK2V617F, the CALR insertion/deletion mutations (indels) are the second most frequent driver and present in 20-30% ET and primary MF. Two major indels, a 52-bp deletion (type 1, p.L367fs*46) and a 5-bp insertion (type 2, p.K385fs*47), account for 80% of the CALR mutations. In addition, there have been more than 100 other indels, which can be classified as type 1- and type 2-like mutations based on the length of negatively-charged amino acid (AA) stretch at the C-terminal side of mutated CALR. Patients with type 1/type 1-like CALR mutations exhibit more incidence of MF while type 2/type 2-like mutations are associated with higher platelet counts in ET (Petra et al, Leukemia, 2016). In previous studies, mice carrying type 1/type 1-like mutations, including knock-in (KI) models, showed mild ET- or MF-like hematopoiesis. Although wild-type (WT) CALR AA sequences are highly conserved between human and mouse, there have been no KI models of type 2/type 2-like CALR mutations. Here, we generated 2 lines of KI mice carrying type 2-like Calr mutations, 2-bp insertion (CR2i, p.K378fs*53) and 10-bp deletion (CR10d, p.K375fs*52), using the CRISPR/Cas9 method. Both KIs removed KDEL, altered AA charges and increased values for isoelectric point, which are similar to type 2/type 2-like mutations in MPN patients. Compared with WT mice, peripheral platelets (1277 ± 228 vs 1560 ± 344 x 109/L, p = 0.004) and leukocytes (14.4 ± 3.7 vs 18.7 ± 4.9 x 109/L, p = 0.006) were increased in CR10d mice, whereas blood cell counts were not different between CR2i and WT mice. In FACS, both CR10d (p = 0.04) and CR2i (p = 0.04) mice exhibited an increased myeloid-cell ratio in bone marrow (BM). Splenomegaly was not present, but histopathological study showed a significant increase and accumulation of large megakaryocytes in BM and spleen of both KI mouse lines. BM fibrosis was not present in any sample. Therefore, CR10d and CR2i mice mimicked ET-like and unclassifiable MPN-like hematopoiesis, respectively. Next, we studied the basis of MPN-like hematopoiesis in CR10d and CR2i mice. Colony forming-cell assay in the presence of cytokines showed reduced growth of CFU-E, especially in CR2i mice (p = 0.01) compared with WT mice, while there was no difference in growth of CFU-Mk between CR10d or CR2i mice and WT mice. TPO-independent colony growth was not observed in both KI mice. Correspondingly, FACS showed comparable expression of phospho-STAT3 (pSTAT3) in BM cells between CR10d or CR2i mice and WT mice in the absence of TPO. However, pSTAT3 was significantly upregulated both in CR10d and CR2i mice compared with WT mice in the presence of TPO, suggesting that high sensitivity of HSCs or progenitor cells to TPO contributes to MPN phenotype in these mice. Thus, we investigated HSC function by a competitive repopulation assay, in which we transplanted a mixture of BM cells from KI mice (Ly5.2) and Ly5.1 mice at a 1:1 ratio into lethally irradiated Ly5.1 mice, showed reduced repopulating capacity, especially in CR2i mice. In the second transplant recipients, cells derived from either CR2i or CR10d mice were markedly diminished, suggesting the reduced self-renewal capacity of an HSC carrying a type 2/type 2-like Calr mutation. Finally, we performed RNAseq for FACS-sorted HSC-enriched lineage-Sca1+Kit+ (LSK) cells, which revealed that approximately 70% of genes among differentially expressed genes were commonly upregulated or downregulated in CR2i and CR10d mice, suggesting a similarity in gene expression profile of LSK cells of these KI mouse lines. As a result, there were several pathways commonly affected in both CR2i and CR10d mice in gene set enrichment analysis, including upregulation of JAK/STAT pathway (FDRq = 0.060 in CR2i and 0.111 in CR10d). On the other hand, targets of polycomb recessive complex 2, which are important for HSC functions in MPNs (Ueda et al, Blood Adv, 2017), were downregulated in both KI mouse lines (FDRq = 0 in both CR2i and CR10d), possibly explaining the reduced repopulating capacities of CR2i and CR10d HSCs. In conclusion, our data indicate that type 2/type 2-like Calr mutation can cause MPN-like hematopoiesis. For disease progression, further mechanism may be required. Disclosures Yokokawa: Actelion Pharmaceuticals Ltd: Other: Donated Fund Laboratory. Ikeda:Kyokuto Pharmaceutical: Research Funding; Hokuyo Denki: Research Funding; Novartis Pharma: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding.

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A NEW TYPE-2-LIKE CALR MUTATION IN ESSENTIAL THROMBOCYTHEMIA

International Journal of Case Reports ◽

10.28933/ijcr-2021-02-1805 ◽

2021 ◽

pp. 204

Author(s):

Keyword(s):

Myeloproliferative Neoplasms ◽

Driver Mutations ◽

Chronic Myeloproliferative Neoplasms ◽

Younger Age ◽

New Type ◽

Leukocyte Counts ◽

Calr Mutations ◽

Calr Mutation

CALR mutations, together with JAK-2 and MPL ones, are recognized as “driver” mutations in Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Most frequent CALR mutations are Type-1 deletions (45-55% of cases) and type-2 insertion (32-42% of cases). These mutations are usually associated with younger age, higher platelet counts, lower leukocyte counts, lower hemoglobin levels and a higher incidence of transformation from ET to MF. Recognizing and describing cases with different mutations can be useful to create a database that might help clinicians to include these patients in risk categories and to guide the appropriate therapeutic choices. We report a case of a 77-years old woman who presented a new type-2 like CALR mutation.

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Type I Calreticulin Mutations Result in Hyperactivation of Its Acetyltransferase Function and Iron Metabolism, Inducing a Susceptibility to Ferroptosis

Blood ◽

10.1182/blood-2021-153480 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3593-3593

Author(s):

Harrison S Greenbaum ◽

Maria Evers ◽

Alex Rosencrance ◽

Luke Maxwell ◽

Katarzyna Kurylowicz ◽

...

Keyword(s):

Iron Metabolism ◽

Transferrin Receptor ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Type I ◽

Lipid Peroxidation Product ◽

Calr Mutations ◽

The Impact

Abstract Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). Of these, approximately half are classified as type 1 and 30% as type 2, characterized by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5) respectively. Although both share identical mutant C-termini and are able to bind and activate MPL, type 1 and type 2 CALR mutations display different clinical and prognostic presentation: type 1 mutations are associated primarily with a fibrotic phenotype and increased proclivity towards fibrotic transformation, while type 2 mutations are more common in ET. Molecularly, type 1 and type 2 mutations result in differential C-domain amino acid sequences with the potential to affect the function of the protein. Various well known functions of CALR, including calcium binding ability and protein folding capacity, have begun to be explored in the context of CALR mutations; however, the impact of CALR mutations on its acetyltransferase ability, which was only discovered in 2006, remains unknown. Here, we show that in accordance with our structural models, mutant CALR not only retains its acyltransferase ability, but type 1 CALRdel52 mutations specifically lead to increased activation of its acetyltransferase ability, revealing a new gain of function phenotype for CALRdel52 mutations. As a result, type 1 CALR mutations lead to increased acetylation of CALR's acetyltransferase targets downstream, such as glutathione-S-transferase and cytochrome P450 reductase, which affects the outputs of these pathways downstream. Exploratory RNA-Seq on CALR-mutated cells revealed a concurrent upregulation of transferrin receptor mediated iron metabolism by CALRdel52. We subsequently validated this finding and show that CALRdel52 cells display differential iron metabolism. Given the upregulation of the transferrin receptor and the increased acetyltransferase ability affecting proteins involved in reactive oxygen species pathways (ROS), ferroptosis-an iron-dependent form of cell death characterized by the accumulation of lipid peroxides-emerged as a potential therapeutic target for CALRdel52 mutated cells. To test this, we first assessed basal proclivity to ferroptosis by measuring the lipid peroxidation product, classic ferroptotic marker 4-HNE (4-hydroxynonenal) as well as both ROS and global lipid peroxide levels in cells expressing wild-type CALR, CALRdel52, and CALRins5. We found that all of these ferroptotic markers were significantly increased in CALRdel52 cells. Therapeutic modulation of these pathways such as iron supplementation resulted in targeting of CALRdel52 cells and ferroptosis induction. This work is the first to examine the acetyltransferase ability of mutant CALR and reveal downstream phenotypic differences based on this ability that set the groundwork for a host of unexplored cellular consequences. Moreover, this study unites the novel understanding of the acetyltransferase function of mutant CALR with changes in transferrin receptor mediated iron metabolism to reveal not only how CALRdel52 induces a ferroptotic proclivity, but the potential of this sensitivity for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

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Calr Mutational Status in Egyptian Patients with Myeloproliferative Neoplams

Blood ◽

10.1182/blood-2019-128194 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5388-5388

Author(s):

Eman A. Soliman ◽

Samah I. El-Ghlban ◽

Abdelaleem H. Abdelaleem ◽

Sherin Abdel-Aziz ◽

Sameh Shamaa ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Response To Therapy ◽

Hcv Infection ◽

Number Of Patients ◽

Significant Difference ◽

Egyptian Patients ◽

Calr Mutations

It has been known that the insertion/deletion mutation of CALR gene is the second deriver mutation in myeloproliferative neoplasm (MPN) of essential thrompocythemia (ET) and primary myelofibrosis (PMF). As the molecular workup has been incorporated for the prospective screening and diagnosis of MPN in our Oncology Center. An Egyptian 87 cohort of patients with non-mutated JAK2 (58 ET and 29 MF) were investigated using polymerase chain reaction (PCR) as a pilot study. We found that 37 out of 87 patients (42%) were carrying CALR mutations (30 out of ET (52%) and 7 out of MF (24%)). Sanger sequencing was used to determine the type of CALR mutations in all positive patients and we found that 13 out of 37 (35%) had type 1/type 1-like and 36 out of 37 (97%) with type 2/type 2-like. This CALR mutation profile in Egyptian patients appear different from the western status as type2/type 2-like is the highest in our patients (97%) versus 35-45% and type1/type 1-like was 35% versus 55-65% compared to western results. Meanwhile, the clinical course and phenotype of our cohort of patients is not similar to that in western as there is no significant difference of overall survival between type1/type1-like and type2/type2-like. This finding might be due to the different environmental and genetic backgrounds of Egyptian population. A part of it might be related to the HCV infection as 12 out of 37 (32%) had HCV infection. Further study is in progress on a large number of patients to correlate that with the clinicopathological status, response to therapy and the mechanistic pathway of oncogenic transformation. Disclosures No relevant conflicts of interest to declare.

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Runx1-Cbfb Interacts with CHD7, a Chromatin Modifying Enzyme with a Potential Role in Hematopoiesis

Blood ◽

10.1182/blood.v118.21.1300.1300 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1300-1300

Author(s):

Jingmei Hsu ◽

Chung-Tsai Lee ◽

Scott Gerber ◽

Shuqian Yu ◽

Nancy A. Speck

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Heart Defects ◽

Genetic Alterations ◽

Transactivation Domain ◽

Hematopoietic Stem

Abstract Abstract 1300 Mutations in RUNX1 and CBFB are among the most common genetic alterations in hematologic malignancies, including acute myeloid and lymphoid leukemia (AML, ALL), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome and myeloproliferative neoplasms. Loss of Runx1-CBFb causes a failure of hematopoietic stem cell emergence during embryogenesis. Critical roles for Runx1-CBFb in adult hematopoiesis include hematopoietic stem and progenitor homeostasis, and lymphoid and megakaryocytic differentiation. We took an unbiased co-immunoprecipitation and mass spectrometry approach to identify Runx1-CBFb co-regulators in T cells, and identified chromodomain helicase binding protein 7 (CHD7) as a potential interacting partner. CHD7 is an ATP-dependent chromatin remodeling protein that primarily occupies enhancer and promoter regions. Autosomal dominant mutations in CHD7 cause CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). It was shown that CHD7 interacts with Sox2, and its occupancy correlates with H3K4me1/2 modifications and P300 binding at enhancer regions, and H3K4me3 marks at promoters. We confirmed the interaction of endogenous Runx1 and CHD7 in T cells. We demonstrate that the Runx1 transactivation domain, which is critical at all stages of hematopoiesis, is required for the CHD7 interaction. To elucidate an in vivo function for CHD7 in hematopoiesis, we generated a conditional pan-hematopoietic Chd7 deletion in mice using a floxed Chd7 allele and Vav1-Cre. Deletion of Chd7 in hematopoietic cells appears to cause no lineage specific defects. However, CHD7 deficient bone marrow cells had a competitive advantage in T cell reconstitution as compared to wild type cells, suggesting a role for CHD7 in restraining T cell numbers in the adult. Determining how CHD7 exerts its functions should shed light on underlying mechanisms in hematopoietic stem cell formation, T cell development, and hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

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A Novel Pathogenic CALR Exon 9 Mutation in a Patient with Essential Thrombocythemia

Laboratory Medicine ◽

10.1093/labmed/lmz064 ◽

2019 ◽

Vol 51 (3) ◽

pp. 306-309

Author(s):

Jee-Soo Lee ◽

Ho Young Kim ◽

Miyoung Kim ◽

Young Kyung Lee

Keyword(s):

Essential Thrombocythemia ◽

Frameshift Mutation ◽

Myeloproliferative Neoplasms ◽

Case Reports ◽

Individual Case ◽

First Case ◽

Exon 9 ◽

Calr Mutations

Abstract The clinical phenotypes and prognoses of CALR-mutant myeloproliferative neoplasms depend on the mutation type. The 2 most common mutations, type 1 (52-bp deletion) and type 2 (5-bp insertion), account for 85% of CALR-mutated neoplasms. The former confers a myelofibrotic phenotype, and the latter is associated with a low risk of thrombosis and an indolent clinical course. Individual case reports for patients with novel pathogenic CALR mutations are rare. Herein, we present the first case in the literature, to our knowledge, of a 63-year old ethnic Korean man with essential thrombocythemia who was diagnosed with a novel +1-bp frameshift mutation in CALR, which was predicted to exhibit a type 2–like phenotype.

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Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2018-99-111512 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4319-4319 ◽

Cited By ~ 1

Author(s):

Wei Wang ◽

Tiansu Wang ◽

Andriana G. Kotini ◽

Camelia Iancu-Rubin ◽

Ronald Hoffman ◽

...

Keyword(s):

Research Funding ◽

Ex Vivo ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Mutant Type ◽

C Terminus ◽

Calr Mutations

Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

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