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  • 1
    In: Blood, American Society of Hematology, Vol. 138, No. Supplement 1 ( 2021-11-05), p. 628-628
    Abstract: Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. Here, we show that type 1 CALRdel52 but not type 2 CALRins5 mutations lead to activation of and dependency on the IRE1α-XBP1 pathway of the unfolded protein response (UPR). Mechanistically, we found that the loss of calcium binding residues in the type 1 mutant CALR protein directly impairs its calcium binding ability, which in turn leads to depleted ER calcium and subsequent activation of the IRE1α-XBP1 pathway. Using cell lines and primary MPN patient samples, we identified two novel transcriptional targets of XBP1 specific to type 1 CALRdel52-expressing cells - the anti-apoptotic protein BCL-2 and the calcium efflux channel IP3R. We show that BCL-2 acts downstream of XBP1 to promote survival in the face of depleted ER calcium, while IP3R is up-regulated downstream of XBP1 to promote continued ER calcium efflux in order to sustain IRE1α-XBP1 pathway activation and survival. We found that genetic or pharmacological inhibition of IRE1α-XBP1 signaling induced cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells. Moreover, we show that in vivo inhibition of IRE1α significantly abrogates type 1 mutant CALR-driven disease in a bone marrow transplantation model, but has no effect on type 2 mutant CALR-driven disease. This work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be exploited for therapeutic gain. Moreover, this study answers an enduring question regarding the functional consequence of the loss of calcium binding sites on the type 1 mutant CALR protein, and demonstrates how type 1 CALR mutant-expressing cells rewire the UPR, downstream calcium signaling, and apoptotic pathways to drive MPN. Figure 1 Figure 1. Disclosures Koschmieder: BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Shire: Honoraria, Other; Karthos: Other: Travel support; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Geron: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Abbvie: Other: Travel support; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); Alexion: Other: Travel support; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Baxalta: Membership on an entity's Board of Directors or advisory committees, Other; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support); CTI: Membership on an entity's Board of Directors or advisory committees, Other; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; AOP Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: (e.g. travel support), Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Image Biosciences: Other: Travel support.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
    In: Blood, American Society of Hematology, Vol. 134, No. Supplement_1 ( 2019-11-13), p. 1679-1679
    Abstract: Introduction: Interferon alpha (IFNa) is a cytokine with anti-viral and anti-tumoral properties which, either in its unmodified or pegylated form, is successfully used in the treatment of patients with myeloproliferative neoplasms (MPN), including polycythemia (PV), essential thrombocythemia (ET) and early primary myelofibrosis (PMF). Despite its efficacy including molecular responses and thus its potential disease modifying capabilities, IFNa carries the risk of significant adverse events, including liver toxicity, autoimmunity, and depression. This has prompted the development of interferons with improved features including better tolerability. However, biomarkers for response have been lacking, mostly due to the heterogeneity of cells analyzed and the difficulty in obtaining them from the bone marrow. Methods: Therefore, we have set up a clonogenic assay from Ficoll-isolated mononuclear cells of the peripheral blood of patients (PBMC) with MPN (n=51, including 17 PV, 14 ET, and 14 PMF, 1 MDS/MPN, 2 Post-PV-MF, 2 Post-ET-MF, 1 MPNu) to analyze the in vitro IFNa response (either using 0.5 µg/ml ropeginterferon alfa-2b [ropegIFNa] or 500 U/ml recombinant human IFN-alpha2b [hIFNa] or no treatment control) in the cells that drive malignant clonogenic growth in the patients. After 10-14 days, the number of colonies was assessed. For each patient and condition, twenty-five of the resulting colonies were then genotyped for zygosity of JAK2V617F and three colonies per condition were analyzed for STAT1 RNA expression using RT-qPCR, an important transcriptionally induced IFNa target gene. The number of mutated alleles was determined (zero for wildtype, one for heterozygous, two for homozygous colonies), and, based upon the change in mutant alleles after in vitro hIFNa treatment, samples were categorized into responders (mutant alleles decreased) and non-responders (mutant alleles unchanged or increased). All patients provided written informed consent, and the study was approved by the local ethics committee. Results: To assess potential differences between the two interferons used, a 14-day proliferation assay was performed in Set-2 cells, showing more sustained inhibition of proliferation by ropegIFNa than hIFNa (p=0.007), confirming the longer half-life of ropegIFNa. In the clonogenic assay, different colonies were observed depending on the MPN subtype, ranging from exclusive CFU-E (PV) to exclusive CFU-G and CFU-M (PMF) types of colonies. Both hIFNa and ropegIFNa significantly inhibited colony growth as compared to the control and reduced the colony number to 72.7 % (hIFNa) and 58.5% (ropegIFNa) of the untreated control, with ropegIFNa showing significantly stronger effects than hIFNa (p=0.0137). Interestingly, there were marked differences in the amount of JAK2 alleles in the colonies between the patients (n=24 analyzed), with 16 patients showing a reduction of up to 22% of the allele burden upon in vitro treatment with hIFNa (responders), while 8 patients showed no reduction or even an increase of up to 15% (non-responders) (p=0.0001). Basal STAT1 expression in the colonies (three colonies per patient and treatment) was significantly lower in responders than non-responders (p=0.0200). After treatment with hIFNa, STAT1 expression was induced to similar levels in both responders and non-responders (p=0.6620). As a result, STAT1 fold induction was significantly higher in responders than non-responders (p=0.0013). Interestingly, there was no correlation of the responses with current clinical treatment of the patients (previous interferon exposure did not prevent responses) or with the MPN subtype, confirming clinical reports that patients with myelofibrosis can respond to interferon. Conclusion: In conclusion, clonogenic cells from the peripheral blood of MPN patients can be used to assess their molecular response to IFNa. In vitro, ropegIFNa induced stronger effects than non-pegylated IFNa. The responses were heterogeneous at the molecular level, but, when combined with RNA expression analysis, we were able to dissect molecular responders from non-responders. The mechanisms for these differences and their clinical impact are currently being studied. Disclosures Gezer: AMGEM: Membership on an entity's Board of Directors or advisory committees. Isfort:Mundipharma: Other: Travel reimbursement; Amgen: Other: Travel reimbursement; Hexal: Other: Travel reimbursement; BMS: Honoraria; Ariad: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement; Roche: Other: Travel reimbursement; Alexion: Other: Travel reimbursement. Brümmendorf:Ariad: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Novartis: Consultancy, Research Funding; Merck: Consultancy. Koschmieder:Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Foundation: Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AOP Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers-Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2019
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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  • 3
    In: Blood, American Society of Hematology, Vol. 137, No. 16 ( 2021-04-22), p. 2139-2151
    Abstract: We studied a subset of hematopoietic stem cells (HSCs) that are defined by elevated expression of CD41 (CD41hi) and showed bias for differentiation toward megakaryocytes (Mks). Mouse models of myeloproliferative neoplasms (MPNs) expressing JAK2-V617F (VF) displayed increased frequencies and percentages of the CD41hi vs CD41lo HSCs compared with wild-type controls. An increase in CD41hi HSCs that correlated with JAK2-V617F mutant allele burden was also found in bone marrow from patients with MPN. CD41hi HSCs produced a higher number of Mk-colonies of HSCs in single-cell cultures in vitro, but showed reduced long-term reconstitution potential compared with CD41lo HSCs in competitive transplantations in vivo. RNA expression profiling showed an upregulated cell cycle, Myc, and oxidative phosphorylation gene signatures in CD41hi HSCs, whereas CD41lo HSCs showed higher gene expression of interferon and the JAK/STAT and TNFα/NFκB signaling pathways. Higher cell cycle activity and elevated levels of reactive oxygen species were confirmed in CD41hi HSCs by flow cytometry. Expression of Epcr, a marker for quiescent HSCs inversely correlated with expression of CD41 in mice, but did not show such reciprocal expression pattern in patients with MPN. Treatment with interferon-α further increased the frequency and percentage of CD41hi HSCs and reduced the number of JAK2-V617F+ HSCs in mice and patients with MPN. The shift toward the CD41hi subset of HSCs by interferon-α provides a possible mechanism of how interferon-α preferentially targets the JAK2 mutant clone.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2021
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Location Call Number Limitation Availability
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