CALR-mutated Myeloproliferative Neoplasms Research Articles

The Role of Mutated Calreticulin in the Pathogenesis of BCR-ABL1-Negative Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are characterized by increased proliferation of myeloid lineages in the bone marrow. Calreticulin (CALR) 52 bp deletion and CALR 5 bp insertion have been identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF). There is not much data on the crosstalk between mutated CALR and MPN-related signaling pathways, such as JAK/STAT, PI3K/Akt/mTOR, and Hedgehog. Calreticulin, a multifunctional protein, takes part in many cellular processes. Nevertheless, there is little data on how mutated CALR affects the oxidative stress response and oxidative stress-induced DNA damage, apoptosis, and cell cycle progression. We aimed to investigate the role of the CALR 52 bp deletion and 5 bp insertion in the pathogenesis of MPN, including signaling pathway activation and functional analysis in CALR-mutated cells. Our data indicate that the JAK/STAT and PI3K/Akt/mTOR pathways are activated in CALR-mutated cells, and this activation does not necessarily depend on the CALR and MPL interaction. Moreover, it was found that CALR mutations impair calreticulin function, leading to reduced responses to oxidative stress and DNA damage. It was revealed that the accumulation of G2/M-CALR-mutated cells indicates that oxidative stress-induced DNA damage is difficult to repair. Taken together, this study contributes to a deeper understanding of the specific molecular mechanisms underlying CALR-mutated MPNs.

Aberrant Activity of the Calcium Sensor STIM1 Underlies Congenital Platelet Disorders and Myeloproliferative Neoplasms

MB and VA equal contributors AB and STO co-corresponding authors This study was initiated following evaluation of a 32 year-old woman who presented with a history of thrombocytopenia identified in childhood who subsequently developed features of myelofibrosis (MF). A bone marrow biopsy demonstrated hypercellularity in conjunction with megakaryocyte hyperplasia and marked reticulin fibrosis. Molecular testing for JAK2, CALR, and MPL mutations was negative. Given the unusual association between congenital thrombocytopenia and MF in this patient, exome sequencing was performed, revealing a heterozygous R304W mutation in the coiled coil (CC) domain of STIM1. Activating mutations in the CC and EF hand domains of STIM1 have been associated with Stormorken syndrome, a rare congenital platelet disorder associated with abnormal store operated calcium entry (SOCE). We subsequently identified a second patient with MF associated with a STIM1 activating mutation. This individual was found to have severe thrombocytopenia at birth, and exome sequencing revealed a heterozygous S88G mutation in the EF hand of STIM1. A bone marrow biopsy obtained at 6 months of age revealed atypical megakaryocytes and grade 1-2 MF. A repeat biopsy at age 2 showed persistence of stable MF. The unusual finding of MF in these two patients suggested the possibility of altered calcium signaling as a shared mechanism driving congenital platelet disorders such as Stormorken syndrome and myeloproliferative neoplasms (MPNs) including MF. In support of this notion, we identified elevated STIM1 expression in MF vs normal megakaryocyte progenitors, as well as in platelets from patients with essential thrombocythemia (ET) vs healthy controls. Additionally, we found that STIM1 expression was significantly elevated in CD34+ hematopoietic stem/progenitor cells (HSPCs) from both ET and MF patients vs healthy controls. Notably, STIM1 expression was increased in both JAK2 and CALR-mutant MF patients. Collectively, these findings provide evidence of aberrant STIM1 expression in MPN patient cells. To determine the functional role of STIM1 in MPN disease development, colony assays and patient-derived xenograft (PDX) experiments were performed with MF patient CD34+ cells subjected to CRISPR ablation of STIM1. Strikingly discordant results were observed with JAK2 vs CALR-mutant patient samples. Abrogation of STIM1 in CALR-mutant CD34+ cells led to decreased colony formation, and NSGS mice engrafted in parallel with STIM1-targeted cells exhibited decreased human CD45+ cell engraftment in conjunction with prolonged survival. These findings suggest an important role for STIM1 in CALR-mutant MPN disease phenotypes. In contrast, targeting of STIM1 in JAK2-mutant CD34+ cells led to increased colony formation and exacerbated disease phenotypes in vivo as manifested by enhanced human CD45+ cell engraftment, worsened splenomegaly, and early lethality. Similar results were obtained in experiments utilizing pharmacologic inhibitors of SOCE activity. Taken together, these findings indicate that the consequences of aberrant STIM1 activity may be context-dependent relating to specific MPN driver mutations. To expand these observations, we identified a separate cohort of 9 family members in Italy with Stormorken syndrome and confirmed STIM1 EF hand mutations. In ex vivo megakaryocytic differentiation assays, cells from affected individuals exhibited a defect in proplatelet formation. These observations were corroborated by initial analyses of a newly generated Stim1 R304W conditional knock-in mouse which recapitulated the characteristic thrombocytopenia found in patients with Stormorken syndrome. In summary, this study represents the first demonstration of MF development in patients with Stormorken syndrome, thereby uncovering a previously unrecognized hallmark of altered calcium signaling via aberrant STIM1 activation underlying Stormorken syndrome and MPNs. Our findings suggest distinct mechanisms relating to the interaction between JAK2 vs CALR mutation and altered STIM1 activity. Further studies of these relationships may have important ramifications for potential therapeutic approaches targeting these pathways.

Arterial and Venous Thromboembolic Complications in 832 Patients with BCR-ABL-Negative Myeloproliferative Neoplasms.

Arterial (ATE) and venous (VTE) thromboembolic complications are common causes of morbidity and mortality in BCR-ABL-negative myeloproliferative neoplasms (MPNs). However, there are few studies that include all MPN subtypes and focus on both MPN-associated ATE and VTE. In our single-center retrospective study of 832 MPN patients, a total of 180 first thromboembolic events occurred during a median follow-up of 6.6 years (range: 0-37.6 years), of which 105 were VTE and 75 were ATE. The probability of a vascular event at the end of the follow-up period was 36.2%, and the incidence rate for all first ATE/VTE was 2.43% patient/year. The most frequent VTE localizations were deep vein thrombosis with or without pulmonary embolism (incidence rate: 0.59% patient/year), while strokes were the most frequent ATE with an incidence rate of 0.32% patient/year. When comparing the group of patients with ATE/VTE (n = 180) and the group without such an event (n = 652) using multivariate Cox regression analyses, patients with polycythemia vera (hazard ratio [HR]: 1.660; [95% confidence interval [CI] 1.206, 2.286]) had a significantly higher risk of a thromboembolic event than the other MPN subtypes. In contrast, patients with a CALR mutation had a significantly lower risk of thromboembolism compared with JAK2-mutated MPN patients (HR: 0.346; [95% CI: 0.172, 0.699]). In summary, a high incidence of MPN-associated VTE and ATE was observed in our retrospective study. While PV patients or generally JAK2-mutated MPN patients had a significantly increased risk of such vascular events, this risk was reduced in CALR-mutated MPN patients.

Mutation-Driven S100A8 Overexpression Confers Aberrant Phenotypes in Type 1 CALR-Mutated MPN.

Numerous pathogenic CALR exon 9 mutations have been identified in myeloproliferative neoplasms (MPN), with type 1 (52bp deletion; CALRDEL) and type 2 (5bp insertion; CALRINS) being the most prevalent. Despite the universal pathobiology of MPN driven by various CALR mutants, it is unclear why different CALR mutations result in diverse clinical phenotypes. Through RNA sequencing followed by validation at the protein and mRNA levels, we found that S100A8 was specifically enriched in CALRDEL but not in CALRINS MPN-model cells. The expression of S100a8 could be regulated by STAT3 based on luciferase reporter assay complemented with inhibitor treatment. Pyrosequencing demonstrated relative hypomethylation in two CpG sites within the potential pSTAT3-targeting S100a8 promoter region in CALRDEL cells as compared to CALRINS cells, suggesting that distinct epigenetic alteration could factor into the divergent S100A8 levels in these cells. The functional analysis confirmed that S100A8 non-redundantly contributed to accelerated cellular proliferation and reduced apoptosis in CALRDEL cells. Clinical validation showed significantly enhanced S100A8 expression in CALRDEL-mutated MPN patients compared to CALRINS-mutated cases, and thrombocytosis was less prominent in those with S100A8 upregulation. This study provides indispensable insights into how different CALR mutations discrepantly drive the expression of specific genes that contributes to unique phenotypes in MPN.

CALR-mutated cells are vulnerable to combined inhibition of the proteasome and the endoplasmic reticulum stress response.

Cancer is driven by somatic mutations that provide a fitness advantage. While targeted therapies often focus on the mutated gene or its direct downstream effectors, imbalances brought on by cell-state alterations may also confer unique vulnerabilities. In myeloproliferative neoplasms (MPN), somatic mutations in the calreticulin (CALR) gene are disease-initiating through aberrant binding of mutant CALR to the thrombopoietin receptor MPL and ligand-independent activation of JAK-STAT signaling. Despite these mechanistic insights into the pathogenesis of CALR-mutant MPN, there are currently no mutant CALR-selective therapies available. Here, we identified differential upregulation of unfolded proteins, the proteasome and the ER stress response in CALR-mutant hematopoietic stem cells (HSCs) and megakaryocyte progenitors. We further found that combined pharmacological inhibition of the proteasome and IRE1-XBP1 axis of the ER stress response preferentially targets Calr-mutated HSCs and megakaryocytic-lineage cells over wild-type cells in vivo, resulting in an amelioration of the MPN phenotype. In serial transplantation assays following combined proteasome/IRE1 inhibition for six weeks, we did not find preferential depletion of Calr-mutant long-term HSCs. Together, these findings leverage altered proteostasis in Calr-mutant MPN to identify combinatorial dependencies that may be targeted for therapeutic benefit and suggest that eradicating disease-propagating Calr-mutant LT-HSCs may require more sustained treatment.

Molecular Pathogenesis of Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are chronic hematological malignancies characterized by increased proliferation of MPN stem and myeloid progenitor cells with or without bone marrow fibrosis that typically lead to increased peripheral blood cell counts. The genetic and cytogenetic alterations that initiate and drive the development of MPNs have largely been defined, and we summarize these here. In recent years, advances in understanding the pathogenesis of MPNs have defined a long-preclinical phase in JAK2-mutant MPN, identified genetic loci associated with MPN predisposition and uncovered mechanistic insights in CALR-mutant MPN. The integration of molecular genetics into prognostic risk models is well-established in myelofibrosis and ongoing studies are interrogating the prognostic implications of concomitant mutations in ET and PV. Despite all these advances, the field is deficient in clonally selective therapies to effectively target the MPN clone at any stage of disease, from pre-clinical to advanced. Although the biological understanding of the pathogenesis of MPNs has progressed quickly, substantial knowledge gaps remain, including in the molecular mechanisms underlying MPN progression and myelofibrotic transformation. An ongoing goal for the MPN field is to translate advances in biological understanding to improved treatments for patients.

Effects of CALR-Mutant Type and Burden on the Phenotype of Myeloproliferative Neoplasms.

Somatic CALR mutations occur in approximately 70% of patients with JAK2 V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). We evaluated the effects of the CALR mutant type and burden on the phenotype of CALR-mutated myeloproliferative neoplasms (MPN). Of the 510 patients with suspected or diagnosed MPN, all 49 patients detected with CALR mutations were diagnosed with ET (n = 32) or PMF (n = 17). The CALR mutant burden was significantly higher in PMF than in ET (45% vs. 34%), and type 1-like and type 2-like mutations were detected in 49% and 51% patients, respectively. Patients with MPN and type 2-like mutation showed a significantly higher median platelet count than those with type 1-like mutation. Particularly, patients with ET and type 2-like mutation had no thrombotic events, despite higher platelet counts. The effect of CALR mutant burden differed depending on the mutant type. A higher mutant burden tended to be associated with a cytopenic phenotype (i.e., lower hemoglobin levels and platelet counts) in patients with the type 1-like mutation and a proliferative hematological phenotype (i.e., higher platelet and neutrophil counts) in patients with the type 2-like mutation. This study suggests that the disease phenotype of MPN may be altered through CALR mutant burden and mutant type.

Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPNs

AbstractCalreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPNs). Although the biological mechanism by which CALR mutations cause MPNs has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPNs. To identify unique genetic dependencies in CALR-mutant MPNs, we performed a whole-genome clustered regularly interspaced short palindromic repeats (CRISPR) knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (among others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor 2-deoxy-glucose (2-DG) and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared with wild-type cells and normalization of key MPNs disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow as compared with bone marrow derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPNs.

S191: CALRETICULIN-MUTATED HEMATOPOIETIC CELLS ARE VULNERABLE TO THE COMBINED INHIBITION OF THE PROTEASOME AND THE IRE1A-XBP1 AXIS OF THE UNFOLDED PROTEIN RESPONSE

Background: Mutations in the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). These mutant CALR proteins have impaired chaperone function. Concordant with this, transcriptional upregulation of the unfolded protein response (UPR) has been reported in patients with CALR-mutated MPN. However, it is not understood how CALR-mutated cells counter-balance ER stress resulting from impaired chaperone function. Despite the frequency of CALR mutations in MPN, there are currently no treatment strategies to preferentially target CALR-mutant cells over healthy cells. Aims: To determine the mechanisms by which mutant CALR cells alleviate ER stress and to exploit these mechanisms selectively by pharmacological intervention in vivo. Methods: Long-term HSCs (LT-HSCs) from CalrΔ52 knockin mice were isolated for RNA-Seq. CalrΔ52 bone marrow (BM) was differentiated ex vivo, subsequently enriched for megakaryocytes, and subjected to quantitative proteomic analysis. Pathway analyses of CALR-mutant megakaryocyte progenitors (MkPs) were performed on Genotyping of Transcriptome (GoT) data from patients with Essential Thrombocytosis (ET). CalrΔ52 VAVCre mice and chimeric transplanted CalrΔ52 mice were treated with a proteasome inhibitor, an IRE1a inhibitor, or both. Results: To identify dysregulated pathways in CalrΔ52/Δ52 LT-HSCs, we performed RNA-seq and found that Xbp1s downstream of IRE1a and the proteasome pathway was significantly upregulated in CalrΔ52/Δ52 animals. We confirmed elevated XBP1s protein level in heterozygous CalrΔ52 knockin mice by flow cytometry. Since elevated and abnormal megakaryopoiesis are hallmarks of CALR-mutant MPN, we performed quantitative proteomics on megakaryocyte-enriched cells from CalrΔ52 mice. We confirmed upregulation of both proteasome and ER chaperone proteins in Calr-mutant cells. To verify these findings in human MPN, we interrogated a published GoT data set and found that in addition to XBP1s, the proteasome pathway was also differentially upregulated in CALR-mutated patient MkPs compared to the wildtype MkPs (p<2e-16). To investigate the functional consequences of IRE1a or proteasome inhibition, we treated CALR-mutant hematopoietic cell lines with KIRA6 (IRE1a inhibitor) and/or bortezomib (proteasome inhibitor) and found CALR-mutant cells to be differentially sensitive as compared to isogenic controls. We subsequently found that treatment with bortezomib leads to the accumulation of misfolded and ubiquitinylated proteins in mutant Calr BM, resulting in pro-apoptotic priming. In vivo treatment of CalrΔ52/+ mice with bortezomib resulted in a significant reduction in platelets. Finally, we combined IRE1a and proteasome inhibition in a chimeric BM transplantation in vivo model using CD45.2 CalrΔ52/+ MxCre GFP and CD45.1 wild-type competitor cells. Following combination therapy, we found a significant reduction in Calr-mutant donor chimerism, specifically in LT-HSCs and platelets, accompanied by reductions in LT-HSC frequency and platelet count. Summary/Conclusion: In summary, we have found that disrupted proteasis in CALR-mutated MPN cells results in a dependency on the IRE1-XPB1 axis of the UPR and proteasome activity. Using pre-clinical MPN models, we found that combined inhibition of both pathways in vivo normalizes important features of MPN and preferentially targets CalrΔ52 over wild-type cells. These findings highlight combined inhibition of IRE1 and the proteasome as a promising therapeutic strategy in CALR-mutant MPN.

Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms.

Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.

Genomic profiling of a randomized trial of interferon-α vs hydroxyurea in MPN reveals mutation-specific responses

AbstractAlthough somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα–treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.

Induced Pluripotent Stem Cells Enable Disease Modeling and Drug Screening in Calreticulin del52 and ins5 Myeloproliferative Neoplasms

Mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and primary myelofibrosis patients. To address the contribution of the human CALR mutants to the pathogenesis of myeloproliferative neoplasms (MPNs) in an endogenous context, we modeled the CALRdel52 and CALRins5 mutants by induced pluripotent stem cell (iPSC) technology using CD34+ progenitors from 4 patients. We describe here the generation of several clones of iPSC carrying heterozygous CALRdel52 or CALRins5 mutations. We showed that CALRdel52 induces a stronger increase in progenitors than CALRins5 and that both CALRdel52 and CALRins5 mutants favor an expansion of the megakaryocytic lineage. Moreover, we found that both CALRdel52 and CALRins5 mutants rendered colony forming unit–megakaryocyte (CFU-MK) independent from thrombopoietin (TPO), and promoted a mild constitutive activation level of signal transducer and activator of transcription 3 in megakaryocytes. Unexpectedly, a mild increase in the sensitivity of colony forming unit-granulocyte (CFU-G) to granulocyte-colony stimulating factor was also observed in iPSC CALRdel52 and CALRins5 compared with control iPSC. Moreover, CALRdel52-induced megakaryocytic spontaneous growth is more dependent on Janus kinase 2/phosphoinositide 3-kinase/extracellular signal-regulated kinase than TPO-mediated growth and opens a therapeutic window for treatments in CALR-mutated MPN. The iPSC models described here represent an interesting platform for testing newly developed inhibitors. Altogether, this study shows that CALR-mutated iPSC recapitulate MPN phenotypes in vitro and may be used for drug screening.

Immune Checkpoint Blockade Enhances Mutated Calreticulin-Induced T Cell Immunity in Myeloproliferative Neoplasms

▪Somatic mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. Each CALR mutation leads to a shift in the reading frame resulting in the formation of an altered protein with an identical 36-amino acid sequence in the C-terminus. We hypothesized that the recurrence and uniformity of the novel sequence would provide the mutated CALR an attractive candidate as a MPN-specific tumor antigen that might elicit anti-tumor immune responses. Therefore, we investigated the immunogenicity of the altered CALR by employing in silico peptide binding prediction algorithms to interrogate the binding affinities of mutated CALR peptides to human leukocyte antigens (HLA). These analyses indicated that the mutated epitopes can bind to class I and II HLA alleles with high affinity supporting the hypothesis that mutant CALR could be immunogenic in vivo . We then tested the immunogenicity of the overlapping peptides spanning the mutated region in vitro by utilizing intracellular staining (ICS) and Enzyme-Linked ImmunoSPOT (ELISPOT) assays. Naïve T cells derived from the peripheral blood mononuclear cells (PBMNCs) isolated from healthy donors displayed effector functions after priming with the mutated peptides but not with the corresponding wild type peptides . Incubation withthe mutated CALR peptides induced T cells proliferation, upregulation of CD137 (4-1BB) and production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Moreover, as suggested by in silico peptide-HLA binding predictions, both CD8+ and CD4+ T cell precursors displayed increased IFN-γ production in response to stimulation with mutated CALR peptides. There was an overall 4±3-fold increase in IFN-γ production by CD4+ T cells and a 2.5±0.5-fold increase by CD8+ T cells after stimulation with mutated peptides as compared to control self-antigens (n = 14 healthy donors out of which 11 showed CD4+ and 5 showed CD8+ T cell responses). These results demonstrate that mutated CALR elicits T cell responses in vitro supporting its potential immunogenic functions in vivo .We next investigated the presence of mutant CALR-specific T cell immunity in MPN patients with CALR mutations. PBMNCs from a total of 18 CALR-mutated MPN patients with ET (7), MF (5) or MF arising from ET (6) were evaluated. MPN T cells were then stimulated with the pooled mutant CALR peptides and T cell responses were assessed by ELISPOT. Out of 18 patients, increased IFN-γ production upon stimulation was observed in only 2 patients (one with ET and one with ET-MF). The less than robust MPN T cell responses suggested that immune suppressive mechanisms might be operating. To this end, we evaluated the expression of cell surface inhibitory receptors, programmed cell death (PD)-1 and the cytotoxic T-lymphocyte-associated antigen (CTLA)-4, by T cells from MPN patients. Both CD8+ and CD4+ T cells from MPN patients exhibited increased levels of PD-1 and CTLA-4 when compared to the healthy controls (HC). For instance, 9.3±8.2% of MPN patients CD4+ T cells expressed PD-1 (n = 12) as compared to 2.5±1.7% of HC CD4+ T cells (n=10) (p value = 0.0482). Similarly, there were twice as many PD-1 expressing CD8+ T cells in MPN patients as compared to HC. We next assessed MPN T cell responses against mutated peptides in the presence of neutralizing PD-1 or CTLA-4 antibodies. PD-1 inhibition restored T cell immunity in cultures derived from 3 MPN patients and CTLA-4 inhibition restored response in one patient (n=16). Of note, following the PD-1 or CTLA-4 inhibition, the number of IFN-γ producing cells and the amount of IFN-γ produced on a per-cell-basis increased upon stimulation with mutant-CALR peptides as compared to control peptides. These results indicate that the limited MPN T cell responses to CALR mutated peptides were due to T cell exhaustion caused, at least in part, by activation of the PD-1/CTLA-4 pathway.In summary, our studies establish mutated CALR as a MPN-specific tumor antigen thereby providing a rationale for the development of immunotherapies targeting mutated CALR in patients. Moreover, our data indicate that immune checkpoint signaling might be responsible for T cell exhaustion in some MPN patients thereby supporting the use of checkpoint blockade inhibitors for treatment of these patients. DisclosuresMascarenhas:Incyte: Other: Clinical Trial Steering Committee , Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Merck: Research Funding; Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding. Bhardwaj:Merck & Co., Inc.: Membership on an entity's Board of Directors or advisory committees; Dendreon Corporation: Membership on an entity's Board of Directors or advisory committees; Dynavax Technologies Corporation: Consultancy; Crucell: Patents & Royalties; Neostem, Inc.: Patents & Royalties.

Activated IL-6 signaling contributes to the pathogenesis of, and is a novel therapeutic target for, CALR-mutated MPNs

Calreticulin (CALR), an endoplasmic reticulum–associated chaperone, is frequently mutated in myeloproliferative neoplasms (MPNs). Mutated CALR promotes downstream JAK2/STAT5 signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Here, we provide evidence of a novel mechanism contributing to CALR-mutated MPNs, represented by abnormal activation of the interleukin 6 (IL-6)-signaling pathway. We found that UT7 and UT7/mpl cells, engineered by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to express the CALR type 1–like (DEL) mutation, acquired cytokine independence and were primed to the megakaryocyte (Mk) lineage. Levels of IL-6 messenger RNA (mRNA), extracellular-released IL-6, membrane-associated glycoprotein 130 (gp130), and IL-6 receptor (IL-6R), phosphorylated JAK1 and STAT3 (p-JAK1 and p-STAT3), and IL-6 promoter region occupancy by STAT3 all resulted in increased CALR DEL cells in the absence of MPL stimulation. Wild-type, but not mutated, CALR physically interacted with gp130 and IL-6R, downregulating their expression on the cell membrane. Agents targeting gp130 (SC-144), IL-6R (tocilizumab [TCZ]), and cell-released IL-6 reduced proliferation of CALR DEL as well as CALR knockout cells, supporting a mutated CALR loss-of-function model. CD34+ cells from CALR-mutated patients showed increased levels of IL-6 mRNA and p-STAT3, and colony-forming unit–Mk growth was inhibited by either SC144 or TCZ, as well as an IL-6 antibody, supporting cell-autonomous activation of the IL-6 pathway. Targeting IL-6 signaling also reduced colony formation by CD34+ cells of JAK2V617F-mutated patients. The combination of TCZ and ruxolitinib was synergistic at very low nanomolar concentrations. Overall, our results suggest that target inhibition of IL-6 signaling may have therapeutic potential in CALR, and possibly JAK2V617F, mutated MPNs.

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.

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.

Overexpression of PD-L1 Correlates with JAK2-V617F Mutational Burden and Is Associated with Chromosome 9p Uniparental Disomy in MPN

Myeloproliferative neoplasms (MPN) are characterized by clonal hematopoiesis, hyperproliferation of myeloid cells, hyperinflammation and immune deregulation. The three classical BCR-ABL1-negative MPN are essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The disease is driven by JAK2, CALR or MPL somatic mutations in most patients. Drug resistance is a major problem in MPN. Recent data suggest that MPN cells display certain immune checkpoint molecules that may contribute to resistance, including PD-L1. Antibodies targeting the PD1/PD-L1 axis are highly promising anti-cancer drugs. Their potential use in MPN is being explored but it is unclear which MPN subtypes are most suitable for testing in clinical trials. The aim of our project was to assess PD-L1 expression in disease-initiating neoplastic stem cells (SC) and differentiated cells of MPN patients and to develop therapeutic approaches capable of blocking PD-L1 expression in MPN SC. In a first step, PD-L1 expression was assessed by RNA-sequencing of granulocytes of 106 MPN patients and 15 healthy donors (HD). The cohort included 56 PMF, 33 ET and 17 PV patients. For 102 patients data from Human Genome-wide Affymetrix 6.0 SNP arrays were available. We observed a ~5-fold higher expression of PD-L1 mRNA in patients with PV compared to other MPN (P&amp;lt;.01) or HD (P&amp;lt;.01). JAK2-V617F positive ET patients had higher expression of PD-L1 compared to CALR-mutated ET (p&amp;lt;.005) and the same was observed in PMF (p&amp;lt;.01). Other mutations (TET2, DNMT3A) detected by NGS did not affect PD-L1 expression. Since PD-L1 and JAK2 are located on chromosome 9p24, we looked into our previously published dataset of 400 MPN patients analyzed by SNP arrays and found that in all 195 patients with 9p uniparental disomy (UPD) the aberrations covered both genes. As PD-L1 is more centromeric it could represent the second target of 9pUPD which can precede the acquisition of JAK2-V617F in MPN. Granulocytes in JAK2-V617F positive patients with 9pUPD expressed significantly higher levels of PD-L1 compared to patients without 9pUPD (P&amp;lt;.0001; Figure 1A). Moreover, the JAK2-V617F mutational burden significantly correlated with PD-L1 expression (R=.52, P&amp;lt;.0001; Figure 1B). This correlation was lost when cases with 9pUPD were excluded from the analysis (R=.03, P=.9), indicating that the UPD is relevant for PD-L1 upregulation. To investigate PD-L1 surface expression on MPN SC we analyzed CD34+CD45dimCD38- cells isolated from fresh bone marrow (BM) samples of another 51 MPN patients and 7 HD by flow cytometry (FC). MPN patients showed a significantly higher surface expression of PD-L1 on CD34+CD45dimCD38- cells compared to HD (p&amp;lt;.001; Figure 1C). PD-L1 levels on the SC surface were elevated in both JAK2- and CALR-mutated MPN patients compared to HD (p&amp;lt;.001 and P&amp;lt;.005, respectively). PD-L2 was neither expressed in MPN granulocytes nor on MPN SC. CD4+ and CD8+ T-cells from BM samples of 17 MPN patients expressed the PD-L1 receptor PD-1 as assessed by FC. We cultured ex vivo primary MPN cells from 7 JAK2-V617F positive patients and showed that PD-L1 expression on MPN SC spontaneously decreases in culture, that interferon-gamma (IFN-γ) can promote expression of PD-L1 on these cells, and that ruxolitinib and the BRD4-degrader dBET6 block IFN-γ-induced PD-L1 expression in CD34+CD45dimCD38- MPN SC (P&amp;lt;.05). Together, we show that PD-L1 is overexpressed on the surface of disease-initiating MPN SC, that PD-L1 mRNA is overexpressed in granulocytes of MPN patients and that PD-L1 overexpression in granulocytes correlates with the JAK2-V617F mutational burden. In patients with JAK2-V617F positive MPN, 9pUPD leads to further PD-L1 upregulation either through increasing the mutant JAK2 gene dosage, loss of wt-JAK2,or amplification of PD-L1 allele with higher expression. Our data suggest the possibility that 9pUPD and the subsequent elevation of PD-L1 expression may provide an immune escape mechanism and may contribute to positive selection of JAK2-V617F homozygous SC. Ruxolitinib and dBET6 downregulate PD-L1 expression on MPN SC suggesting a role for the JAK2 and BRD4-MYC pathway. As recent studies revealed an immunogenic potential of JAK2 and CALR mutants, overcoming the disease-mediated immune escape may be of particular importance. Further preclinical and clinical studies are now required to examine the value of PD1/PD-L1 inhibitors in patients with MPN. Disclosures Gisslinger: Celgene: Honoraria; MyeloPro Diagnostics and Research: Honoraria; AOP Orphan Pharmaceuticals AG: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; PharmaEssentia: Honoraria; Janssen-Cilag: Honoraria; Roche: Honoraria. Kralovics:AOP Orphan Pharmaceuticals AG: Honoraria; PharmaEssentia: Honoraria; Qiagen: Honoraria; Novartis: Honoraria; MyeloPro Diagnostics and Research: Current equity holder in private company. Valent:Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

CALR mutations in myeloproliferative neoplasms: An unfolding story.

CALR mutations in myeloproliferative neoplasms: An unfolding story.

Pathological characteristics of megakaryocytes in myeloproliferative neoplasms and their correlation with driver gene mutations

目的探讨骨髓增殖性肿瘤（MPN）巨核细胞病理特征及其与起始基因突变的相关性。方法收集2012年2月至2017年10月于中国医学科学院血液病医院就诊的160例初诊MPN患者，根据世界卫生组织（WHO）2016年MPN诊断标准对患者骨髓活检组织切片进行重新评估。结果160例患者中男72例（45.0％），女88例（55.0％），中位年龄59（13～87）岁。重新评估后真性红细胞增多症（PV）39例，原发性血小板增多症（ET）33例，纤维化前/早期原发性骨髓纤维化（pre-PMF）37例，明显期原发性骨髓纤维化（overt PMF）37例，真性红细胞增多症后骨髓纤维化（post-PV MF）2例，原发性血小板增多症后骨髓纤维化（post-ET MF）1例，MPN-未分类（MPN-U）11例。按PV、ET、pre-PMF及overt PMF疾病亚型顺序，密集成簇分布、少分叶核及裸核巨核细胞逐渐增加，红系增生程度正常及增高的比例逐渐降低，1级及以上网状纤维、胶原及骨硬化的比例逐渐升高。相关性分析示密集成簇分布、少分叶核及裸核巨核细胞占比与红系增生程度呈负相关，与粒系增生程度及纤维化程度呈正相关。对pre-PMF及overt PMF患者病理特征的相关性分析示密集成簇分布及裸核巨核细胞占比与纤维化程度呈正相关。JAK2V617F突变MPN患者红系增生程度明显增高（P＝0.022），CALR突变MPN患者疏松成簇、密集成簇、骨小梁旁分布及裸核巨核细胞明显增多（P＝0.055，P＝0.002，P＝0.018，P＝0.008），1级及以上网状纤维、胶原及骨硬化比例增高（P＝0.003，P<0.001，P＝0.001）。伴JAK2V617F突变pre-PMF及overt PMF患者骨髓增生程度较高（P＝0.037），伴CALR突变患者巨大体积及密集成簇分布巨核细胞明显增多（P＝0.059，P＝0.055），1级及以上胶原及骨硬化的比例明显增高（P＝0.011，P＝0.046）。结论不同亚型MPN患者骨髓巨核细胞病理改变特征各异，其病理异常特征与纤维化水平相关。CALR突变可能与巨核细胞病理异常特征及骨髓纤维化水平相关。

Incorporation of the 2008 WHO into 2008/2020 ECMP/CLMP Classification for Staging and Prog-nosis Assessment of JAK2, MPL, TPO, MPL and CALR Mutated Myeloproliferative Neoplasms: From Dameshek to Vainchenker and Michiels 1940 - 2020: A Historical Appraisal and Novel Therapeutic Implications

Incorporation of the 2008 WHO into 2008/2020 ECMP/CLMP Classification for Staging and Prog-nosis Assessment of JAK2, MPL, TPO, MPL and CALR Mutated Myeloproliferative Neoplasms: From Dameshek to Vainchenker and Michiels 1940 - 2020: A Historical Appraisal and Novel Therapeutic Implications