Transformation Of Myeloproliferative Neoplasms Research Articles

MPN Transformation Is Characterized By Heterogeneous Shifts in Lineage Character

Introduction: The Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are characterized by the clonal proliferation of differentiated hematologic cells. However, there is a limited understanding of how perturbations at the transcriptional level may contribute to accelerated or blast-phase disease, together referred to here as transformation. Here we characterize transcriptomic characteristics and cell type composition of MPNs along the transition to transformed disease. Methods: RNAseq and clinical data were collected on 261 patients who were part of BeatAML, including those with MPN, de-novo AML, and transformed MPN. This was combined with data from 56 transformed MPN patients seen at MSK, 11 of whom also had paired chronic-phase data available. Differential expression analysis was performed with DESeq2; both GSEA and GSVA were used to evaluate gene-set enrichment and pathways of biological relevance. A custom RNA-seq lineage deconvolution algorithm was used to estimate hematopoietic stage proportions and was used for unsupervised hierarchical clustering of cases. Kaplan-Meier methods were used to estimate survival among transformed MPN lineage subtypes. Results: The initial analysis included 207 de novo AML cases and 75 MPN-BP cases from BeatAML and MSK on which RNAseq and molecular data were integrated across cohorts. Unsupervised analysis was performed using hierarchical clustering applied to batch corrected, variance stabilized RNA-seq count data. Transformed MPN cases were enriched in 2 of 6 clusters (N = 10, 33.3%; N = 36, 54.5%). Notably, the molecular characteristics in these clusters are varied, suggesting that multiple genomic perturbations may lead to similar pathways of transformation. Gene set variation analysis (GSVA) demonstrated a coalescence of transcriptomic perturbation into specific pathways for transformed MPNs, such as hematopoietic stem cell gene sets (q-value = 0.00018). On the contrary, AML cases typically demonstrated upregulation in later stages of hematopoiesis, such as monocytes (q-value =0.00016). In evaluating specific genes differentially expressed between transformed MPNs compared to de novo AML and tAML patients, microRNA MIR29B1 was particularly upregulated in MPN-BP (logFC = 9.52, q < 0.01), while the proto-oncogene CT45A1 had decreased expression in transformed MPN compared to other AMLs (logFC = -7.47, q < 0.01). Paired GSEA of the 11 patients with myelofibrosis MF and transformed disease showed that transformed disease was enriched for CD34-positive HSCs (NES = 2.64, q-value < 0.0001) and E2F transcription factors (NES 2.66, q-value < 0.0001), as well as doxorubicin resistance (NES 2.40, q-value < 0.0001). Given enrichment of the CD34 positive HSC geneset, we performed our RNA-seq based lineage deconvolution on each of the paired samples ( Figure 1). MPNs appeared to transform in a heterogenous fashion reminiscent of the biological diversity in AML. In aggregate, transformation was associated with decreased monocytic (p = 0.002), natural killer (NK) (p = 0.042) character and expansion of common lymphoid progenitor character (p = 0.022). Lineage composition changes varied, with certain samples showing HSC expansion (ID: 54910, ID: 46567) and others exhibiting LMPP expansion (ID: 29485, 2456). Figure 2 defines seven distinct clusters of transformed MPN patients, driven by their lineage components. While these clusters are transcriptionally distinct, we again see that they are not defined by their genomic or cytogenetic alterations. We evaluated for a difference in overall survival (OS) between each cluster and intermediate-risk de novo AML. We found significantly worse OS among patients in three MPN-BP clusters - MPP (p =0.008), LMPP (p = 0.038), and GMP (p < 0.001) - represented as clusters 2, 5, and 7 in Figure 2. Compared to intermediate risk AML, transformed MPNs had significantly worse OS (HR: 1.79 95% CI: 1.18, 2.71, p = 0.0057). Discussion: To our knowledge, this is the first ever description of the transcriptional characteristics of MPN-BP, its comparison to de novo AML, and the first paired comparison of transcriptional and lineage characteristics for patients with chronic-phase MPNs that subsequently transformed. Our study provides valuable insights into the transcriptomic landscape of transformed MPN including both accelerated and blast phase disease, and draws novel contrasts to de novo AML.

Trp53-Loss in Hematopoietic Stem Cells Drives the Evolutionary Process of Leukemic Transformation in a Jak2V617F-Driven Myeloproliferative Neoplasms Mouse Model

Myeloproliferative neoplasms (MPNs) are clonal disorders resulting from genetic lesions in hematopoietic stem cells (HSCs). The development of post-MPN AML is treatment resistant and associated with very poor outcomes. This leukemic transformation is due to TP53 inactivation in approximately 50% of cases. Existing mouse models with double mutant Jak2V617F and Trp53 have demonstrated that Trp53-lossalone is sufficient to induce leukemic transformation in Jak2V617F-driven MPN. These models were generated either by crossing Jak2V617F transgenic mice with Trp53R172H knock-in or Trp53 knock-out mice, or by retroviral expression of Jak2V617F in Trp53 knock-out bone morrow (BM) cells. These germline mutated models do not accurately model the sequential somatic Trp53 mutations that arise in Jak2V617F MPN, or track their clonal expansion in vivo. Here, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice to generate a novel mouse model of high molecular risk MPN that transforms to AML. We isolated lineage -/low Sca1 +c-Kit + (LSK) cells from BM of donor mice expressing a conditional allele of mutant Jak2 from its endogenous locus ( Jak2fl-V617F) in combination with an inducible Cas9-IRES-GFP allele and CreER-fusion allele, both from the Rosa26 locus ( Rosa26CreER/lsl-Cas9-IRES-GFP). LSKs were transduced with lentivirus expressing a sgRNA targeting exon 4 of Trp53 (sgTrp53), located in the DNA-binding domain of Trp53. These transfected sgTrp53 LSKs, along with control groups transduced with either empty vector (EV) or a sgRNA targeting Catsper1 (sgCatsper1, off target cutting control), were transplanted into lethally irradiated recipients. Successful engraftment and CreER activity, induced by a 2-week period of Tamoxifen chow, were confirmed by flow cytometry. Validation of Trp53-editing was performed using amplicon sequencing, revealing the most common mutations were frameshift mutations. A subset of these frameshift mutations exhibited an extremely high frequency, conferring a selective advantage within the cell populations in sgTrp53 mice. We further examined the levels of p53 induced by nutlin-3a (an MDM2 inhibitor) in edited BM cells from three groups of mice and found that Trp53 editing leads to a reduction in p53 levels and prevents the loss of viability in response to MDM2 inhibition. This indicates that CRISPR-Cas9 mediated Trp53 mutations result in a loss of function. SgTrp53 mice recapitulated the evolution of leukemic transformation of MPN, showing a PV phenotype for 8 weeks post CreER activation, characterized by elevated hematocrit but normal leukocytes and platelet counts, consistent with the phenotype exhibited by the EV and sgCatsper1 recipients. Subsequently, after 12 weeks, this MPN phenotype transformed to acute leukemia. This disease progression was associated with significantly shortened survival of sgTrp53 recipients, compared to the sgCatsper1 or EV control groups. Histopathologic analysis further supported these findings, showing leukocytosis and the presence of immature blast cells in the PB, spleen and BM of sgTrp53 mice, whereas such cells were absent in the control groups. Notably, the frequency of blast cells was correlated with the frequency of Trp53 frameshift mutations, consistent with what has been reported in MPN patients. Additionally, CRISPR-Cas9 mediated Trp53-loss primarily affected the erythroid lineage, driving expansion of erythroblasts, megakaryocyte-erythroid progenitors (MEP), and Lineage -/low Sca1 -c-Kit + in sgTrp53 mice. The erythroblasts showed aberrant co-expression of CD41 and CD71. Through secondary transplants with unfractionated spleen cells, whole BM cells, or sorted MEP cell populations, we confirmed that the leukemia driven by CRISPR-cas9 mediated Trp53-loss in the Jak2V617F-driven PV is transplantable, and that the MEP compartment in leukemic sgTrp53 mice contains the leukemia initiation population. In total, we used CRISPR-Cas9 to induce Trp53-loss in HSCs of Jak2V617F mice, faithfully replicating the process of clonal expansion and evolution observed in the leukemic transformation of MPN. These mouse models of secondary AML are valuable for testing new therapies targeting disease initiating stem cells. Moreover, our models represent the distinct stages of MPN progression, offering a platform to evaluate preventive and therapeutic interventions at various stages of this disease.

Enkurin: a novel marker for myeloproliferative neoplasms from platelet, megakaryocyte, and whole blood specimens.

Impaired protein homeostasis, though well established in age-related disorders, has been recently linked with the pathogenesis of myeloproliferative neoplasms (MPNs). However, little is known about MPN-specific modulators of proteostasis, thus impeding our ability for increased mechanistic understanding and discovery of additional therapeutic targets. Loss of proteostasis, in itself, is traced to dysregulated mechanisms in protein folding and intracellular calcium signaling at the endoplasmic reticulum (ER). Here, using exvivo and invitro systems (including CD34+ cultures from patient bone marrow and healthy cord/peripheral blood specimens), we extend our prior data from platelet RNA sequencing inpatients with MPN and discover select proteostasis-associated markers at RNA and/or protein levels in each of platelet, parent megakaryocyte, and whole blood specimens. Importantly, we identify a novel role in MPNs for enkurin (ENKUR), a calcium mediator protein originally implicated only in spermatogenesis. Our data reveal consistent ENKUR downregulation at both RNA and protein levels across specimens from patients with MPN and experimental models (including upon treatment with thapsigargin, an agent that causes protein misfolding in the ER by selective loss of calcium), with a concomitant upregulation of a cell cycle marker, CDC20. Silencing of ENKUR using short hairpin RNA in CD34+-derived megakaryocytes further confirms this association with CDC20 at both RNA and protein levels and indicates a likely role for the PI3K/Akt pathway. Together, our work sheds light on enkurin as a novel marker of MPN pathogenesis and indicates further mechanistic investigation into a role for dysregulated calcium homeostasis and ER and protein folding stress in MPN transformation.

Expression of Jak2 R1063H Leads to Altered Epor/Tpor Signaling, Changes in HSC Fitness and Represents Increased Risk of Thrombosis

Non-canonical weakly activating germline JAK2 variants may predispose progenitor cells to acquire JAK2 V617F during the evolution of myeloproliferative neoplasms (MPN) (Lanikova et al. Blood 2016;128(18):2266) and represent risk factors for MPN transformation into AML (Benton et al. Cancer 2019;125:1855). We have previously described and functionally characterized the JAK2 R1063H germline variant, cooperating with the JAK2 E846D variant in a case of hereditary MPN with erythrocytosis and megakaryocytic atypia (Kapralova et al. Blood 2016;128:1418) or with the acquired JAK2 V617F causing increased JAK2 signaling and higher disease severity in patients with essential thrombocythemia (Mambet et al. Blood 2018;132:2695). The mouse model bearing Jak2 R1063H mutation was created using microinjection of synthesized ssODNs containing the R1063H mutation, sgRNA mRNA and Cas9 protein into C57BL6/N-derived zygotes. In order to intensify a possible effect of the mutation, we bred and analyzed the knock-in Jak2 R1063H homozygous mice. The mice are born in normal (expected) frequency and display increased platelets (PLT) counts when compared to wild-type (wt) mice during life-span. Despite increased PLT counts, the plasma thrombopoietin level is normal whereas erythropoietin levels are increased without marked erythrocytosis. The preliminary co-immunoprecipitation experiments with human erythropoietin and thrombopoietin receptors (EPOR/TPOR) and JAK2 variants suggest increased TPOR and decreased EPOR binding with JAK2 R1063H kinase, possibly explaining the hematological phenotype. Also experiments with humanized mouse model of EPOR (Divoky et al. Proc Natl Acad Sci USA 2001;98:986) crossed with Jak2 R1063H mice supported these findings, where anemia due to hypoactivation of wt human EPOR signaling is more severe in Jak2 R1063H mice than in a control group and mild thrombocytosis is still present in the double mutant mice. The Jak2 R1063H model displays increased number of long-term hematopoietic stem cells (HSC) suggesting increased cycling in this population at young age (3 months) and shift to increased short-term HSC and multipotent progenitors with aging (12 months). The immunophenotypically-defined myeloid progenitor cells (LK, Lin-/Sca1-/cKithigh) are statistically increased in Jak2 R1063H mice due to disproportionately increased megakaryocytic/erythroid progenitors (MEP) over other myeloid progenitors at 3 months of age. The LK compartment is compromised in old Jak2 R1063H mice due to substantial myeloid bias by disproportionately increased CMP cells and marked decreased MEP cells, explaining the more pronounced anemia of aging in these mice. Some of these features are reminiscent of aging-associated myeloid and megakaryocytic bias, increased LKs, and relative exhaustion of progenitors with an erythroid lineage-priming program in aged bone marrow (BM) of a mouse model of primary familial and congenital polycythemia (PFCP) due to gain-of-function EPOR (Kralova et al, Am J Hematol 2022; doi: 10.1002/ajh.26658). However, the gain-of-function EPOR-introduced changes into the BM microenvironment are not associated with augmented hypoxia signaling and systemic inflammation and do not represent permanent challenges that would contribute to an increased incidence of myeloid malignancies in PFCP. In this regard, it will be interesting to compare these characteristics with the possible predisposing potential of Jak2 R1063H; further analysis of proliferative history of Jak2 R1063H HSC populations and expression profiling are ongoing. In the cohort of Jak2 R1063H mice, we also observed a relatively high frequency of sudden death in mice with median overall survival of 215 days (5/15) in comparison with the control group (0/15). We therefore measured the levels of D-dimers as a marker of thrombosis and found it statistically increased in the Jak2 R1063H group. Interestingly, in a WES study of patients with venous thromboembolism, the JAK2 R1063H was identified in one case, being considered a probable disease-causing variant (Lee at el. Blood Adv 2017;1:1224). In conclusion, our results confirm a direct effect of germline Jak2 R1063H mutation on EpoR/TpoR signaling but also on HSC biology, suggesting that the variant may represent a risk factor for BM remodeling in the process of myeloid cell transformation. Grant support: Czech Health Research Council NU21/03/00338.

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

P05 Blast transformation of essential thrombocythemia presenting as hip monoarthritis

P05 Blast transformation of essential thrombocythemia presenting as hip monoarthritis

Exome Sequencing Of Paired Chronic-Blast Phase Samples In Myeloproliferative Neoplasms Reveals High Frequency Of Rare Germline Sequence Variants In TYK2 and Novel Blast Phase-Restricted Mutations

The molecular underpinnings of leukemic transformation in myeloproliferative neoplasms (MPN) are not well characterized. We used Whole Exome Sequencing (WES) as a screening tool to gain further insight into the genomic origin of MPN progression to blast phase disease. WES analysis was performed in 6 JAK2V617F(+) MPN patients for which paired samples from chronic and blast phase disease were available. Respective clinical data were incorporated into the evaluation, and variations of interest were validated by conventional Sanger sequencing. Single-nucleotide-polymorphism database (dbSNP) was used to exclude/include established germline aberrations. Using WES filtering for non-synonymous variations as predicted deleterious, we detected a significant number of genetic abnormalities in both chronic and blast phase of MPN; these could be functionally linked to “tumorigenesis” (157 genes), “cancers and tumors” (159 genes) and “hematological neoplasia” (48 genes). In both phases of MPN, we could find mutations in genes already known to be linked with the development and/or transformation of MPN (ie, MPL, TET2, EZH2, IDH1, NRAS, LNK, TP53, WT1). Notably, eight genes were identified exclusively at onset of leukemic transformation and could be classified as potential driver mutations with damaging translational impact; these novel mutations could be grouped into “transcriptional regulators” (CUX2, ORC2, KDM6B, CNOT3, TBL1XR1), “NOTCH-signaling pathway” (NOTCH2NL, NOTCH1) and “tyrosine kinases” (STAP2). Moreover, we also identified several single-nucleotide polymorphisms encoding amino acid substitutions (ie, non-synonymous changes) in receptor as well as cytoplasmatic tyrosine kinase genes. Interestingly, the JAK-family member TYK2 showed the largest number of rare non-synonymous polymorphisms (A165T, V362F, G363S, I684S) leading to missense mutations in 67% (4/6) of the MPN patients; in contrast, none of 30 control samples with other types of cancer showed sequence variances in the TYK2 gene. Deficiency of TYK2 has shown to impair tumor surveillance and increase the susceptibility for neoplasia including AML. The present study demonstrates recurrent germline sequence variants in the non-receptor tyrosine kinase TYK2, and novel functionally-relevant somatic mutations with potential pathogenetic contribution to leukemic transformation in MPN. Disclosures:No relevant conflicts of interest to declare.

Understanding the Secondary Transformation of JAK2 Myeloproliferative Neoplasms: Genomic Analysis

Myeloproliferative neoplasms (MPNs) comprise of a group of clonal stem cell disorders characterized by proliferation of red blood cells (RBC; polycythemia vera or PV), platelets (essential thrombocytosis or ET) or white blood cells (WBC; primary myelofibrosis or PMF). Biggest challenge with MPNs is the leukemic transformation that occurs in 1-2%, 4-5%, and 20-30% of patients over a 10-year period in ET, PV, and PMF, respectively. These secondary leukemia patients are associated with dismal prognosis, with median survival of 6 months. Mutations in JAK2 have been observed in the majority of MPN patients, and though JAK2 mutation seems to be the phenotypic driver of MPNs, recent evidence suggest clonality and mutational events antecedent to JAK2 are responsible for leukemic transformation. To better understand the impact of somatic mutations that contribute to transformation of MPN to leukemia, and the clonal architecture of these mutations, we conducted this study. The collection of bone marrow samples, saliva samples and clinical data of 48 clinically JAK2 positive MPN patients (i.e. JAK2 V617F PCR positive) was carried out at Seoul National University Hospital, and this study was approved by the institutional review board. Written informed consents were obtained from all patients in accordance with the Declaration of Helsinki. The bone marrow samples obtained at chronic MPN state were subjected to target sequencing. Target genes providing diagnostic or prognostic information in myeloid malignancies were selected, and the final gene panel consisted of 2228 amplicons covering 47 genes. Amplified targets were sequenced on the Illumina TruSeq Amplicon. The mean coverage depth was 1056X. For 3 patients with sequential paired samples at MPN diagnosis and at secondary transformation, whole genome sequencing (WGS) was performed using Illumina HiSeq2000. Fisher's exact test was used to compare the mutation frequency of single genes in different MPN subgroups, while Levene's test was used to for comparison of mutation frequency of gene groups. Two-sided t-test was used to evaluate the statistical significance. There were 18 ET patients (37.5%), 21 PV patients (43.8%) and 9 PMF patients (18.8%), and the median age at MPN diagnosis was 63 years for the total cohort. Not surprisingly, PMF was associated with most secondary transformations (P=0.030). About 89.6% of the patients carried somatic mutations other than JAK2. The most frequently observed mutations included TP53, PTCH1, ASXL1, TET2, CALR, EZH2, KMT2A, MPL and SETBP1. Interestingly, majority of the patients carrying ASXL1 were PV. PMF patients more frequently carried mutated splicing genes per patient (p=0.005), while PV patients seem to carry more mutated epigenetic regulator genes per patient, especially the chromatin modifiers (P=0.077). In addition, somatic mutations in TP53 and NF1 are associated with increased risk of secondary transformation and these mutations seem to be present already in the chronic MPN state at a low allelic burden. The WGS results showed the number of mutations at chronic MPN state versus at secondary transformation did not significantly differ, suggesting novel mutation rate is low in MPN and the secondary transformation is indeed driven by expansion of already existent small clones. Efforts to effectively incorporate next generation sequencing into MPN diagnosis and follow-up will be helpful for recognition of patients at high risk of secondary transformation, and can ultimately lead to better clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Combination of myeloproliferative neoplasm driver gene activation with mutations of splice factor or epigenetic modifier genes increases risk of rapid blastic progression.

Myeloproliferative neoplasms (MPN) comprising polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) follow a bi-phasic course of disease with fibrotic and/or blastic progression. At presentation in the chronic phase, currently there are only insufficient tools to predict the risk of progression in individual cases. In this study, chronic phase MPN (16 PMF, 11 PV, and 11 MPN unclassified) with blastic transformation during course of disease (n=38, median follow-up 5.3years) were analyzed by high-throughput sequencing. MPN cases with a comparable follow-up period and without evidence of blast increase served as control (n=63, median follow-up 5.8years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) found at presentation were not significantly associated with blastic transformation. By contrast, mutations of SRSF2, U2AF1, and IDH1/2 at first presentation were frequently observed in the progression cohort (13/38, 34.2%) and were completely missing in the control group without blast transformation during follow-up (P=.0007 for SRSF2; P=.0063 for U2AF1 and IDH1/2). Unlike frequent ARCH/CHIP alterations (TET2, ASXL1, DNMT3A), mutations in SRSF2, IDH1/2, and U2AF1 when manifest already at first presentation provide an independent risk factor for rapid blast transformation of MPN.

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

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

Bone marrow niche dysregulation in myeloproliferative neoplasms.

The bone marrow niche is a complex and dynamic structure composed of a multitude of cell types which functionally create an interactive network facilitating hematopoietic stem cell development and maintenance. Its specific role in the pathogenesis, response to therapy, and transformation of myeloproliferative neoplasms has only recently been explored. Niche functionality is likely affected not only by the genomic background of the myeloproliferative neoplasm-associated mutated hematopoietic stem cells, but also by disease-associated ‘chronic inflammation’, and subsequent adaptive and innate immune responses. ‘Cross-talk’ between mutated hematopoietic stem cells and multiple niche components may contribute to propagating disease progression and mediating drug resistance. In this timely article, we will review current knowledge surrounding the deregulated bone marrow niche in myeloproliferative neoplasms and suggest how this may be targeted, either directly or indirectly, potentially influencing therapeutic choices both now and in the future.

Should Transplantation Still Be Considered for Ph1-Negative Myeloproliferative Neoplasms in Transformation?

BCR-ABL-negative myeloproliferative neoplasms (MPNs) in transformation have a dismal prognosis, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the sole curative therapeutic option. We retrospectively analyzed 53 molecularly annotated patients treated at Saint Louis Hospital, Paris, diagnosed with MPN in transformation between 2008 and 2018. The median patient age was 65 years, and the median interval between MPN diagnosis and MPN transformation was 46 months. The median overall survival (OS) of the entire cohort after transformation was 7.1 months. OS was better for patients treated with hypomethylating agents (HMAs) or with chemotherapy compared than for those treated by best supportive care or single-agent targeted therapy (median, 9.1 months versus 1.5 months; P < .001). Patients treated with chemotherapy more often achieved complete remission compared with those treated with HMAs (68% versus 29%; P=.02), and were more often candidates for transplantation (59% versus 14%; P=.02), but the median OS was similar in the 2 groups. We then compared the outcomes in transplant recipients and nonrecipients using the Mantel-Byar methodology and found that allo-HSCT did not improve survival. In multivariate analysis, independent factors in prognosis were performance status at transformation (P < .01), initial treatment with HMAs or chemotherapy (P=.02), and the ability to achieve complete remission during follow-up (P < .01). Our data demonstrate that the indication for allo-HSCT for high-risk MPN should be discussed before transformation, because transplantation rescues few patients after transformation.

High Mobility Group A1 Chromatin Regulators: Key Epigenetic Switches and Therapeutic Targets Required for Leukemic Transformation in JAK2 Mutant MPN

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by hyperactive JAK/STAT signaling and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML). However, mechanisms driving progression remain elusive and therapies are ineffective after leukemia develops. The High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive solid tumors where they portend adverse outcomes. HMGA1/2 genes are also up-regulated in hematologic malignancies and MPN with disease progression. In murine models, Hmga1/2 overexpression drives clonal expansion and deregulated proliferation while Hmga1 overexpression is sufficient for lymphoid leukemic transformation. We therefore sought to: 1) test the hypothesis that HMGA1/2 proteins are rational therapeutic targets required for leukemic transformation in MPN, 2) elucidate mechanisms mediated by HMGA1/2 during disease progression, and, 3) identify therapeutic approaches to disrupt HMGA function and intercept the transition from chronic disease to aggressive leukemia. Methods: We compared HMGA1/2 in JAK2V617F mutant AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cells and generated murine models of PV-AML with heterozygous Hmga1 or Hmga2 deficiency. To dissect molecular mechanisms underlying HMGA, we compared RNA-Seq from MPN-AML cell lines after gene silencing. Finally, to identify therapies to target HMGA pathways, we integrated the RNA-Seq data with the Broad Connectivity Map (cMAP). Results: There is a marked up-regulation in HMGA1/2 in CD34+ cells from PV patients after transformation to AML and in leukemic blasts from our PV-AML mouse model. Conversely, silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemia development in mice. Further, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality, although Hmga2 deficiency has no effect. RNA-Seq analyses from human MPN-AML cell lines revealed that HMGA1 up-regulates transcriptional networks involved in cell cycle progressions (E2F targets, mitotic spindle, G2M checkpoint, MYC targets) while repressing immune pathways (inflammation, interferon gamma) and oxidative phosphorylation. HMGA2 up-regulates similar pathways, but represses TNFalpha signaling. cMAP identified inhibitors of histone deacetylation and cell cycle progression as potential agents to target HMGA1 pathways; DNA synthesis inhibitors were predicted to target HMGA2 pathways. Cytotoxicity assays demonstrate that epigenetic therapy with HDAC inhibitors synergizes with Ruxolitinib in JAK2 mutant MPN cells after transformation to leukemia. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels after leukemic transformation. Further, silencing HMGA1/2 disrupts leukemogenic phenotypes in vitro and prevents the development of leukemia in mice. In addition, heterozygous deficiency of Hmga1 prolongs survival in a fulminant MPN-AML model. Mechanistically, RNA-Seq analyses revealed that HMGA amplifies transcriptional networks involved cell cycle progression, which can be targeted with epigenetic therapies. Our findings further underscore the key role for HMGA as an epigenetic switch required for leukemic transformation in MPN and opens the door to novel therapeutic approaches to intercept the transition from chronic indolent disease to aggressive leukemia. Disclosures No relevant conflicts of interest to declare.

Immune Checkpoint Blockade Enhances Shared Neoantigen-Induced T-cell Immunity Directed against Mutated Calreticulin in Myeloproliferative Neoplasms.

Somatic frameshift mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN). All patients carrying these mutations (CALR + MPN) share an identical sequence in the C-terminus of the mutated CALR protein (mut-CALR), with the potential for utility as a shared neoantigen. Here, we demonstrate that although a subset of patients with CALR + MPN develop specific T-cell responses against the mut-CALR C-terminus, PD-1 or CTLA4 expression abrogates the full complement of responses. Significantly, blockade of PD-1 and CLTA4 ex vivo by mAbs and of PD-1 in vivo by pembrolizumab administration restores mut-CALR-specific T-cell immunity in some patients with CALR + MPN. Moreover, mut-CALR elicits antigen-specific responses from both CD4+ and CD8+ T cells, confirming its broad applicability as an immunogen. Collectively, these results establish mut-CALR as a shared, MPN-specific neoantigen and inform the design of novel immunotherapies targeting mut-CALR. SIGNIFICANCE: Current treatment modalities for MPN are not effective in eliminating malignant cells. Here, we show that mutations in the CALR gene, which drive transformation in MPN, elicit T-cell responses that can be further enhanced by checkpoint blockade, suggesting immunotherapies could be employed to eliminate CALR + malignant cells in MPN.This article is highlighted in the In This Issue feature, p. 1143.

PS1471 MYELOPROLIFERATIVE NEOPLASMS IN KOREAN CHILDREN AND YOUNG ADULTS

Background:Myeloproliferative neoplasms (MPNs) are a rare, heterogeneous group of clonal hematopoietic stem cell disorders, characterized by aberrant proliferation of one or more myeloid lineages leading to an overproduction of both mature and immature cells. Three major components of classical BCR‐ABL‐negative MPNs are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (pMF). They may be transformed to either secondary MF (sMF) or secondary acute myeloid leukemia (sAML). In addition, they can have second cancers during follow up (FU). These classical MPNs primarily occur in middle and advanced age adults, and are exceptionally rare in pediatric age.Aims:We conducted an epidemiologic study to elucidate clinical course of MPNs in children and young adults under 30 years of age focusing on disease transformation as well as thromembolism and second cancers.Methods:We analyzing data from the Health Insurance Review and Assessment Service (HIRA) database which includes almost all hospital‐based patients’ information in Korea.Results:A total of 352 patients who were diagnosed as one of the three kinds of MPNs from Jan. 2008 to Dec. 2016 (242 patients for ET, 104 for PV, and 6 for pMF) were included. Six pMF patients were excluded from further analysis. The incidence was 0.148 per 100,000 for ET and 0.064 per 100,000 for PV. Among 242 ET patients, the male‐female ratio was 119 to 123, and median age at diagnosis was 22 years (range 13–29). Four ET patients (1.65%) underwent any transformation during the study period (median FU duration 49.5 months); ET→PV (i.e., initially diagnosed ET but changed to PV later; N = 2), and ET→sMF (N = 2). Among 104 PV patients, the male‐female ratio was 96 to 8, and median age at diagnosis was 24 years (range 11–29). No PV patients were transformed into sMF during the FU period (median 45.9 months). sAML did not develop in both ET and PV patients. Solid cancers occurred in 5 ET patients and 3 PV patients. Cumulative incidence of arterial and venous thromboembolism were 6.98% and 1.71% in ET, 16.69% and 4.77% in PV, respectively. Five‐year overall survival and transformation‐free survival (to PV, MF and cancer) were 98% and 96.12% in ET, 100% and 97.29% in PV, respectively.Summary/Conclusion:This is the first population‐based epidemiologic study in Korean children and young adults with MPNs. The incidence of MPNs is very low in this age group. ET is more common than PV, whereas pMF is very rare. Transformations of ET to PV or MF occurred in a small number of patients. Considering long latency to transformation in MPNs, continuous observation is needed. In addition, second solid tumors should also be carefully observed. Further research is necessary in this patient group, including studies involving other complications such as bleeding.

PS1450 THE PHARMACOLOGICAL IGF1R‐IRS1/2 INHIBITOR OSI‐906 REDUCES ERYTHROID PROGENITORS AND INHIBITS STAT3 IN JAK2V617F KNOCKIN‐MYELOPROLIFERATIVE NEOPLASM MICE

Background:The recurrent V617F mutation in JAK2 confers growth factor‐independent proliferation for hematopoietic cells and plays an important role in the pathogenesis of myeloproliferative neoplasms (MPN). Absence of complete clinical remission in most MPN patients treated with the JAK1/2 inhibitor ruxolitinib highlights the need of new therapeutic strategies. Recent evidence indicates that IGF1R/IRS pathway is a potential target in MPN. In mice, AIRAPL down‐regulation induces MPN transformation by upregulation of IGF1R signaling. Linsitinib (OSI‐906) is a potent highly selective IGF1R/IR inhibitor, with anti‐neoplastic effects in solid tumors, but underexplored in hematological neoplasms. We have previously noticed that OSI‐906 reduces cell viability and inhibited IRS1/2, IGF1R, AKT1/2/3 and P70S6K in Ba/F3 JAK2V617F cells (EHA 2018 ‐ Abstract #3288).Aims:To investigate the in vivo and ex vivo effects of OSI‐906 treatment in MPN phenotype and progenitors cells using JAK2V617F knockin‐MPN mice.Methods:For MPN phenotype induction, 5 × 106 bone marrow cells from JAK2V617F knockin mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton‐Dickinson) by flow cytrometry in peripheral blood. Mice with &gt;70% CD45.2 cells were randomized and treated with vehicle (25 mM tartaric acid solution, n = 5) or OSI‐906 (50 mg/kg, n = 4), both were administered oral by gavage, five days per week, from days 1 to 36. In day 37, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen and bone marrow were evaluated by CD71 and Ter119 markers. The immature subpopulation LSK (Sca1+/c‐Kit+), the myeloid progenitor (Sca1−/c‐Kit+), multipotent progenitors (MPP) (CD48+/C150−), long‐term (LT‐HSC) (CD48−/CD150+) and short‐term (ST‐HSC) (CD48−/150−) hematopoietic stem cells were evaluated by flow cytometry. All experiments were approved by the Animal Ethics Committee. Mann‐Whitney test or ANOVA test and Bonferroni post‐test was used. Alternatively, JAK2V617F knockin mice were euthanized between 10–16 weeks of age and the bone marrow was isolated, treated ex vivo with vehicle or OSI‐906 (≥20 μM); and submitted to clonogenicity and to protein expression/activation studies.Results:OSI‐906 treatment in vivo was well‐tolerated by JAK2V617F‐kockin MPN mice, but did not reduce the spleen volume. OSI‐906 significantly reduced erythroid progenitors (p = 0.03) and increased myeloid progenitors (p = 0.01) in bone marrow when compared with vehicle. Hemoglobin, hematocrit and the number of MPP, ST‐HSC, or LT‐HSC were not modulated by OSI‐906 treatment. Bone marrow cells from JAK2V617F knockin‐MPN mice treated ex vivo with OSI‐906 presented absence of colony formation, and decreased of protein expression/activation of IGF1R, AKT1/2/3, ERK1/2, and STAT3.Summary/Conclusion:OSI‐906 exhibited promising results ex vivo, including inhibition of IGF1R signaling pathways and progenitor cells proliferation. OSI‐906 treatment in vivo in the current protocol reduced erythroid progenitors in bone marrow from JAK2V617F knockin‐MPN mice. The lack of better results in vivo reinforces the need to adjust treatment regimen to this disease model.

Blast Transformation in Myeloproliferative Neoplasms: Risk Factors, Biological Findings, and Targeted Therapeutic Options.

Myeloproliferative neoplasms represent a heterogenous group of disorders of the hematopoietic stem cell, with an intrinsic risk of evolution into acute myeloid leukemia. The frequency of leukemic evolution varies according to myeloproliferative neoplasms subtype. It is highest in primary myelofibrosis, where it is estimated to be approximately 10–20% at 10 years, following by polycythemia vera, with a risk of 2.3% at 10 years and 7.9% at 20 years. In essential thrombocythemia, however, transformation to acute myeloid leukemia is considered relatively uncommon. Different factors are associated with leukemic evolution in myeloproliferative neoplasms, but generally include advanced age, leukocytosis, exposure to myelosuppressive therapy, cytogenetic abnormalities, as well as increased number of mutations in genes associated with myeloid neoplasms. The prognosis of these patients is dismal, with a medium overall survival ranging from 2.6–7.0 months. Currently, there is no standard of care for managing the blast phase of these diseases, and no treatment to date has consistently led to prolonged survival and/or hematological remission apart from an allogeneic stem cell transplant. Nevertheless, new targeted agents are currently under development. In this review, we present the current evidence regarding risk factors, molecular characterization, and treatment options for this critical subset of myeloproliferative neoplasms patients.

Janus kinase 2 variants associated with the transformation of myeloproliferative neoplasms into acute myeloid leukemia.

Canonical Janus kinase 2 (JAK2) V617F and exon 12 mutations in myeloid neoplasms are well described. There are limited reports of other JAK2 variants of potential clinical relevance. This study was designed to survey JAK2 variants in patients with myeloproliferative neoplasms (MPNs) and acute myeloid leukemia (AML) and to determine their contributions to disease pathogenesis. Next-generation sequencing of the coding region of JAK2 and 27 other genes was performed on bone marrow DNA samples. The study population was classified into 3 cohorts: chronic MPNs only (the MPN cohort); MPNs transformed into AML (the MPN>>AML cohort); and AML only, with MPN>>AML patients excluded (the AML cohort). Testing was performed for 2154 patients, and non-V617F/non-exon 12 JAK2 sequence variants were identified in 114 (5.3%). They included 35 unique JAK2 variants across all functional domains. Sixteen of the 114 JAK2 variants occurred without somatic mutations in the remaining 27 genes. JAK2 variants were detected at a higher frequency in the MPN>>AML cohort (15.3%) in comparison with the MPN (4.6%; P<.001) and AML cohorts (5.2%; P<.001). Detected variants occurred at higher than expected frequencies in patients with MPNs and AML in comparison with the population, and N1108S had a significantly increased prevalence in patients with AML. A JAK2 variant in addition to JAK2 V617F (n=13) in myelofibrosis was associated with an increased cumulative risk of transformation into AML (P=.003). Specific JAK2 variants detected in MPNs may be predictors for transformation into AML.

Abstract B072: Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms

Abstract Somatic mutations in the 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. CALR mutations are 1-bp frameshift mutations that result in the formation of a novel C-terminus with an identical 36-amino acid (aa) sequence that is shared by all CALR+ MPN patients. We hypothesized that the recurrence and uniformity of the novel C-terminus mark the mutated (mut)-CALR an attractive candidate as an MPN-specific tumor neoantigen that might elicit anti-tumor immune responses across patients who express this mutation. To assess the immunogenicity of mut-CALR neopeptide, we interrogated the binding affinity of epitopes from the altered C-terminus using in silico peptide binding prediction algorithms. The analysis showed that multiple epitopes could bind to class I and II human leukocyte antigens (HLA). We investigated whether we could elicit T-cell responses against mut-CALR neopeptide from CALR+ MPN patients. Peripheral blood mononuclear cells (PBMCs) from 18 MPN patients with ET (7), MF (4) or MF arising from ET (7) were stimulated in vitro with pooled overlapping long peptides (OLPs; 14-15 aa) spanning the mutated or wild type (WT) CALR C-terminus and T-cell responses were assessed by enzyme-linked immunospot assay (ELISPOT). There was a significant increase in interferon (IFN)-γ production upon stimulation with mut-CALR OLPs, whereas the corresponding WT sequence did not induce a T-cell response. Next, we evaluated whether mut-CALR-specific T-cells were undergoing exhaustion. Examination of the expression of checkpoint receptors on T-cells ex vivo determined that T-cells from peripheral blood of CALR+ MPN patients exhibited increased expression of multiple cell surface inhibitory molecules compared to healthy donor T-cells, including programmed cell death (PD)-1 and cytotoxic T lymphocyte-associated antigen (CTLA)-4. In order to address whether PD-1 and CTLA-4 overexpression suppressed mut-CALR-specific T-cell responses in CALR+ MPN patients, PD-1 or CTLA-4 signaling on T-cells was blocked through addition of monoclonal blocking antibodies. T-cell responses against mut-CALR peptides were recovered in 3 CALR+ MPN patients that were previously nonresponsive to mut-CALR stimulation. To gain further insights into checkpoint receptor-mediated T-cell suppression in CALR+ MPN patients, we investigated mut-CALR-specific T-cell responses in a CALR+ MPN patient receiving PD-1 inhibitor pembrolizumab, in our ongoing clinical trial (NCT03065400). We monitored the changes in the frequencies of peripheral blood T-cells in this patient before and after pembrolizumab treatment by both flow cytometry and T-cell receptor (TCR) sequencing. Prior to therapy, this patient had low proportion of T-cells (4.33% of PBMCs) and 32.3% of the T-cells expressed PD-1. After 2 cycles of pembrolizumab, the T-cell proportion greatly increased (26% of PBMCs) and remained high when measured after 6 cycles (18% of PBMCs). Sequencing of TCR Vβ chains of peripheral blood T-cells revealed that T-cells exhibited greater clonality after pembrolizumab treatment. Furthermore, we evaluated changes in mut-CALR-specific T-cell responses in this CALR+ MPN patient prior to and during pembrolizumab administration. At baseline, peripheral blood T-cells from this patient did not respond to stimulation with mut-CALR peptides. After pembrolizumab treatment, however, mut-CALR-specific T-cell responses were evident, as shown by increased IFN-γ production. Together, these results establish mut-CALR as a novel MPN-specific tumor shared-neoantigen and provide a strong rationale to study its immunogenic properties to inform the design of novel immunotherapies targeting mut-CALR in MPN patients carrying the mutation. Citation Format: Cansu Cimen Bozkus, Vladimir Roudko, John P. Finnigan, John Mascarenhas, Ronald Hoffman, Camelia Iancu-Rubin, Nina Bhardwaj. Immune checkpoint blockade enhances mutated calreticulin-induced T-cell immunity in myeloproliferative neoplasms [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B072.