Transformation Of Myeloproliferative Neoplasms Research Articles

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.

High Mobility Group A1 Chromatin Remodeling Proteins Amplify Inflammatory Networks to Drive Leukemic Transformation in Chronic Myeloproliferative Neoplasia in Humans and JAK2V617F Transgenic Mouse Models

Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression.Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis.Results:HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1.Conclusions:HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. DisclosuresNo relevant conflicts of interest to declare.

Large Genomic Alterations Occurring in the Transition from Chronic to Blast Phase of Chronic Myeloproliferative Neoplasms

INTRODUCTION Five to 20% of patients with myeloproliferative neoplasms (MPN), including Essential Thrombocythemia (ET), Polycythemia Vera (PV) and Primary Myelofibrosis (PMF), transform to an aggressive secondary acute myeloid leukemia (sAML). While several studies reported the association of some mutations with the risk of leukemic transformation (Vannucchi AM et al, Leukemia 2013), the mechanisms that contribute to transformation from MPN to sAML remain largely poor characterized.METHODS. We collected annotated samples from 15 chronic-phase (CP) MPN patients (pts), 6 pts with accelerated phase (AP; PB blasts 10-19%) and 12 pts with sAML; for the latter, paired samples (chronic/blast phase-BP) were available. CP and BP samples were separated by a mean of 77 (12 to 216) months interval. We used Illumina whole exome sequencing (WES) to identify copy number variations (CNV) in all samples. In the paired samples set, we also performed long reads genome sequencing by the Oxford Nanopore technology, a uniform process that generates sequences randomly and independently, without classical sources of bias such as GC-content and mappability. Data analysis for CNV detection was performed by a novel devised computational package (Nano-GLADIATOR; Magi A, Bartalucci N et al, Genome Biology, submitted) allowing the analysis of individual samples without the need of paired-analysis.RESULTS. The mean number of CNV detected in CP, AP and BP samples was respectively 130.7±49, 132±42 and 177.4±61 (P=0.03 of BP vs CP). CNV were represented by gain of genomic material in 63.6%, 68.2% and 64.1% of CP, AP and BP. Considering the length of all CNV, expressed as base pairs (bp), we found that 96.8% of CNV in CP were focal alterations spanning <1 Mega bases (Mb) while only 2.9% and 0.3% were larger than 1Mb and 30Mb, respectively. Conversely, alterations involving >1Mb and >30Mb in BP samples were 14.4% (P=0.05) and 2.1% (P=0.04), and corresponding figures in AP were respectively 5.3% (ns vs CP, P=0.05 vs BP) and 0.7% (P=0.04 vs CP). Considering CNV >1Mb only, 53.7% were gains in CP compared with 68.1% in AP and 63.6% in BP, while losses were 46.3%, 31.9% and 36.4% respectively. Furthermore, alterations involving all the short (p) and long (q) -arm or the whole chromosome were found in 77% of BP compared to 36% of AP and 11% only of CP samples (P<0.01), overall indicating that larger alterations are enriched in BP. We used Nanopore platform to analyze paired CP and BP samples. We found that the total number of bp involved in CNV was 380x106 and 2x109 in CP and BP, respectively, with an estimated frequency of 0.01 and 0.07 altered base every 1 base of normal genome. The mean length of CNV was 5.6x106 bp and 23.7x106 bp respectively in CP and BP samples. There was a total of 29 new CNV acquired in BP samples compared to CP, involving 10 different chromosomes. We identified recurrent alterations as double or single deletion of 100,000 bp in chromosome 8 (55.5% of BP), deletion of a 510.000 to 610.000 bp region of chromosome 4q (40% of BP) and an amplification of 32,535,000 bp in chromosome 21q in 34% of BP samples. Alterations involving chromosomes 4 and 8 detected in BP samples were already present at paired CP samples in 75% and 80% of cases, while in the remaining 25% and 20% they were acquired at BP. The same abnormalities were absent from the 15 unpaired CP samples (WES data) whereas they were present in 4/6 and 3/6 of AP patients, respectively.CONCLUSION All together, this data indicate that genomic instability is a hallmark of leukemic transformation of MPN and for the first time identify regions that may be recurrently associated with disease progression from CP to BP, potentially representing novel biomarkers. These finding require confirmation in larger series. DisclosuresNo relevant conflicts of interest to declare.

Genetic Alterations in Essential Thrombocythemia Progression to Acute Myeloid Leukemia: A Case Series and Review of the Literature.

The genetic events associated with transformation of myeloproliferative neoplasms (MPNs) to secondary acute myeloid leukemia (sAML), particularly in the subgroup of essential thrombocythemia (ET) patients, remain incompletely understood. Deep studies using high-throughput methods might lead to a better understanding of genetic landscape of ET patients who transformed to sAML. We performed array-based comparative genomic hybridization (aCGH) and whole exome sequencing (WES) to analyze paired samples from ET and sAML phases. We investigated five patients with previous history of MPN, which four had initial diagnosis of ET (one case harboring JAK2 p.Val617Phe and the remaining three CALR type II p.Lys385fs*47), and one was diagnosed with MPN/myelodysplastic syndrome with thrombocytosis (SF3B1 p.Lys700Glu). All were homogeneously treated with hydroxyurea, but subsequently transformed to sAML (mean time of 6 years/median of 4 years to transformation). Two of them have chromosomal abnormalities, and both acquire 2p gain and 5q deletion at sAML stage. The molecular mechanisms associated with leukemic progression in MPN patients are not clear. Our WES data showed TP53 alterations recurrently observed as mutations (missense and frameshift) and monoallelic loss. On the other hand, aCGH showed novel chromosome abnormalities (+2p and del5q) potentially associated with disease progression. The results reported here add valuable information to the still fragmented molecular basis of ET to sAML evolution. Further studies are necessary to identify minimal deleted/amplified region and genes relevant to sAML transformation.

Dual Targeting of Oncogenic Activation and Inflammatory Signaling Increases Therapeutic Efficacy in Myeloproliferative Neoplasms

Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production invivo. Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis invivo.

The Genomic Landscape of Leukemic Transformation of Myeloproliferative Neoplasm

The Genomic Landscape of Leukemic Transformation of Myeloproliferative Neoplasm

Abstract 3812: JAK2 and PTPN11 mutations as potential biomarkers for BCL-xL inhibition as monotherapy and in combination therapy for acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a heterogeneous disease where many distinct functional mutations have been identified. While mutations in FLT3, NRAS, NPM1, and IDH genes have been well characterized in de novo AML, a subset of AML associated with leukemic transformation of chronic myeloproliferative neoplasms (MPNs) are enriched in mutated JAK2 and PTPN11. MPNs are a set of disorders characterized by the chronic and abnormal overproduction of blood cells which can ultimately progress to AML. Targeted therapy against mutated JAK2 and PTPN11 with small molecule inhibitors against the JAK-STAT and RAS-ERK pathways, respectively, are being actively investigated in AML. However, acquired resistance and non-durable responses are already being demonstrated in monotherapy treatment. Thus, AML that has arisen from leukemic transformation of MPNs remains an area of unmet medical need. This need is underscored by the fact that these patients suffer from poor outcomes and low response rates to standard chemotherapy. Together, these results indicate the need for rational combinations in this population. In this study, we sought to better define the dependencies of AML cells on BCL-2 family members by screening AML cell lines with selective small molecule inhibitors to the BCL-2 family members. The screen revealed that the majority of AML cell lines depend on BCL-2, or BCL-2 together with MCL1 for survival. Intriguingly, a subset of AML cell lines is exquisitely sensitive to BCL-xL inhibition. Treating these cell lines with BCL-xL-selective inhibitor or with navitoclax, a dual BCL2/BCL-xL inhibitor induces apoptosis. These BCL-xL inhibitor-sensitive cell lines express high levels of BCL-xL, but not BCL-2 and MCL1. Genomic analysis of BCL-xL inhibitor-sensitive AML cell lines revealed that this subset is also enriched in JAK2V16F and PTPN11 mutations. We further demonstrate positive combination effect between BCL-xL inhibitor or navitoclax with JAK inhibitor (ruxolitinb) and two MEK inhibitors (trametinib and AZD6244) in cell lines with JAK2V16F and PTPN11 mutations, respectively. Together, these results demonstrate the potential utility of a BCL-xL inhibition as a combination partner in JAK2 and PTPN11 mutated cancers. Importantly, JAK2 and PTPN11 mutations in addition to expression of BCL-xL, BCL-2, and MCL1 could also be further tested as potential biomarkers for the utility of BCL-xL inhibition based treatments for patients with these mutations. Citation Format: Richard J. Bellin, Valerie Hilgenberg, Relja Popovic, Tamar Uziel, Lloyd T. Lam. JAK2 and PTPN11 mutations as potential biomarkers for BCL-xL inhibition as monotherapy and in combination therapy for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3812. doi:10.1158/1538-7445.AM2017-3812

Leukemic transformation in patients with myeloproliferative neoplasms: a population-based retrospective study.

This study determined the epidemiology of developing leukemic transformation in patients with myeloproliferative neoplasms (MPN). We utilized the Surveillance, Epidemiology and End Results 13 database to identify 83 cases of leukemic transformation in MPN (n = 9335). The 5-year cumulative incidence of leukemic transformation was higher in male versus female (2.17 vs 1.09%; p < 0.001), and in myelofibrosis (2.19%; 95% CI: 1.36-3.34%), compared with essential thrombocythemia (0.37%; 95% CI: 0.19-0.65%) and polycythemia vera (0.72%; 95% CI: 0.46-1.07%; p < 0.001). Patients had a median survival of 2 months after leukemic transformation, worse in older patients and without any impact of prior MPN subtypes. Myelofibrosis has a higher risk of leukemic transformation. Overall survival is dismal regardless of MPN subtypes.

Somatic mutations of calreticulin in myeloproliferative neoplasms.

Recurrent somatic mutations in calreticulin (CALR) gene that encodes a molecular chaperone residing in the endoplasmic reticulum were identifiedin 2013 in a subset of patients with myeloproliferative neoplasms (MPNs). All of these mutations found in patients were either small insertion or deletion in a narrow region on exon 9 of CALR gene, and caused +1 frameshift in the reading frame for the translation of the carboxyl-terminus of CALR. Because of this unique feature, the CALR mutation is believed to be a gain-of-function mutation. However, there was essentially no rationale model to implicate the involvement of mutant CALR in the pathogenesis of MPN or other malignancies. Based on the recent findings, this review summarizes a novel molecular mechanism by which this mutant molecular chaperone constitutively activates the cytokine receptor to induce cellular transformationin MPNs.

Aside from the Allelic Burden of JAK2-V617F, Acquisition of Mutations in Myeloid Gene Panel Is Associated with the Transformation of Myeloproliferative Neoplasm into Secondary AML

Introduction:The discoveries of JAK2-V617F as well as MPL and CARL mutations have greatly clarified the underlying genetics of Philadelphia negative myeloproliferative disease (MPN). Mutation status on these three genes, especially JAK2-V617F, can characterize over 90% of MPN patients. However, the heterogeneity of MPN in terms of its AML transformation and treatment response remains unclear. To assess the difference in mutational status between MPN patients who progress to secondary AML and those who do not, we aim to examine longitudinal samples taken from multiple time points using next generation sequencing.Patients and Methods:Bone-marrow (BM) samples were collected from 19 MPNpatientsfrom2003 to 2012atChonnamNational UniversityHwasunHospital.The diagnosis of MPN was established according to the revised criteria of the World Health Organization.Longitudinal samples were taken at the time of diagnosis and at a follow-up as well as its T-cell fractions (CD+3) isolated from the peripheral blood using MAC separation column.Targeted sequencing was performed using an Agilent custom probe set of a panel of 84 myeloid genes. We multiplexed and sequenced the samples using an IlluminaHiseq2000.Results:The mean on-target coverage for the 57 sequenced samples was 861.4x. We detected a total of 48 somatic mutations in 25 genes in 17 patients (89%) throughout the course of the disease. Five of the 25 genes were recurrently mutated (JAK2, IDH2, ASXL1, SRSF2 and, TP53). As expected, JAK2-V617F was the most commonly observed (15/17 patients). One of the patients without JAK2-V617F carried a MPL mutation and another was triple negative.At the time of follow-up, 12 patients had chronic MPN (11 stable disease and 1 spleen response) withRuxolitinib treatment for a median duration of 373 days (range 255 - 729).Among 7 progressed patients, 5 patients had additional mutation at diagnosis of MPN other than JAK2 or MPL: MPN-13 (DNMT3A and ASXL1), MPN-14 (SRSF2 and IDH2), MPN-15 (IDH2), MPN16 (U2AF1), MPN-17 (IDH2 and SRSF2) as shown in figures.On the other hand, 4/12 non-progressed patients carried additional mutations: MPN-01 (ZRSR2), MPN-05 (ASXL1, CBL, FGR, KMT2D and TET2), MPN-10 (ASXL1, CDH13, EED, EZH2, MN1, NF1 and TRRAP), MPN-11 (FOXP1). In summary, only ASXL1 and JAK2-V617F were recurrently mutated among the non-progressed group.At the time of leukemic transformation, 6 out of 7 patients acquired new mutations:MPN-13 (SETBP1 and TP53), MPN-14 (RUNX1, ASXL1, and IDH1), MPN-15 (TP53 and CASP8), MPN-16 (TP53), MPN-18 (CEBPA), and MPN-20 (ASXL1). In the remaining case (MPN-17), increase of JAK2-V617F VAF was also observed. In summary, TP53 mutation was the most common mutation to acquire by the time of leukemic transformation (n=3). Other mutations acquired by this stage were in SETBP1, RUNX1, IDH1, CEBPA, and ASXL1. We did not observe any significant difference in the allelic burden increase of JAK2-V617F from diagnosis to follow-up between the non-progressed and progressed groups (Figure A).Conclusion:There is no significant difference in mutation burden increase of JAK2-V617F between patients who progressed to secondary AML and patients that did not. Acquisition of mutations other than JAK2-V617F at both diagnosis and at follow-up is associated with the risk of transformation to secondary AML. Mutation profiling using a myeloid gene panel at timed follow-up after MPN diagnosis can be more helpful than monitoring JAK2-V617F status in these patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low-Burden TP53 Mutations Occur in Chronic Phase of Myeloproliferative Neoplasms Regardless of Hydroxyurea Administration, Disease Type, and JAK2 Status

▪Myeloproliferative neoplasms (MPN) are characterized by abnormal proliferation of myeloid lineages and a tendency toward leukemic transformation. Inactivation of tumor suppressor TP53 has been repeatedly associated with MPN transformation into secondary acute myeloid leukemia (sAML), but no fully expanded TP53 mutated clones in chronic phase of MPN were reported. The link between TP53 mutations and widely used cytoreductive treatment by hydroxyurea (HU) still remains controversial. To which extent TP53 mutations represent a risk of disease progression is not known. We aimed to search for low-burden TP53 mutated subclones in chronic-phase-MPN patients and to correlate presence of these clones with therapy, disease course, and other clinical features.In total we analyzed 220 patients by ultra-deep next generation sequencing (NGS). We detected TP53 mutations in 39 (18 %) patients with variant allelic frequency of 0.1-16.3 % at the first examination. The analysis of 136 patients treated with hydroxyurea or other drugs (anagrelide (ANA), interferon α (IFN)) for more than 4 years, as well as a group of 84 patients untreated by cytoreductive drugs, showed that TP53 mutations occurrence in chronic phase is independent of hydroxyurea use, disease type, and JAK2/CALR/MPL status. Mutations were found in 17/72 (24 %) HU treated patients, in 11/64 (17 %) patients treated by other drugs, and in 11/84 (13 %) untreated patients. Median size of mutated clones was 0.5 % and was not influenced by previous treatment. In 10 patients we found more than one mutation.In patients harboring TP53 mutations, retrospective samples were examined if available to explore the clonal evolution of TP53 mutated clones. The respective TP53 mutations were found in 13/20 cases analyzed; out of them, in 4 cases the mutation was found even in a diagnostic sample. Follow-up samples were examined in 28 patients with TP53 mutations and the mutation burden changed during the monitored time in majority of patients; however, the expansion into dominant clone was observed in one patient only. When all data from retrospective and prospective analyses taken together (30 patients), the median follow-up was 7.2 years. TP53 mutation burden tended to increase in 14 patients. In 6 patients the mutation burden remained stable and in 4 patients it fluctuated. In 6 patients the mutated clone size decreased; out of them in 2 originally very low-burden mutations (0.2 %) were not detectable in samples taken 15 and 3.4 years later. We did not observe any correlations of different patterns in TP53 mutation changes with therapy or other clinical characteristics (disease type, driver mutation, time from diagnosis, treatment response).Further, we assessed the TP53 mutation impact on overall survival and leukemic transformation. The mutations did not negatively affect disease progression or overall survival either from diagnosis or from mutation identification. sAML developed in 2 patients with TP53 mutations 17.9 (treated with IFN) and 8.3 (treated with HU) years from diagnosis. In the latter patient, the sAML developed from a different clone as it was TP53-wt, JAK2-wt, although 2 TP53 mutated clones within JAK2 mutated clone were detected in chronic phase. On the other hand, we have observed another interesting case where the TP53 mutation burden grew rapidly from 10 % up to nearly 100 % during the follow-up. In contrast to published data, this patient did not show any clinical signs of disease progression for 2 years after the expansion and died of MPN unrelated cause.In summary, using highly sensitive method we showed that low-burden TP53 mutations are present in MPN chronic phase. Neither their presence nor their size is associated with previous therapy and has impact on overall survival or leukemic transformation. Monitoring of TP53 mutations during the disease course showed that their clonal development is rather variable; nevertheless, TP53 minor mutations may represent a pool for future clonal evolution.Supported by MZ CR-RVO (FNBr, 65269705), MUNI/A/1028/2015, H2020 692298, MZO AZV 15-31834A, 15-30015A, MEYS LQ1601 and LM2015064. DisclosuresGisslinger:Baxalta: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria. Mayer:AOP Orphan Pharmaceuticals: Research Funding; Novartis: Research Funding. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Molecular Mechanism of Mutant CALR-Mediated Transformation.

Elf and colleagues used an elegant series of functional and biochemical assays to investigate the molecular mechanism of mutant calreticulin (CALR)-driven cellular transformation in myeloproliferative neoplasms (MPN). Mutant CALR is sufficient to induce MPN in mouse transplantation experiments, and transformation is dependent upon physical interaction mediated by the positive electrostatic charge of the mutant CALR C-terminal domain and the thrombopoietin receptor MPL.

Ptch2 loss drives myeloproliferation and myeloproliferative neoplasm progression.

JAK2V617F(+) myeloproliferative neoplasms (MPNs) frequently progress into leukemias, but the factors driving this process are not understood. Here, we find excess Hedgehog (HH) ligand secretion and loss of PTCH2 in myeloproliferative disease, which drives canonical and noncanonical HH-signaling. Interestingly, Ptch2(-/-) mice mimic dual pathway activation and develop a MPN-phenotype with leukocytosis (neutrophils and monocytes), strong progenitor and LKS mobilization, splenomegaly, anemia, and loss of lymphoid lineages. HSCs exhibit increased cell cycling with improved stress hematopoiesis after 5-FU treatment, and this results in HSC exhaustion over time. Cytopenias, LKS loss, and mobilization are all caused by loss of Ptch2 in the niche, whereas hematopoietic loss of Ptch2 drives leukocytosis and promotes LKS maintenance and replating capacity in vitro. Ptch2(-/-) niche cells show hyperactive noncanonical HH signaling, resulting in reduced production of essential HSC regulators (Scf, Cxcl12, and Jag1) and depletion of osteoblasts. Interestingly, Ptch2 loss in either the niche or in hematopoietic cells dramatically accelerated human JAK2V617F-driven pathogenesis, causing transformation of nonlethal chronic MPNs into aggressive lethal leukemias with >30% blasts in the peripheral blood. Our findings suggest HH ligand inhibitors as possible drug candidates that act on hematopoiesis and the niche to prevent transformation of MPNs into leukemias.

Abstract A46: Real time niche tracking of cancer stem cell cycle kinetics using a novel bi-cistronic lentiviral reporter.

Abstract Human leukemia stem cells (LSC) are quiescent self-renewing cells that drive leukemic transformation of myeloproliferative neoplasms and myelodysplastic syndromes. Currently, there are few methods to quantitatively visualize cell cycle progression in (LSC) and to interrogate stem cell cycle gene regulatory networks in live cells. Human LSC are characterized as quiescent self-renewing cells and current methods require sequential transduction and clonal selection. These methods and conditions are not feasible for studying primary patient samples and LSC, which are generally limited in cell numbers. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI2 (fluorescent ubiquitination-based cell-cycle indicator) probes. We characterized the fidelity of the cell cycle by confirming normal cell division and distinct nuclear staining of either green or red fluorescence in both transiently and stably transduced 293 cells. Further, analysis by flow cytometry of 293 cell confirmed that S/G2/M and G1/G0 phases of cell cycle correlate with expression of fluorescent reporters expressed from the bicistronic lentiviral vector and recapitulate FACS based analysis using traditional Ki67 and 7AAD staining. Recently, using this lentiviral biscistronic reporter we succeeded in tracking cell cycle progression in CD34+ cells from CML primary patient samples co-cultured with SL/M2 cells. Time-lapse confocal imaging of these samples reveals distinct differences within the population of CML cells. In summary using this new Dual FUCCI2 lentiviral reporter makes it possible to track real time cell cycle kinetics in cancer stem cells. Future studies will include measuring LSC, cell cycle gene regulatory networks in live cells at different stages of the cell cycle. Citation Format: Gabriel Pineda, Kathleen M. Lennon, Florence Lambert-Fliszar, Gennarina L. Riso, Catriona Jamieson. Real time niche tracking of cancer stem cell cycle kinetics using a novel bi-cistronic lentiviral reporter. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A46.

Does the level of WT1 expression predict the outcome in Philadelphia-negative myeloproliferative neoplasms?

Despite the clinical importance of the leukemic transformation of chronic myeloproliferative neoplasms (MPNs), very little is known about markers that predict leukemic transformation. We studied WT1 expression in 37 MPN patients diagnosed as bcr-abl negative and JAK2 (V617F) positive with a molecular genetic test, and 23 healthy controls. WT1 expression is higher in MPN patients compared with normal controls (p=0.002). According to the WT1 expression levels, patients were divided into two groups: high (≥0.205) and low (0-0.205) WT1 expression. Two out of six patients with a high WT1 expression level transformed to myelodysplastic syndrome at a 42- and 46-month follow-up, respectively. Our results suggest that the overexpression of WT1 may play an important role in the leukemic transformation of MPNs.

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm

AbstractThe most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F+ transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F+ mice, JAK2V617F+Mpl−/− mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl+/− mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F+Tpo−/− mice, although thrombocytosis was reduced compared with JAK2V617F+ mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F+ MPNs, highlighting an entirely novel target for therapeutic intervention.

Abstract LB-159: Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter

Abstract Currently, there are few methods to visualize cell cycle progression in leukemia stem cells (LSC). Human LSC are characterized as quiescent self-renewing cells that drive leukemic transformation of myeloproliferative neoplasms and myelodysplastic syndromes. Additionally, current methods require sequential transduction and clonal selection. These methods and conditions are not feasible to study primary patient samples and LSC, which are generally limited in sample size. To alleviate this challenge, we generated a lentiviral biscistronic vector encoding FUCCI (fluorescent ubiquitination-based cell-cycle indicator) probes. Time-lapse confocal imaging of transduced 293 cells and SKNO-1 cells exhibited normal cell division and distinct nuclear staining of either green or red fluorescence. Further, analysis by flow cytometry of 293 cell confirms that S/G2/M and G1/G0 phases of cell cycle correlate to expression of fluorescent reporters expressed from bicistronic lentiviral vector and recapitulate FACS plots using traditional Ki67 and 7AAD staining. The development of this new reagent offers a new and more effective method to track cell cycle progression in limited mammalian cell populations. Future studies will utilize primary patient CD34+CD38- selected leukemic stem cell populations. Citation Format: Gabriel Pineda, Florence Lambert-Fliszar, Gennarina L. Riso, Kathleen M. Kane, Catriona Jamieson. Characterization of cell-cycle progression using a novel bi-cistronic lentiviral FUCCI reporter. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-159. doi:10.1158/1538-7445.AM2014-LB-159

Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms

Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype.

Focus on the epigenome in the myeloproliferative neoplasms

The discovery of mutations activating JAK-STAT signaling in the majority of patients with myeloproliferative neoplasms (MPNs) led to identification of tyrosine kinase activation as a predominant mechanism driving MPN pathogenesis. Despite this, the existence of additional genetic events that modify the MPN phenotype, predate JAK2 mutations, and/or contribute to leukemic transformation of MPNs has been suggested. Recently, mutations in several epigenetic modifiers have been described in patients with MPNs, including mutations in ASXL1, DNMT3A, EZH2, IDH1, IDH2, and TET2. Moreover, the mutant JAK2 itself has been shown recently to affect histone posttranslational modifications directly. Here we review the biological and clinical implications of epigenetic alterations in the pathogenesis of MPNs.

Guest editorial: Genetic and epigenetic alterations in hematopoietic malignancies

It is now recognized that multiple gene alterations are required for the development of leukemia, as is the case with solid tumors. About a decade ago, these gene alterations were classified into two groups, called class I and class II mutations [1]. Class I mutations include activating mutations of tyrosine kinase receptors such as FLT-3 and c-Kit, tyrosine kinases such as JAK2, oncogenes such as Ras as well as inactivating mutations of tumor suppressors such as p53 and NF-1. Class I mutations induce cell proliferation or block apoptosis. Class II mutations, which include inactivating mutations of transcription factors such as AML1/Runx1 and chromosome modifying enzymes such as MLL, block hemopoietic differentiation. Class II mutations are often caused by chromosomal translocations resulting in fusion proteins such as AML1-ETO, PMLRARa and a variety of MLL-fusions. Combinations of class I and II mutations but not either class I or class II mutations alone induced acute leukemia in mouse models. In addition, both class I and class II mutations frequently coexist in patients’ leukemic cells. Thus, it was assumed that combinations of class I and class II mutations are required for leukemogenesis. On the other hand, class I mutations alone seem to induce myeloproliferative neoplasm (MPN) while class II mutations are associated with myelodysplastic syndromes (MDS), and additional mutations would lead to leukemic transformation of MPN and MDS (Fig. 1). However, recently developed high-speed sequencing and SNP arrays have revealed a variety of novel mutations in hematologic malignancies. Those include mutations of epigenetic regulators, signaling adaptors and proteins involved in splicing [2]. Among these mutations, those in adaptors such as c-Cbl and Lnk may be categorized into class I mutations because mutations of these adaptors with negative signaling roles would lead to autonomous signaling of cytokine receptors. In addition, one of epigenetic regulators mutations, MLLfusions, was categorized with class II mutations. Thus, the ‘‘class I and class II story’’ need to be modified. In this series of Progress in Hematology, four articles review genetic and epigenetic alterations in hematological malignancies. Drs. Tomoki Naoe and Hitoshi Kiyoi briefly summarize the class I and class II story, and then offer an overview of newly identified mutations of epigenetic regulators. They attempt to integrate these mutations, which they call class III, into the class I and class II story. They also discuss driver and passenger mutations as well as background mutations randomly acquired in hematopoietic cells with age and stress [3]. When we think about epigenetic aspects of diseases, MDS is the most prominent because DNA hypomethylating agents are found to be effective in a substantial numbers of patients with MDS. Drs. Jumpei Yamazaki and Jean-Pierre Issa review the basics of epigenetics in cancer and MDS as well as the therapeutic use of DNA hypomethylating agents in MDS and AML. This review update the history as well as the current state of MDS therapy, including a combination therapy of DNA hypomethylating agents and histone deacetylating enzyme (HDAC) inhibitors. Concerning MPN, a class I mutation alone such as BcrAbl and JAK2V617F (activating mutant of JAK2) induces MPN-like diseases in mouse models, and most patients with MPN harbor either Bcr-Abl or JAK2-V617F. Therefore, it was assumed that these mutations are the sole mutations related to MPN and were able to induce MPN by T. Kitamura (&) Division of Cellular Therapy/Division of Stem Cell Signaling, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan e-mail: kitamura@ims.u-tokyo.ac.jp