ALBERT

Description: Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematological disorders characterized by proliferation of mature blood cells. Recently, several agents that influence epigenetic modifications, such as histone deacetylase inhibitors (HDACi), as well as JAK2 inhibitors, have been investigated for high-risk MPNs. For example, an HDACi, panobinostat has shown significant efficacy including nearly complete response in PMF (Mascarenhas et al, BJH, 2013), but molecular targets of HDACi remain largely unknown. The High Mobility Group AT-hook 2 (HMGA2) is a non-histone chromatin protein that modulates transcriptions of various genes and contributes to chromatin modification and epigenetic regulation including DNA methylation (Fusco et al, Nat Rev Cancer, 2007; Sun et al, PNAS 2013). Let-7 micro RNAs (miRNAs) negatively regulate expression of HMGA2 through 3’UTR of HMGA2 mRNA, although HMGA2 mRNA consists of both the major variant containing 3’UTR with let-7-specific sites (variant 1) and some minor variants without 3’UTR. We previously reported that overexpression of HMGA2 due to transgenic expression of Hmga2 cDNA without 3’UTR caused proliferative hematopoiesis with providing a clonal advantage to hematopoietic stem cells in mice (Ikeda et al, Blood, 2011). We also showed a deregulation of HMGA2 mRNA expression due to reduced let-7 miRNA level in granulocytes from patients with almost all of PMF and over 20% of PV and ET (Harada-Shirado et al, Blood [Abst], 2013), being associated with splenomegaly, elevated serum LDH values, and methylation of p16 promoter. Thus, we hypothesized that HMGA2 may be a candidate gene as a therapeutic target in MPNs. Since association of HDAC with HMGA2 has been reported in cord blood-derived cells (Lee et al, Cell Mol Life Sci, 2011), we here studied effects of the panobinostat on expressions of HMGA2 and let-7 in HMGA2-expressing myeloid cells including PMF-derived CD34+ cells. First, we found significantly higher HMGA2 mRNA levels in CD34+ cells from 2 PMF patients compared with CD34+ cells from 2 healthy individuals (P

Description: Graft-versus-host disease (GVHD) and thrombotic microangiopathy (TMA) are severe complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Host endothelial cells are targets of alloreactive donor cytotoxic T lymphocytes or various proinflammatory cytokines following allo-SCT, and endothelial damage plays an important role in the pathogenic mechanism(s) of TMA associated with GVHD. However, the detailed mechanism(s) of TMA as well as GVHD have not yet been fully elucidated. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family; Tyro3, Axl, and Mer. Gas6 and the TAM receptor tyrosine kinases have been reported to be associated with systemic inflammatory and autoimmune disorders, as well as hemostatic abnormalities. We hypothesized that Gas6-TAM pathway signaling contributed to endothelial dysfunction in the pathogenesis of TMA associated with GVHD. ELISA showed that the serum levels of Gas6 were markedly increased in the all-SCT patients with grades 2 or 3 GVHD at 21-35 days after stem-cell engraftment. The increased serum levels of Gas6 were also correlated with the elevated D-dimer and plasmin-α2 plasmin inhibitor complex values in the allo-SCT patients. Immunostaining showed that the expression of Gas6 and Mer was significantly upregulated in the intestinal acute GVHD lesion. The platelet aggregation test showed that UNC2250 (5 μM), a selective Mer tyrosine kinase inhibitor markedly suppressed increased platelet aggregation caused by exogenous Gas6 and collagen. Using Western blot analysis, exposure of human umbilical vein endothelial cells (ECs) to, Gas6 or 25-50% (vol/vol) serum isolated from patients with GVHD caused the downregulation of thrombomodulin (TM) and plasminogen activator inhibitor type-1 (PAI-1) as well as the upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as assessed by Western blot analyses, which were inhibited by UNC2250 or Mer siRNA. Furthermore, exogenous Gas6 or the addition of serum from patients with GVHD to ECs induced apoptosis of ECs with activation of caspase-3 and poly (ADP-ribose) polymerase, which were inhibited by UNC2250 or Mer siRNA. These results indicate that the Gas6-Mer axis may be crucial for platelet activation, endothelial damage and subsequent hypercoagulation in the pathogenesis of TMA associated with GVHD after allo-SCT. Furthermore, in the murine models of allo-SCT, we observed hepatic GVHD with hepatocellular apoptosis, necrosis and fibrosis and TMA characterized by thrombosis formation in the microvasculature of liver and kidney. The intravenous administration of 3 mg/kg UNC2250 markedly inhibited hepatic GVHD and TMA formations of liver and kidney in murine models of allo-SCT at 21 days after allo-SCT. The present study provides new evidence that the Gas6-Mer axis may be involved in the mechanism(s) underlying the progression of the pathogenesis of TMA associated with GVHD after all-SCT. We suggest that Gas6-Mer axis may be an attractive therapeutic target for treating TMA associated with GVHD caused by endothelial damage. Disclosures No relevant conflicts of interest to declare.

Description: MDS/MPN comprise a group of uncommon myeloid neoplasms with clinical and hematological features overlapping MDS and MPN. Pathogenesis remain elusive. HMGA2 on chromosome 12q plays a crucial role in self-renewal and proliferation of fetal hematopoietic stem cells (HSCs), but not adult HSCs because of repression by binding of MIRLET-7 with the 3'-untranslated region (3'UTR) of HMGA2 (Copley et al, Nat Cell Biol, 2013). Trimethylation of histone H3 at lysine 27, which is catalyzed by EZH2 - a member of polycomb recessive complex 2 (PRC2) - also suppresses HMGA2 (Sashida et al, JEM, 2016). We have demonstrated that expression of Hmga2 cDNA lacking 3'UTR caused proliferative hematopoiesis mimicking MPN in young mice and led to anemia with ineffective erythropoiesis and leukocytosis like MDS/MPN in old mice (Ikeda et al, Blood, 2011; Ueda et al, Blood Adv, 2017), suggesting that HMGA2 may contribute to the pathogenesis of MDS/MPN. In fact, we found high HMGA2 mRNA levels in granulocytes of MPN including 〉90% of patients with myelofibrosis (MF) and about 30% with essential thrombocythemia (ET), and either reduced MIRLET-7c levels or PRC2-related mutations were correlated with high HMGA2 mRNA in MF, while reduced MIRLET-7c with no particular mutation was correlated with high HMGA2 mRNA in ET (Harada-Shirado et al, BJH, 2015; Blood Adv, 2017). However, in MDS/MPN, only one case series has reported high HMGA2 mRNA due to the 12q rearrangement that removed the 3'UTR of HMGA2 in a few patients (Odero et al, Leukemia, 2005). So far, expression of HMGA2 has not been studied in MDS/MPN without the 12q rearrangement. To clarify the expression of HMGA2 in MDS/MPN in relation to clinical and other genetic findings, we investigated HMGA2 mRNA and MIRLET-7c levels by real-time RT-PCR and sought PRC2-related mutations by targeted deep sequencing in peripheral leukocytes of 11 patients with MDS/MPN [5 chronic myelomonocytic leukemia (CMML), 2 atypical chronic myeloid leukemia (aCML), and 4 unclassifiable MDS/MPN (MDS/MPN-u)]. In addition, to identify the features specific for MDS/MPN, we also evaluated 10 patients with low-risk MDS [MDS with multilineage dysplasia (MDS-MLD)]. No MDS/MPN or MDS-MLD patients had 12q rearrangement. In MDS/MPN, high HMGA2 mRNA [〉1.0 relative to HPRT1 mRNA above mean + 2SD of healthy volunteers, per our previous study (Blood Adv, 2017)] was found in each subtype: 2 of 5 (40%) in CMML, 1 of 2 (50%) in aCML, and 3 of 4 (75%) in MDS/MPN-u. Therefore, more than half of patients with MDS/MPN (6 of 11, 54.5%) showed high HMGA2 mRNA levels. In contrast, high HMGA2 mRNA was found in only 1 of 10 (10%) patients with MDS-MLD (P = 0.06). Reduction in MIRLET-7c was seen in 5 of 11 (45.5%) MDS/MPN and 3 of 10 (30.0%) MDS-MLD (P = 0.16). Among these 5 MDS/MPN patients with low MIRLET-7c, only 2 patients had high HMGA2 mRNA. Thus, MIRLET-7c reduction did not correlate with deregulation of HMGA2 mRNA in MDS/MPN as frequently as in MPN. In contrast, HMGA2 mRNA levels were high in all 3 patients with MDS/MPN carrying EZH2 mutations, suggesting that loss of EZH2 might augment HMGA2 in MDS/MPN. Laboratory findings of MDS/MPN patients with high HMGA2 mRNA were not different from those without high HMGA2 mRNA. However, MDS/MPN patients with both high HMGA2 mRNA and an EZH2 mutation showed more severe anemia compared with those with high HMGA2 mRNA without an EZH2 mutation (hemoglobin level: 9.6±2.0 vs. 11.0±2.3 g/dL, P = 0.05). In conclusion, we demonstrated that MDS/MPN cases highly express HMGA2 mRNA even without 12q rearrangement, more often than MDS-MLD. Unlike MPN, HMGA2 mRNA levels did not correlate with reduced MIRLET-7c, but probably with mutations in EZH2, which may also contribute to anemia in MDS/MPN. Disclosures No relevant conflicts of interest to declare.

Description: MPN harbors altered hematopoietic stem cell (HSC) function, resulting in skewed hematopoiesis and extramedullary hematopoiesis with splenomegaly. Mutations such as JAK2V617F and insertion/deletion of CALR exon9 have been established as phenotypic drivers of MPN. In addition, mutations in epigenetic modifiers and aberrant expressions of microRNAs play a crucial role in disease progression and clonal expansion. We have shown that almost all patients with myelofibrosis (MF) highly express HMGA2 (Harada-Shirado et al, BJH, 2015) and that transgenic mice expressing HMGA2 without 3'UTR including let7 binding sites (∆Hmga2 : H) (Ikeda et al, Blood, 2011) develop mild MPN. Moreover, we (Sashida et al, JEM, 2016) and other groups (Shimizu et al, JEM, 2016; Yang et al, Blood, 2016) showed that loss of EZH2 induces endogenous expression of HMGA2 and provokes severe MF in mice carrying JAK2V617F. Thus, we hypothesized that HMGA2 plays a central role in the disease progression of MPN. To clarify this, we generated ∆Hmga2/JAK2V617F transgenic mice (HJ), which developed severe leukocytosis, thrombocytosis, anemia, giant splenomegaly and shorter survival period, but did not progress fibrosis compared with transgenic mice carrying JAK2V617F alone (J) (Ueda et al, ASH, 2015). Compared with J, HJ also showed an increased bone marrow (BM) lineage-Sca1+Kit+(LSK) cells and growth advantage in competitive serial BM transplants. Encouraged by these findings, we further investigated the mechanism that HMGA2 exacerbates disease phenotype, and elucidated up-stream and down-stream factors of HMGA2. First, we sought cause of aggressive phenotypes. In BM cells of HJ mice, STAT3 and STAT5 were drastically upregulated in both expression and phosphorylation. Despite severer anemia, formations of EPO-independent erythroid colonies and proportions of CD71+Ter119+ erythroblasts in BM were not different between HJ and J. To explain this discrepancy, we measured spontaneous apoptosis. Erythroblasts were more susceptible to apoptosis in HJ as well as aged H, compared with J, suggesting that HMGA2 contributes to apoptosis under stressed condition. Next, we compared gene expression profiles of BM LSK cells between HJ and J by RNA sequence to seek genes altered by addition of HMGA2 expression to JAK2V617F. To clarify the role of endogenous expression of HMGA2 induced by EZH2 deletion in hematopoiesis with JAK2V617F in addition to direct effects of EZH2 deletion, we also studied RNA sequence in BM LSK cells of Ezh2-/-JAK2V617F-Tg mice (EJ). Upregulated genes were highly overlapped in between HJ and EJ. Relative to J, 200 genes including oncogenic Lmo1 were commonly upregulated, while 314 and 739 were exclusively upregulated in HJ and EJ, respectively. Thus, some of the EZH2 targets may be also targets of HMGA2. Pathway analysis revealed that chaperon-related pathways involving SCAP1 and SREBF1/2 were commonly upregulated. On the other hand, TGFB pathway was upregulated in EJ but not in HJ, and it may explain the discrepancy that addition of HMGA2 in JAK2V617F hematopoiesis did not progress fibrosis despite severe phenotype of MPN, but deletion of EZH2 exacerbated fibrosis as well as MF patients with high expression of HMGA2. To verify this finding, we measured expression of HMGA2 and its main suppressor let7, and performed target sequencing in blood samples from 16 MF patients. Because all of them highly expressed HMGA2, we also evaluated selected essential thrombocythemia (ET) patients (N=10 for HMGA2 low, 10 for HMGA2 high). While high HMGA2 expression was consistently correlated with low let-7 expression in ET, only half of MF showed low expression of let7. The other half of MF harbored mutations in components or modifiers of polycomb, including EZH2, ASXL1 and spliceosomes. These findings are compatible with the finding that loss of polycomb function upregulates HMGA2. In fact, the genetic depletion of HMGA2 or administration of HMGA2 inhibitor neuropsin diminished phenotype of EJ mice and prolonged survival. Our findings suggest importance of HMGA2 as a therapeutic target of MPN. Disclosures Komatsu: Shire: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Multiple myeloma (MM) is a lethal hematologic neoplasm characterized by proliferation of malignant plasma cells in part due to abnormal surrounding microenvironment, but the mechanism of disease progression remains largely unknown in MM. Thus, we screened molecules differentially expressed in bone marrow (BM) cells of MM patients using microarray analysis, which revealed significant upregulation of Gas6 (growth arrest-specific gene 6), a vitamin K-dependent protein with a structural homology with protein S. Since upregulation of Gas6 and its tyrosine kinase receptors have been reported to associate with various malignancies, we hypothesized that Gas6 contributed to the pathogenesis of MM. Gas6 was overexpressed in the BM of MM patients and in various MM cell lines. We found that the serum levels of Gas6 protein were markedly increased in 18 symptomatic MM patients compared to 16 healthy volunteers using human Gas6 ELISA. High levels of soluble Gas6 were detected in the supernatants of in vitro cultured MM cell lines RPMI-8226 and AMO-1 using Gas6 ELIZA. Analysis of apoptosis by flow-cytometry (FCM) with annexin V showed that exogenous Gas6 (100-200 ng/mL) inhibited the apoptosis of MM cells. MTT cell proliferation assay showed that exogenous Gas6 induced cell proliferation of MM cells with increased extracellular signal regulated kinase (ERK) phosphorylation. These results suggest that the presence of an autocrine loop of Gas6 and its receptor with ERK signaling enhanced the proliferative and anti-apoptotic effects on MM cells. Bone marrow stromal cell (BMSC) line HS-5 was found to secrete high levels of soluble Gas6 into culture supernatants using Gas6 ELIZA. Reduction of MM cell apoptosis by HS-5 cell-conditioned medium (CM) was suppressed by Gas6 neutralizing antibody (20 microg/mL) using FCM with annexin V. HS-5 cell-CM induced a marked increase in cell proliferation of MM cells, which was completely inhibited by Gas6 neutralizing antibody (0.2-20 microg/mL) using MTT cell proliferation assay. Our results showed a critical role of Gas6 in the pathology of apoptosis inhibition and cell proliferation of MM cells through paracrine mechanisms. Interleukin (IL)-6 is identified as a major growth and anti-apoptotic factor of MM cells through autocrine mechanisms. Conversely, BMSCs provide efficient support for MM cell survival by paracrine IL-6 stimulation. As determined by western blotting and real-time PCR, HS-5 cell-CM induced Gas6 upregulation, which was suppressed by IL-6 neutralizing antibody (1-10 microg/mL in a dose-dependent manner in MM cells. Meanwhile, HS-5 cell-CM induced the increased IL-6 expression, which was reversed by Gas6 neutralizing antibody in MM cells. Our findings indicate that Gas6 possessed both autocrine and paracrine functions similar to IL-6 between MM cells and BMSCs. Intercellular cell adhesion molecule-1 (ICAM-1) enhanced adhesion of MM cells to BMSCs and subsequently resulted in MM disease progression. Our immnoblotts showed that HS-5 cell-CM induced ICAM-1upregulation, and the ICAM-1 upregulation was reversed by Gas6 neutralization antibody, indicating that Gas6 stimulation increased ICAM-1 synthesis and subsequently accelerated the proliferation of MM cells. The present study offers new insights into autocrine and paracrine actions of Gas6 in concert with IL-6 between MM cells and BMSCs on apoptosis inhibition and cell proliferation of MM cells. We suggest that Gas6-related signaling pathways may be an attractive therapeutic target for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often associated with comorbidity or mortality, due to graft-versus-host disease (GVHD), opportunistic infections, and regimen-related toxicities. Recently, several studies have shown that pre-transplant red blood cell (RBC) transfusions may increase risks of such allo-HSCT-related complications, possibly due to iron overload caused by pre-transplant RBC transfusions. However, it is unknown how post-transplant RBC and platelet (PLT) transfusions influence the long-term outcome of allo-HSCT. In this study, to clarify impact of RBC and PLT transfusions at early after engraftment of allo-HSCT, we investigated the clinical and laboratory findings and long-term outcomes of 71 recipients of allo-HSCT including 48 bone marrow transplantation and 23 peripheral blood stem cell transplantation who received leukoreduced and irradiated RBC and/or PLT from days 31 to 50, after exclusion of patients who did not achieve complete chimera (〉90% donor cells) before day 30 or engraftment (neutrophil count 〉 0.5 x 109 /L) up to day 35; whose disease relapsed until day 60; and who did not survive until day 100. In these 71 patients, 33 (46.5%) patients received RBC and/or PLT transfusion, while 38 (53.5%) received no transfusion from days 31 to 50. Among groups of those patients who received RBC (RBC-transfused patients), PLT (PLT-transfused patients), and both (RBC/PLT-transfused patients) transfusions, pre-transplant findings, including age, sex, sources, serum ferritin (Ft) levels, conditioning regimens, and blood group and HLA compatibilities were not significantly different. However, even at day 100, significant decreases of both Hb concentrations and platelet counts, but not leukocyte counts, were observed in RBC-transfused patients, PLT-transfused patients, and RBC/PLT-transfused patients (Table 1), although vast majority of them no longer required transfusion after day 50. Moreover, serum Ft and C-reactive protein (CRP) levels were higher at day 100 in RBC-, PLT-, and RBC/PLT-transfused patients versus other patients (Table 1). Risk of clinically significant chronic GVHD (cGVHD) by transfusions of both RBC and PLT from days 31 to 50 did not reach statistical significance [hazard risk (HR) = 2.75, p = 0.06], but cumulative incidence of cGVHD was significantly greater in RBC/PLT-transfused patients compared with other patients (p = 0.03). Risk of cGVHD was also correlated with elevated CRP levels (HR = 3.28, p = 0.02) rather than serum Ft levels (HR = 1.40, p = 0.6) at day 100 in univariable analysis, although multivariable analysis did not show significant differences in CRP levels (p=0.07). Moreover, non-relapse mortality was greater in patients who received RBC transfusion compared other patients (p = 0.02), although overall survival (p=0.1) and cumulative incidence of relapse (p=0.4) were not different. In conclusion, both RBC and PLT transfusions early after engraftment of allo-HSCT may be correlated with following elevations of CRP and serum Ft levels and contribute to adverse long-term outcomes of allo-HSCT with an increase of cGVHD. Disclosures No relevant conflicts of interest to declare.