ALBERT

Description: Reversible acetylation of lysine residues on histone tails is associated with changes to chromatin structure and plays a key role in regulation of gene expression. In this process, histone hypoacetylation is generally associated with gene silencing and pharmacological inhibition of histone deacetylases (HDACs) leads usually to activation of gene expression. Decreased histone acetylation is a hallmark of cancer cells and increased HDAC expression or their mistargetting to specific gene promoters has been associated with a variety of tumors. In the past we have identified and cloned class IIa HDAC9. The HDAC9 gene is located in chromosome 7p21, which is frequently amplified in B-cell tumours such as mantle cell lymphoma (MCL) and in B-cell non-Hodgkin’s lymphoma cell lines. Consistently, initial analysis of patient samples and/or publicly available microarray data highlighted high levels of HDAC9 expression in chronic lymphocytic leukemia, folicullar lymphoma and MCL. Within the normal lymphoid system, HDAC9 is co-expressed with BCL-6 in germinal center B-cells (∼60% of cells). HDAC9 is also expressed in marginal zone B cells and a fraction of CD38 or CD27 positive subepithelial tonsilar cells. In order to examine the role of HDAC9 in the lymphoid development and pathogenesis of lymphoid malignancies we used Ig heavy chain enhancer (Eμ), which drives gene expression from early stages of B-cell development, to ectopically express HDAC9 in transgenic mice. Hemizygous and homozygous mice expressing Flag epitope tagged human HDAC9 (fHDAC9) transgene display throughout their lifespan altered B-cell development. Immunophenotypic analysis of B-cells isolated from bone marrow (BM) revealed an absence of cells expressing the pre-B/immature-B cell markers normally associated with C-E Hardy’s fractions. In vitro functional clonogenic assays for IL-7 responsive BM-derived B-cell progenitors demonstrated an increase (∼50%) in colony numbers in the transgenic BM. Moreover, morphologic and flow cytometric analyses of the transgenic colonies, but not those derived from normal BM, revealed the presence of granulocyte/macrophage colony forming units expressing the HDAC9 transgene, suggesting a lympho-myeloid lineage switch. This correlates with the finding that extramedullary myelopoiesis occurs in a fraction of mice presenting splenomegaly (44%). Furthermore, a subgroup of homozygous Eμ-fHDAC9 mice (n=16) developed tumours (81%) at middle age, and present with enlarged lymph nodes (6%) and abnormal hematopoietic elements in peripheral blood and BM. Taken together these data suggest that HDAC9 plays a role in B-cell maturation and its ectopic expression in early B-cells leads to perturbation of normal B-cell development, possibly predisposing transgenic mice to tumorigenesis.

Description: Research Objectives: The Centers for Medicare and Medicaid Services (CMS) uses hospital readmissions as an indicator of quality of care during the index hospitalization. Patients with hematologic malignancies are at high risk of abnormal blood glucose levels, in particular hyperglycemia. Hyperglycemia during hematopoietic stem cell transplantation (HCT) is associated with adverse outcomes such as infection, delayed engraftment, and prolonged length of stay. Little is known about the association between pre-stem cell infusion glucose patterns as a prognostic indicator of patient outcomes. Methods: This single institution retrospective study using electronic health records (EHR) investigated the association of mean glucose levels prior to stem cell infusion with length of stay (LOS), 30 and 90 day readmissions, and time to engraftment in 739 patients undergoing bone marrow transplant between January 1, 2009 and December 31, 2013. Data was sorted and reported with the assistance of our BMT data management team and our institution's Data Warehouse who procured information from EPIC, EPIC's predecessor, and hospital-linked databases. Data were analyzed using descriptive statistics, Kruskal-Wallis, and Chi-square analyses. Results: Mean age was 57+ 14. The majority of population was male (55%) and was racially and ethnically mixed (White 38%, African American 23%, Hispanic 6%, Asian 7%, Other 21%). Twenty two percent of patients had a diagnosis of diabetes (ICD9 250.X). A review of mean glucose levels prior to stem cell infusion in 619 evaluable patients identified five unique patterns (see Figure), characterized by glucose levels 7 days prior to infusion (normal/moderate/high) and trend over time (stable/increasing): normal/stable (n=445), high/stable (n=16), moderate/increasing (n=75), high/ increasing (n=28), and moderate/stable (n=55). Kruskal-Wallis and Chi-square analyses showed that a normal/ stable pattern of daily mean blood glucose prior to stem cell infusion was associated with significantly lower LOS (p

Description: Self-renewal is a fundamental property essential for both normal and malignant hematopoietic stem cells (HSCs). While we and others have previously shown that activation of Hoxa9 or β-catenin enhances HSC self-renewal, its inactivation has modest impacts on adult HSCs and their malignant counterparts (Cobas et al., 2004; Lawrence et al., 2005; Siriboonpiputtana et al., 2017; Smith et al., 2011; So et al., 2004; Zhao et al., 2007), suggesting the presence of complementary pathways capable of mediating self-renewal in the absence of either protein. However, simultaneous inactivation of other key Hox genes involved in hematopoietic self-renewal including Hoxb3/b4 did not yield additional hematopoietic phenotypes in the Hoxa9 knockout (KO) background (Magnusson et al., 2007). Similarly, suppression of γ-catenin in β-catenin KO mice did not result in additional hematopoietic defects, and exhibited largely normal hematopoietic functions (Koch et al., 2008). Therefore, the alternative pathways that support hematopoietic self-renewal upon inactivation of Hoxa9 or β-catenin remain largely unknown. Our recent studies examining the regulation of posterior Hoxa loci reveals a key function of long non-coding RNA, HOTTIP in protecting Hoxa9 gene expression and uncovers a co-regulation of canonical Wnt signalling pathways by HOTTIP in HSCs, providing a molecular link between these two previously unrelated pathways. This finding is also consistent with our recent report demonstrating their functional complementation in AML stem cells, where suppression of Hoxa9 sensitizes HSC-derived AML stem cells to β-catenin inhibition and ablates their transformation ability, suggesting a novel crosstalk between β-catenin and Hoxa9in mediating hematopoietic self-renewal. To this end, the current study developed and characterized a novel β-cateninfl/fl Hoxa9-/-Rosa-CreER mouse model. In contrast to single Hoxa9 or β-catenin inactivation where a mild hematopoietic phenotype has been reported in adult HSCs, we found that double inactivation of Hoxa9 and β-catenin resulted in severe hematopoietic defects. In vitroclonogenic assays revealed that bone marrow cells harbouring combined inactivation of Hoxa9/β-catenin generated markedly reduced myeloid colony numbers (〉9-fold reduction compared to either single KO alone) of which mature CFU-G (50%) and CFU-E (50%) were the predominant composition. Colonies generated from isolated LSK (Lin-c-kit+Sca1+) populations were equivalent in quantity but devoid of multipotential progenitors (CFU-GEMM) in contrast to the diverse colony composition of control and single KO cells. Transplantation of β-cateninfl/flHoxa9-/-bone marrow to lethally irradiated recipient mice followed by β-catenin inactivation (4 days tamoxifen treatment) resulted in defects in all HSC and progenitor compartments compared to single KO alone at all time points measured (3wk, 6wk and 12 wk post tamoxifen treatment). Immunophenotypic analysis revealed that the defect originated as early as the LT-HSC stage with a drastic 7-fold reduction in LT-HSCs (Lin-c-kit+Sca1+CD150+CD48-) and 3-fold reduction in ST-HSCs (Lin-c-kit+Sca1+CD150-CD48-). Consistent with a stem cell defect, we also observed significant reductions in downstream myeloid progenitor populations including common myeloid progenitor (CMP) (2-fold), granulocyte-monocyte progenitor (GMP) (2-fold) and megakaryocyte and erythrocyte progenitors (MEP) (7-fold). Mechanistically, Hoxa9 and β-catenin co-regulate expression of Prmt1, a key epigenetic regulator with multifaceted functions in mediating RNA splicing and DNA damage response in hematopoietic cells. To further investigate the role of Prmt1 in hematopoietic development, we generated a novel Prmt1 KO model where Prmt1 can be conditionally inactivated in HSCs. Deletion of Prmt1 alone phenocopied simultaneous inactivation of β-catenin and Hoxa9, resulting in severe reductions in LT-HSC (3.5-fold), ST-HSC (4-fold) and downstream hematopoietic progenitor populations (CMP 4.5-fold, GMP 3.5-fold and MEP 4-fold). Together these data suggest that the posterior Hoxa loci and canonical Wnt pathways are developmentally regulated in a complementary manner as a safeguard mechanism to allow efficient hematopoietic self-renewal, which is largely dependent on intact Prmt1 functions. Disclosures No relevant conflicts of interest to declare.

Description: Objective Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane domain, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of ERK/STAT5 via SRC kinase, activation of PI3K/AKT, phosphorylation of FOXO3A, down-regulation of equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) that may lead to increased DNA damage and defective repair. The present study investigated if the latter can be effectively targeted for the treatment of this AML subtype. Methods Primary samples from patients with FLT3-ITD AML, human cell line carrying FLT3-ITD (MOLM-13) as well as mouse B-lymphoid Ba/F3 cells transduced with human FLT3-ITD were used in this study. Traffic Light Reporter (TLR) assay was used to measure fidelity of double-strand breaks (DSB) repair, either via error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Percentage of repair events by HR or NHEJ were quantified by flow cytometry. DNA DSB were examined by γ-H2AX foci staining using confocal microscopy. Single-cell DSB were quantified by neutral comet assay and analysed by OpenComet software. The tail moment was calculated as the length of comet tail multiplied by the tail DNA %. MOLM-13 was transplanted into NOD/SCID/IL2Rg-/- (NSG) mice by tail vein injection. Treatment comprised cytarabine (25mg/kg, i.p., day 1-5) and doxorubicin (1.5mg/kg, i.v., day 1-3), with or without olaparib (25mg/kg, i.p., day 1-5). Comparisons between groups of numerical data were evaluated using Student's t-test. P-values less than 0.05 were considered statistically significant. Results To investigate the link between FLT3-ITD AML and DNA damage response (DDR), expression of critical DDR genes in primary AML samples was examined by real-time quantitative PCR. The panel of genes included apical kinase ATM, ATR and DNA-PKcs; DNA damage mediators BRCA1,BRCA2 and PARP1; downstream response kinase CHEK1 and CHEK2 and effectors TP53. Among them, BRCA2 was significantly down-regulated in FLT3-ITD AML(Wild-type FLT3=18 samples; FLT3-ITD=13 samples; Average dCt 9.7 in WT vs. 10.6 in ITD; p

Description: Abstract 479 The identification of activating mutations in the FLT3 gene and their impact on prognosis has been crucial to the rationale behind the development of FLT3 inhibitors. While it has been shown that some leukemic cells with high tumorigenic potential exist mostly in a dormant state, it is unclear if this quiescent/non-cycling leukemia-initiating fraction carries the FLT3-ITD mutation and if it is successfully targeted by FLT3 inhibitors. As a paradigm, quiescent Ph+ stem cells in CML have been shown to exhibit resistance to bcr-abl targeted inhibitors. Additionally, results from clinical trials suggest that FLT3 inhibitors reduce the leukemic blast count in peripheral blood but are less successful in the bone marrow where factors regulating hematopoietic stem cell quiescence are active. In order to investigate the non-cycling and cycling human leukemic cell boundary, we devised a biological model that allowed us to distinguish non-cycling AML cells from cycling AML progenitors in human FLT3-ITD positive AML samples. CD34+ cells were isolated from AML samples using magnetic cell sorting, labeled with the cell membrane dye PKH26 to enable tracking of cell division, and cultured on murine stroma for 12 days. Non-cycling AML cells were then separated from cycling cells by FACS sorting and were found to retain a CD34+ primitive phenotype in contrast to expanding leukemic blasts. Fluorescence in situ hybridization analyses revealed that non-cycling cells carried leukemic gene rearrangements (trisomy 8, trisomy 13, t[3;21]and t[16;16] in our cases), and were therefore part of the original leukemic clone. PCR for the FLT3-ITD region showed that in four out of five cases, the FLT3-ITD mutation was present in the non-cycling fraction. To examine the distribution of FLT3-ITD to FLT3 wild type (WT) bearing cells, non-cycling AML cells were FACS sorted, DNA extracted and the PCR products subsequently cloned. Bacterial colonies were sequenced and colony-PCR used to determine the ratio of FLT3-ITD to WT bearing colonies for each patient. These data indicated that at least 25% of non-cycling cells (range 25%-100%) harbored the FLT3-ITD mutation. We then assessed the impact of a potent FLT3-directed inhibitor, TKI258 (Novartis), on leukemic cell expansion and the viability of non-cycling cells. TKI258 has been found to induce apoptosis of FLT3-ITD bearing cells of the human acute monocytic leukemia MV4;11 cell line. In our present study, CD34+ AML blasts from the same FLT3-ITD positive patient samples were grown in vitro in the presence of 0 μM, 0.3 μM (IC50 dose) and 1.25 μM TKI258. In stromal cultures, TKI258 significantly reduced leukemic cell expansion (range 2.13 to 20 fold for untreated cultures and 0.07 to 2.27 fold for 1.25 μM TKI258 treated cultures at day 12, p ≤ 0.05). In methylcellulose colony assays, TKI258 exposure resulted in dose-dependent suppression of colony formation of CD34+ FLT3-ITD positive leukemia cells (60% to 81% reduction in the mean plating efficiency of CD34+ AML cells at 0.3 μM TKI258). Despite this striking anti-proliferative effect, the majority of non-cycling cells from AML patients showed resistance to TKI258 (five out of six cases). In these samples, FLT3-ITD positive non-cycling cells could still be detected after treatment with the equivalent highest clinical dose (1.25 μM) of TKI258. Moreover, at a functional level, limiting-dilution experiments on non-cycling AML cells pre-treated with TKI258 showed no impairment in a modified leukemic cobblestone assay at four weeks compared to untreated non-cycling cells. These results suggest that the majority of non-cycling AML cells that harbor FLT3-ITD are unaffected by a FLT3 inhibitor and may constitute an as yet untargeted disease reservoir. Only one FLT3-ITD AML case showed exquisite sensitivity to TKI258 with elimination of the non-cycling fraction observed from 0.3 μM of TKI258 upwards. Possible explanations for this may include specific mutant receptor sensitivities or generic multi-drug resistance mechanisms operating in dormant cells. Interestingly, TKI258 selectively eradicated an ‘intermediate' dividing progenitor population in two of the insensitive cases, an indication that leukemic progenitors may be rendered sensitive to FLT3 inhibition on transiting the dormancy-cycling boundary. Further studies are needed to determine if these findings are representative of a generation of FLT3 inhibitors or specific for TKI258. Disclosures: No relevant conflicts of interest to declare.