Mouse Bone Marrow Progenitor Cells Research Articles

The effects of age, origin, and biological sex on rodent mast cell (BMMC and MC/9) and basophil (RBL-2H3) phenotype and function

Mast cells initiate allergic inflammatory immune responses and play a role in disease by releasing various inflammatory and immunomodulatory mediators. Several mast cell-lines and primary cultured cells have been used as mast cell models with inconsistent results among research groups. Bone marrow-derived mast cells (BMMC) cultured from mouse bone marrow progenitor cells are often used as a representative model of mucosal mast cell behaviour, however their reported phenotype is variable due to inconsistent culture protocols. RBL-2H3 is a rat basophilic histamine-releasing cell line that has some characteristics of both mast cells and basophils but is not a true representation of either cell type. The murine mast cell line MC/9 is an IL-3-dependent mucosal mast cell model but has limited mast cell characteristics. In this study, we have compared the response of BMMC (derived from C57BL/6 male or female mice), two sources of RBL-2H3 (purchased directly from ATCC and a lab curated culture), and MC/9 (ATCC) at several critical stages to some common stimuli (IgE/Ag, A23187) and analyzed mast cell morphology, expression level of common mast cell surface markers (CD117 and FcεRI), protease expression, and function (growth kinetics, viability, ROS production, degranulation, cytokine release and FcεRI signaling). The objective of this study was to provide insight into the effects of culture conditions, biological sex, and age of the cells on variability among reported phenotypes and, to determine optimal conditions for activation of these cells. Our data show that factors that are often overlooked such as source, age and biological sex of mast cells play an integral role in phenotypic outcomes and may account for the reported variability in their function.

Age and sex influence cultured rodent mast cell and basophil responses to antigen

Abstract Mast cells initiate allergic inflammatory immune responses and play a role in disease by releasing various inflammatory and immunomodulatory mediators. Several mast cell-lines and primary cultured cells have been used as mast cell models with inconsistent results among research groups. Bone marrow-derived mast cells (BMMC) cultured from mouse bone marrow progenitor cells are often used as a representative model of mucosal mast cell behaviour, however their reported phenotype is variable due to inconsistent culture protocols. RBL-2H3 is a rat basophilic histamine-releasing cell line that has some characteristics of both mast cells and basophils but is not a true representation of either cell type. The murine mast cell line MC/9 is an IL-3-dependent mucosal mast cell model but has limited mast cell characteristics. In this study, we have compared the response of BMMC (derived from C57BL/6 male or female mice), two sources of RBL-2H3 (purchased directly from ATCC and a lab curated culture), and MC/9 (ATCC) at several critical stages to some common stimuli (IgE/Ag, A23187) and analyzed mast cell morphology, expression level of common mast cell surface markers (CD117 and FcɛRI), protease expression, and function (growth kinetics, viability, ROS production, degranulation, cytokine release and FcɛRI signaling). The objective of this study was to provide insight into the effects of culture conditions, biological sex, and age of the cells on variability among reported phenotypes and, to determine optimal conditions for activation of these cells. Factors that are often overlooked such as source, age and biological sex of mast cells play an integral role in phenotypic outcomes and may account for the reported variability in their function. National Research Council Canada

Trained immunity induction by the inactivated mucosal vaccine MV130 protects against experimental viral respiratory infections.

SummaryMV130 is an inactivated polybacterial mucosal vaccine that confers protection to patients against recurrent respiratory infections, including those of viral etiology. However, its mechanism of action remains poorly understood. Here, we find that intranasal prophylaxis with MV130 modulates the lung immune landscape and provides long-term heterologous protection against viral respiratory infections in mice. Intranasal administration of MV130 provides protection against systemic candidiasis in wild-type and Rag1-deficient mice lacking functional lymphocytes, indicative of innate immune-mediated protection. Moreover, pharmacological inhibition of trained immunity with metformin abrogates the protection conferred by MV130 against influenza A virus respiratory infection. MV130 induces reprogramming of both mouse bone marrow progenitor cells and in vitro human monocytes, promoting an enhanced cytokine production that relies on a metabolic shift. Our results unveil that the mucosal administration of a fully inactivated bacterial vaccine provides protection against viral infections by a mechanism associated with the induction of trained immunity.

Engineered red blood cells carrying PCSK9 inhibitors persistently lower LDL and prevent obesity.

Low plasma levels of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) are associated with decreased low-density lipoprotein (LDL) cholesterol and a reduced risk of cardiovascular disease. PCSK9 binds to the epidermal growth factor-like repeat A (EGFA) domain of LDL receptors (LDLR), very low-density lipoprotein receptors (VLDLR), apolipoprotein E receptor 2 (ApoER2), and lipoprotein receptor–related protein 1 (LRP1) and accelerates their degradation, thus acting as a key regulator of lipid metabolism. Antibody and RNAi—based PCSK9 inhibitor treatments lower cholesterol and prevent cardiovascular incidents in patients, but their high-cost hampers market penetration. We sought to develop a safe, long-term and one-time solution to treat hyperlipidemia. We created a cDNA encoding a chimeric protein in which the extracellular N- terminus of red blood cells (RBCs) specific glycophorin A was fused to the LDLR EGFA domain and introduced this gene into mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). Following transplantation into irradiated mice, the animals produced RBCs with the EGFA domain (EGFA-GPA RBCs) displayed on their surface. These animals showed significantly reduced plasma PCSK9 (66.5% decrease) and reduced LDL levels (40% decrease) for as long as 12 months post-transplantation. Furthermore, the EGFA- GPA mice remained lean for life and maintained normal body weight under a high-fat diet. Hematopoietic stem cell gene therapy can generate red blood cells expressing an EGFA—glycophorin A chimeric protein as a practical and long-term strategy for treating chronic hyperlipidemia and obesity.

The IL-33 Receptor/ST2 acts as a positive regulator of functional mouse bone marrow hematopoietic stem and progenitor cells

There is a paucity of information on a potential role for the IL-33 receptor/ST2 in the regulation of mouse bone marrow (BM) hematopoietic stem (HSC) and progenitor (HPC) cells. Comparing the BM of st2−/− and wild type (WT) control mice using functional assays, it was found that st2−/− BM cells had poorer engrafting capacity than WT BM in a competitive repopulating assay using congenic mice, with no changes in reconstitution of B-, T- and myeloid cells following transplantation. The BM of st2−/− mice also had fewer granulocyte-macrophage, erythroid, and multipotential progenitors than that of WT BM and these st2−/− HPC were in a slow cycling state compared to that of the rapidly cycling HPC of the WT mice. While functional assessment of HSC and HPC demonstrated that ST2 has a positive influence on regulation of HSC, we could not pick up differences in st2−/− compared to WT BM using only phenotypic analysis of HSC and HPC populations prior to transplantation, again demonstrating that phenotypic analysis of HSC and HPC do not always recapitulate the functional assessments of these immature hematopoietic cells.

Dnmt3a loss and Idh2 neomorphic mutations mutually potentiate malignant hematopoiesis

AbstractMutations in the epigenetic regulators DNMT3A and IDH1/2 co-occur in patients with acute myeloid leukemia and lymphoma. In this study, these 2 epigenetic mutations cooperated to induce leukemia. Leukemia-initiating cells from Dnmt3a−/− mice that express an IDH2 neomorphic mutant have a megakaryocyte-erythroid progenitor–like immunophenotype, activate a stem-cell–like gene signature, and repress differentiated progenitor genes. We observed an epigenomic dysregulation with the gain of repressive H3K9 trimethylation and loss of H3K9 acetylation in diseased mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). HDAC inhibitors rapidly reversed the H3K9 methylation/acetylation imbalance in diseased mouse HSPCs while reducing the leukemia burden. In addition, using targeted metabolomic profiling for the first time in mouse leukemia models, we also showed that prostaglandin E2 is overproduced in double-mutant HSPCs, rendering them sensitive to prostaglandin synthesis inhibition. These data revealed that Dnmt3a and Idh2 mutations are synergistic events in leukemogenesis and that HSPCs carrying both mutations are sensitive to induced differentiation by the inhibition of both prostaglandin synthesis and HDAC, which may reveal new therapeutic opportunities for patients carrying IDH1/2 mutations.

TET1 Modulates DNA Replication in Leukemia Cells Via a Catalytic-Independent Mechanism through Cooperating with KAT8

TET1 was first identified as a fusion partner of the histone H3 Lys4 (H3K4) methyltransferase MLL (mixed-lineage leukemia) in acute myeloid leukemia (AML), and then was discovered as the founding member of the Ten-Eleven-Translocation (TET) family of DNA hydroxylases which are capable of converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Our group has previously demonstrated that TET1 plays an oncogenic role in MLL-rearranged AML (Huang H, et al. PNAS 2013; 110(29):11994-9) and also other TET1-overexpressing AMLs (e.g., t(8;21) AML and AMLs carrying FLT3-ITD and/or NPM1 mutations) (Jiang X, et al. Nature Communications. 2017; 8(1):2099). The expression of the TET1 protein and the global level of its enzymatic product, 5hmC, are significantly up-regulated in MLL-rearranged leukemia, whereas the opposite has been reported in other cancers where TET1 functions as a tumor suppressor. Therefore, a comprehensive identification of all critical targets of TET1 in AML is important for us to better understand the role and underlying molecular mechanism of TET1 in AML. To this end, we cultured murine inducible MLL-ENL cells and performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling in parallel with RNA-seq to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. Gene ontology (GO) analysis of target genes indicated enrichment in genes associated with DNA replication (FDR<0.001) and cell cycle progression (FDR<0.001). Interestingly, the six main minichromosome maintenance genes, including MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, showed marked downregulation when TET1 expression was depleted. We further showed that not only the total levels of the MCM2-7 proteins, but also their binding to chromatin, were decreased upon TET1 knockdown in human AML cell lines. Consistent with the roles of MCMs in cell cycle regulation, knockdown of TET1 led to a significant decrease in the S phase population, attributing to the inhibition on DNA replication, as shown by reduced 5-ethynyl-2'-deoxyuridine (EdU) incorporation into newly synthesized DNA upon TET1 knockdown. Furthermore, DNA combing assays suggest that TET1 knockdown inhibits new origin firing but does not influence replication fork speed. Chromatin immunoprecipitation (ChIP) assays demonstrated that TET1 binds directly to the CpG islands in the promoters of the MCM genes, suggesting that the regulation of these genes by TET1 may occur at the transcriptional level. However, 5hmC sequencing revealed low abundance of 5hmC around these genomic regions, and more importantly, the abundance was not influenced by TET1 knockdown. In addition, catalytically inactive TET1 showed similar effects to wild-type TET1 on promoting cell cycle progression and DNA replication, suggesting that TET1 regulates MCM genes and cell cycle in a manner independent of its catalytic activity. Interestingly, we found that KAT8, an acetyltransferase that specifically catalyzes histone H4 lysine 16 acetylation (H4K16Ac) and was reported to bind to TET1, plays a role in the regulation of TET1 on transcription of MCM genes. Direct binding of KAT8 with TET1 was confirmed in AML cells. Depletion of TET1 reduced H4K16Ac abundance in the MCM promoters where TET1 bind, suggesting that the binding of KAT8 to such genomic regions is owing to its recruitment by TET1. Similar to TET1 knockdown, KAT8 knockdown also decreased MCM genes expression at both RNA and protein levels and resulted in defects in cell cycle progression and DNA replication. Based on the above data, we speculated that combined inhibition of TET1 and KAT8 could exert potent inhibitory effect on DNA replication in TET1-overexpressing AML cells. Indeed, Tet1 knockout sensitized MLL-AF9-transformed mouse bone marrow (BM) progenitor cells to MG149, a KAT8 inhibitor. In addition, TET1 inhibitor (U514321) synergized with MG149 in inhibiting the viability and growth of human AML cells. Furthermore, co-treatment of MG149 and U514321 exhibited synergistic effect on inhibiting colony-forming ability of MLL-AF9-transformed mouse BM progenitors. Collectively, our findings reveal a catalytic-independent novel function of TET1 on regulating DNA replication in AML cells through cooperating with KAT8 and highlight the therapeutic implication of targeting both TET1 and KAT8 in treating TET1-overexpressing AMLs. Disclosures Chen: Genovel Biotech Corp: Other: scientific founder and Chairman.

Deciphering liver environmental signaling pathways for Kupffer cell identity

Abstract Functional specialization of tissue resident macrophages occurs through environmental signals controlling activity and/or expression of transcription factors. Kupffer cells are resident macrophages in the hepatic sinusoids and have critical roles in the innate immune response and iron metabolism. Here, we characterize transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. Nr1h3 encoding LXRα is rapidly and highly induced in repopulating liver macrophages, suggesting its induction plays a crucial role in Kupffer cell differentiation. Restricted deletion of Nr1h3 in Kupffer cells reveal that it is required for shaping the Kupffer cell-specific enhancer landscape. Further, we obtain evidence that combinatorial interactions of DLL4 and TGF-β/BMP produced by sinusoidal endothelial cells and endogenous LXR ligands are required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of RBPJ through Notch signaling plays a key role in activating poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogram the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Using molecules which mimic these liver environment signals, we show that it is possible to induce Kupffer cell-specific genes in mouse bone marrow progenitor cells and human monocytes in vitro. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Modulation of TET2 Activity By Ascorbic Acid and Factors Affecting Lysine Acetylation

Loss-of-function TET2 mutations (TET2mt) strongly associate with myeloid neoplasia. TET2 is an α-ketoglutarate (αKG) and Fe2+-dependent DNA-dioxygenase that hydroxylates 5-methylcytosine-DNA (5mC), producing 5-hydroxymethylcytosine-DNA (5hmC). Ascorbic acid (AA) activates TET2 and other dioxygenases via several direct and indirect mechanisms, including recharging Fe3+ to Fe2+. Recent studies, chiefly in murine models, indicated that increased AA availability may alleviate the decreased of dioxygenase activity in cells affected by hypomorphic mutations via up-modulation of the remaining functional activity, particularly in heterozygous TET2mt, but it may also act via increased compensatory activity of other TET enzymes. Beyond these observations, the mechanisms of AA action in TET2mt bone marrow cells are not well understood. Yet to be identified are factors that modulate the effects of AA.We performed in silico binding prediction validated by surface plasmon resonance assay coupled with confirmatory in vitro and in vivo assays. In cell free system we demonstrated the AA increases the production of 5mC-DNA oxidation products (TDOP, 5hmC/5fmC/5caC) in a mixture of recombinant TET2wt:TET2S1898F 1:1 mimicking heterozygous TET2mt neoplasia. Using cell-free system with recombinant TET2 protein and the aforementioned biophysicochemical analyses, we further demonstrated that AA binds to the TET2 catalytic domain adjacent to Fe2+/aKG binding site and recycle Fe2+ back into the catalysis.In vitro cultures demonstrated that AA increases TDOP and slows proliferation in Tet2+/- and Tet2-/- mouse bone marrow progenitor cells. A similar but less pronounced effect was also observed in shRNA TET2KD MOLM13 and K562 cells in which AA restored the original proliferation rate of these cells which was increased by TET2KD. Finally, using cultures of healthy human marrows (retrovirally transduced with TET2 or scrambled shRNA), we showed that AA decreased the proliferation rate of these cells similar to ectopic overexpression of TET2. When primary MDS bone marrow samples with various TET2mt (n=4) were studied, the effects of AA were less consistent. Consistent with the observation, in SIG-M5 cell line characterized by natural TET2 mutations, AA only modestly increased 5hmC 2-fold. While, in high TET2 expression CMK cell line, AA increased 5hmC 6-fold. In vivo, high-dose oral AA increased TDOP, decreased spleen size and myeloproliferation, and prolonged survival in sublethally-irradiated Tet2+/-Gulo-/- and Tet2+/- mice. However the AA treatment has only a modest effect on the overall survival of these mice.Recent reports indicated that TET2 activity is down regulated by HDAC1/2 by deacetylation at the N-terminus (Zhang et al., Molecular Cell 65, 323-335, 2017) and up regulated by Sirtuin 1 (Class III HDAC) via lysine deacetylation in the catalytic domain (Sun et al., abstract, Blood 2016 128:1053). In search of factors that modulate the effects of TET, experiments were performed to determine if acetylation also modulates the activity of TET in leukemia cells. Inhibition of class I and II but not class III HDACs by trichostatin A (TSA) increased the 5hmC level 1.5-fold. However, when combined with AA, TSA amplifies the 5hmC level nearly 4-fold. Acetylation of lysine in the catalytic site inactivates TET via structural perturbations. Therefore, activation of class III HDACs should activate TET2 and inhibition should inhibit it. Indeed, consistent with this presumption, in leukemia cells, we observed that activation of sirtuin, a class III HDAC, by small molecule SRT1720, significantly increased 5hmC while the inhibitors 3-TYP and sirtnol reduced 5hmC. Context-dependent TET2 acetylation/deacetylation significantly affects its AA-mediated activation, and thus MDS/MPN clones with dysregulation of histone acetyltransferases and/or histone deacetylases may be less responsive to AA alone.In sum, we demonstrate the mechanism and context of AA-mediated TET activation which may guide novel treatment strategies for TET2-deficient neoplasia. Our results show that TET2 activity can be modulated by various mechanisms including acetylation/deacetylation interactions with the effects of AA. DisclosuresNazha:MEI: Consultancy. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Effects of Low-to-Moderate Doses of Gamma Radiation on Mouse Hematopoietic System.

In this study, we investigated the effects of low-to-moderate doses of radiation in mice, given our limited understanding of the health risks associated with these exposures. Here, we demonstrate the different responses of the CD2F1 mouse hematopoietic system to low-to-moderate (0.5, 1, 3 or 5 Gy) doses of gamma radiation. After 3 and 5 Gy of 60Co total-body irradiation (TBI), mouse blood cell counts were decreased and maintained below baseline up to 28-42 days. In contrast, after 0.5 Gy TBI, lymphocyte and monocyte counts increased, and peaked from day 3 to day 14. Radiation doses at 0.5 and 1 Gy did not cause cell death or T-cell subpopulation changes in spleen and thymus, whereas the clonogenicity of mouse bone marrow (BM) progenitor cells was significantly suppressed on the first day after 0.5-5 Gy TBI, and these low levels were maintained up to 42 days. Although a transient recovery in total colony forming units (CFUs) was shown in mouse BM at days 14 and 21 after 0.5 Gy TBI, the early-stage multipotential progenitor colonies (CFU-GEMM) remained at a significantly low level compared to those of the sham-irradiated (0 Gy) controls. Consistently, the level of stem cell factor (SCF) in BM cells was decreased after low-to-moderate TBI. Serum from individual mice was collected after irradiation and 23 cytokines/chemokines were measured; massive releases of cytokines and chemokines were observed at day 3 postirradiation in a dose-dependent manner. When human hematopoietic CD34+ cells were cultured with the serum collected from mice irradiated at different doses, a significant decrease of CFU-GEMM colonies in the CD34+ cells was observed. Our data suggest that low-to-moderate doses of radiation induced cellular responses that are cell type-dependent. The early stage multipotential progenitor cells in mouse BM were the most sensitive cells even to low-dose irradiation compared to spleen and thymic cells, and 0.5 Gy TBI induced hematopoietic cell injury from day 1 to the end of our experiment, day 42 postirradiation. Radiation-induced decrease of SCF in mouse BM and increase in circulating pro-inflammatory factors may be responsible for the enhanced sensitivity of hematopoietic progenitor cells to radiation.

Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells.

Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The principles underlying the techniques for evaluating toxicity to BM progenitors (erythroid, myeloid, and lymphoid) exploit changes in the normal hematopoietic process, biochemical cell surface and intracellular markers, as well as components of the BM microenvironment. Toxicological investigations following in vivo exposures of mice or in vitro exposures of mouse primary BM cell cultures allow the assessment of the developmental and functional integrity of BM cells, cell population shifts, and adverse biochemical effects due to toxicity. Colony forming unit (CFU) assays and flow cytometry are indispensable techniques in these toxicity studies.

Preclinical Activity of Splicing Modulators in U2AF1 Mutant MDS/AML

Mutations in spliceosome genes are detectable in ~50% of patients with myelodysplastic syndromes (MDS), making this cellular pathway the most commonly mutated in MDS and providing a novel target for therapeutic intervention. Spliceosome gene mutations are mutually exclusive, implying they are either redundant in pathogenic function or are not tolerated in a cell when they co-occur. Our group and others identified recurrent heterozygous missense mutations in the splicing factor gene U2AF1 in 11% of MDS patient samples. The most common U2AF1 mutation results in a conversion of serine to phenylalanine at position 34 (S34F) of the U2AF1 protein. We previously reported that expression of mutant U2AF1(S34F) in vivo using doxycycline-inducible U2AF1(S34F) transgenic mice revealed an expansion of hematopoietic bone marrow progenitor cells and leukopenia following transgene induction; both phenotypes are seen in patients with MDS. We also identified mutant U2AF1-specific alterations in pre-mRNA splicing in transgenic mouse bone marrow progenitor cells, primary AML patient samples, and CD34+ cells by RNA sequencing. We hypothesize that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs, providing a new treatment approach for patients with U2AF1 mutations. For our studies, we utilize sudemycins, which are compounds that bind the SF3B1 spliceosome protein and modulate pre-mRNA splicing in non-hematopoietic tissues. We examined the effects of sudemycin treatment on pre-mRNA splicing in primary hematopoietic cells by treating CD34+ cells isolated from human umbilical cord blood with sudemycin in vitro. We performed whole transcriptome (RNA-seq) analysis following 6 hours of sudemycin treatment (1µM) of CD34+ cells and identified robustly altered pre-mRNA splicing patterns that are sudemycin-specific (26,120 splice junctions by DEXSeq, FDR<0.05, n=3), thereby validating that splicing is altered in hematopoietic cells treated with sudemycin. We confirmed a subset of these altered pre-mRNA splicing changes by RT-PCR and gel electrophoresis, as well as by Nanostring assay of RNA. We performed in vitro studies to examine the sensitivity of cells expressing U2AF1(S34F) to sudemycin treatment. Primary human MDS/AML cells with U2AF1(S34F) mutations display increased sensitivity to sudemycin, compared to non-mutant controls in a cell cycle (EdU incorporation) assay (n=3), while treatment with daunorubicin showed no specificity for mutant U2AF1(S34F) samples compared to non-mutant controls. Primary mouse c-Kit+ bone marrow cells transduced with a retrovirus expressing U2AF1(S34F) display a marked increase in apoptosis (by flow cytometry for Annexin V+ staining) in response to increasing concentrations of sudemycin, compared to controls (p<0.001, n=3-5). In addition, in vivo treatment of U2AF1(S34F) transgenic mice with sudemycin resulted in attenuation of hematopoietic progenitor cell expansion by colony forming unit (CFU-C) assay (p<0.01, n=6-11) and by flow cytometry for lineage-, c-Kit+, Sca-1+ (KLS) cells (p<0.001, n=6-11). Ongoing studies are examining the splicing alterations in U2AF1 mutant and wild-type transgenic mouse bone marrow cells treated with vehicle versus sudemycin. Taken together, these data suggest that we may be able to specifically treat hematological cancers with U2AF1 mutations using small molecule pre-mRNA splicing modulators such as sudemycin. DisclosuresNo relevant conflicts of interest to declare.

Identification of TET1⊣miR-22⊣CREB-MYC Signaling Reveals Potent Tumor-Suppressor Role of Mir-22 in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with diverse chromosomal and molecular abnormalities. The majority of AML patients do not survive more than 5 years. Advanced genomic studies reveal that both genetic and epigenetic abnormalities frequently occur in de novo AML. However, it remains a challenge to understand the complicated genetic/epigenetic regulatory networks and identify the functionally important nodes in these networks. There is an urgent need to develop effective therapeutic strategies based on these new insights.The ten-eleven translocation (Tet) proteins are important epigenetic regulators, which can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and lead to DNA demethylation. Among the three TET family members (TET1/2/3), TET2 was identified as a tumor suppressor in myeloid malignancies. Our lab recently reported that TET1 is highly expressed in MLL/KMT2A (Mixed Lineage Leukemia)-rearranged AML, a subtype of AML with poor prognosis. It is a direct target activated by MLL-fusions, and functions as an essential oncogene (Huang et al., PNAS, 2013). However, the function and regulatory pathway(s) of TET1 in AML remain poorly understood.MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play important roles in posttranscriptional gene regulation. Dysregulation of miRNAs is frequently observed in AML. Results of our profiling assays show that miR-22 is widely down-regulated in all major subtypes of de novo AML (Jiang et al., Cancer Cell, 2012), implying a tumor suppressor function. However, an oncogenic role for miR-22 was recently reported in myelodysplastic syndromes (MDS) and breast cancer, in which TET2 was repressed by miR-22 as its direct target gene.Here we show that, amongst a group of miRNAs (e.g. miR-495 and miR-150, etc.) whose expression levels are repressed in AML, miR-22 exhibits the most potent and consistent inhibition on MLL-AF9-induced transformation of mouse bone marrow (BM) progenitor cells. Moreover, forced expression of miR-22 dramatically inhibits cell transformation and leukemogenesis induced by multiple fusion genes, such as MLL-fusions and RUNX1/AML1-ETO9a. Furthermore, the maintenance of various subtypes of AML (e.g., those induced by MLL-fusion, AML1-ETO9a or FLT3-ITD/NPM1c+) is also dependent on the repression of miR-22. Thus, our data demonstrate a potent tumor-suppressor role of miR-22 in AML.Surprisingly, our analysis of three (in-house and outside) large-scale AML datasets revealed that TET2 (and likely also TET3) expression levels exhibited a significant positive correlation, whereas only TET1 exhibited a significant negative correlation (r<-0.32; p<0.001), with miR-22 expression. Our subsequent ChIP/qPCR studies suggest an epigenetic repression on miR-22 transcription mediated by TET1 and its repressive cofactors such as SIN3A and EZH2, through their direct binding to the miR-22 promoter region and subsequent modifications of histone markers such as H3K27Me3 which, in turn, inhibit RNA polymerase II recruitment and, thereby, miR-22 transcription. Besides the TET1-mediated epigenetic repression, the miR-22 locus (within 17p13.3) is also affected by DNA copy loss in 8-20% of AML cases, further highlighting its tumor-suppressor role in AML.Further, through a series of data analyses followed by experimental validations and functional studies, we show that a set of critical oncogenes, including CRTC1, FLT3 and MYCBP, are functionally important direct target genes of miR-22 in AML and thus, miR-22 negatively regulates the CREB and MYC signaling pathways. Our proof-of-concept study shows that miR-22 RNA oligos formulated with dendritic nanoparticles significantly inhibit leukemia progression and extend the overall median survival of MLL-AF9-induced leukemic mice from 29 days to 54 days (n=10 per group, p<0.001, log-rank test) in mouse BM transplantation assays, indicating the therapeutic potential of miR-22 in treating AML.Taken together, our results demonstrate a potent tumor-suppressor role of miR-22 in AML, and suggest the potential clinical application of miR-22-nanoparticles in treating AML. We also identified a TET1⊣miR-22⊣CREB/MYC regulatory pathway, which is critical in AML pathogenesis (see Fig. 1). Our findings also highlight potential distinct genetic/epigenetic mechanisms underlying de novo AML and MDS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selective Targeting Of Inv(16)+ AML Stem Progenitor Cells By Inhibiting HDAC8

Chromosomal inversion inv(16)(p13.1q22) which leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, occurs in over 8% of acute myeloid leukemia (AML) cases. The fusion product CBFβ-SMMHC (CM) inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. The mutations of tumor suppressor p53 occur in approximately half of all cases of human cancer, but TP53 mutations are relatively rare in inv(16) AML. We have previously shown that CM expression leads to reduced acetylation of p53 and impaired p53 target gene activation through formation of aberrant protein complex with p53 and HDAC8 (Blood, 2012,120: A772.). Here, we showed that CM interacts with p53 both in CM transformed mouse primary bone marrow cells as well as in AML stem and progenitor cells from inv(16) patients. When HDAC8 selective pharmacological inhibitor 22d directed against its catalytic sites (ChemMedChem 2012, 7:10, 1815-24;) were used to treat inv(16) mouse primary bone marrow progenitor cells and inv(16)+ CD34+ stem progenitor cells from patients, Ac-p53 levels were remarkably increased as shown by western blot. We further assessed the p53 target genes expression after HDAC8 inhibitor 22d treatment by qRT-PCR assay in inv(16)+ CD34+ stem progenitor cells (n=8), and observed variable levels of activation in p53 targets (Fold activation: p21:2.25-fold, hdm2:1.17-fold, 14-3-3σ: 3.12-fold, puma: 2.39-fold), indicating p53 was re-activated. Similar results were also shown in CM transformed mouse bone marrow progenitor cells. Importantly, we found that 22d treatment significantly inhibit the growth of inv(16)+ AML CD34+ cells (n=9) rather than normal CD34+ cells (n=7) , (AML IC50= 6.509 μM, vs Normal cells IC50=13.83 μM, p=0.0003). Meanwhile, 22d selectively induces apoptosis of inv(16)+ AML stem and progenitor cells while sparing normal HSPCs (AML LD50= 10.24 μM, vs NL LD50= 46.36 μM, p=0.001). To evaluate whether the effect of HDAC8i is mediated by p53, we knocked down p53 with a lentiviral vector expressing shRNA against p53 (or non-silencing shRNA) in AML CD34+ cells, and treated the cells with HDAC8 inhibitor 22d (5-20 µM). We showed that despite the inter-sample variability, knocking down p53 expression in all AML samples tested (n=3) led to reduced HDAC8i-induced apoptosis, suggesting that p53 contributes to the apoptosis effect induced by HDAC8i (22d) in inv(16)+ AML cells. Importantly, by taking advantage of our conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre), which develops AML under induced expression of CBFß-SMMHC (Cancer Cell, 2006, 9:1, 57-68), we were able to perform the ex vivo treatment assay by treating primary leukemic cells (marked with dTomato) with either DMSO (as vehicle control) or with HDAC8 inhibitor 22d (10μM) for 48h, followed by transplantion into congenic mice (control group n=8, treatment group n=7). We observed reduced short-term engraftment of leukemic cells that are treated with 22d (10 μM) at 4 weeks post-transplantation in the peripheral blood (Donor cell%: control group=5.99%, treatment group=0.178%, P=0.0093). Interestingly, engraftment of cord blood CD34+ cells at 16 weeks post-bone marrow transplantation was not reduced after treatment with 22d (10 μM) (human CD45+ %: control=66.2% versus treatment=63.4%, p=0.9), indicating the effect by HDAC8 inhibition is selective for leukemic cells. In conclusion, we have identified a novel mechanism whereby CBFβ-SMMHC inhibits p53 fucntion, and may further implicate inhibition of HDAC8 as a promising approach to selectively target inv(16)+ AML stem and progenitor cells. Disclosures:No relevant conflicts of interest to declare.

MiR-9 is an essential oncogenic microRNA specifically overexpressed in mixed lineage leukemia –rearranged leukemia

MicroRNAs (miRNAs), small noncoding RNAs that regulate target gene mRNAs, are known to contribute to pathogenesis of cancers. Acute myeloid leukemia (AML) is a group of heterogeneous hematopoietic malignancies with various chromosomal and/or molecular abnormalities. AML with chromosomal translocations involving the mixed lineage leukemia (MLL) gene are usually associated with poor survival. In the present study, through a large-scale, genomewide miRNA expression assay, we show that microRNA-9 (miR-9) is the most specifically up-regulated miRNA in MLL-rearranged AML compared with both normal control and non-MLL-rearranged AML. We demonstrate that miR-9 is a direct target of MLL fusion proteins and can be significantly up-regulated in expression by the latter in human and mouse hematopoietic stem/progenitor cells. Depletion of endogenous miR-9 expression by an appropriate antagomiR can significantly inhibit cell growth/viability and promote apoptosis in human MLL-rearranged AML cells, and the opposite is true when expression of miR-9 is forced. Blocking endogenous miR-9 function by anti-miRNA sponge can significantly inhibit, whereas forced expression of miR-9 can significantly promote, MLL fusion-induced immortalization/transformation of normal mouse bone marrow progenitor cells in vitro. Furthermore, forced expression of miR-9 can significantly promote MLL fusion-mediated leukemogenesis in vivo. In addition, a group of putative target genes of miR-9 exhibited a significant inverse correlation of expression with miR-9 in a series of leukemia sample sets, suggesting that they are potential targets of miR-9 in MLL-rearranged AML. Collectively, our data demonstrate that miR-9 is a critical oncomiR in MLL-rearranged AML and can serve as a potential therapeutic target to treat this dismal disease.

Dysregulation of PSTPIP2 Due to Loss of GATA-1 Contributes to Aberrant Megakaryopoiesis

AbstractAbstract 854GATA-1-deficiency causes dysregulation of multiple genes and leads to aberrant megakaryopoiesis. How the dysregulated genes may contribute to abnormal megakaryocyte development and differentiation is largely unknown. In this study, we focused on Pstpip2, a gene that was upregulated in GATA-1low and GATA-1s mutant megakaryocytes. A strong GATA-1 binding site in the intron 1 of Pstpip2 gene was previously identified by GATA-1 ChIP-Seq studies. These observations strongly suggest that Pstpip2 is a GATA-1 target gene. By using ChIP-PCR we showed that GATA-1 indeed bound to Pstpip2 intron 1 region in megakaryocytes. We also observed that Pstpip2 was expressed in myeloid cells and hematopoietic progenitors, but not lymphocytes. Noticeably, two cell lines with GATA-1 deficiencies, G1ME and CMK, which approximate the MEP and megakaryoblast stages, respectively, showed moderate expression of Pstpip2. Moreover, K562 cells displayed significant upregulation of PSTPIP2 and concomitant downregulation of GATA-1 during TPA-induced megakaryocytic differentiation. To study its function in megakaryocytic differentiation, we modulated PSTPIP2 expression by overexpression or shRNA knockdown. Ectopic expression of PSTPIP2 in K562 or CMK cells impaired TPA-induced megakaryocytic differentiation as evidenced by decreased CD41 expression. In contrast, downregulation of PSTPIP2 by shRNA promoted CD41 expression and increased polyploidy. These findings suggest that failure to suppress PSTPIP2 due to loss of GATA-1 may contribute to abnormal megakaryopoiesis.To probe the mechanism by which PSTPIP2 inhibits megakaryocytic differentiation, we tested whether the interaction with its partner PEST-PTP or phosphorylation of key tyrosine residues in the protein by Src kinase is required. We created two mutants, W232A and Y323/333/F, which block its interaction with PEST-PTP or prevent tyrosine phosphorylation, respectively. Similar to WT PSTPIP2, both mutants retained the ability to inhibit megakaryocytic differentiation suggesting that inhibition of megakaryocytic differentiation does not depend on the PEST-PTP interaction or Src kinase phosphorylation. Separately, we have noted that PSTPIP2 also interacts with Fyn, Grb2, and Shc, suggesting that PSTPIP2 may affect signal transduction. Indeed, we found that ERK phosphorylation was downregulated by PSTPIP2. Since THPO/MPL signaling via JAK/STAT is the physiological pathway in megakaryocyte maturation rather than BCR-ABL signaling seen in K562 cells, we next analyzed the effect of PSTPIP2 on MPL signaling in G1ME cells. Consistent with our findings in K562 and CMK cells, both WT and mutant PSTPIP2 inhibited CD41 expression. Interestingly, ectopic expression of WT PSTPIP2 also caused cell cycle arrest at S phase in G1ME cells. PSTPIP2 also suppressed Erk and Shc phosphorylation. Considering the important role of MAPK/ERK signaling in megakaryocyte development, we believe that PSTPIP2 acts as a negative regulator of THPO-MPL signaling to suppress TPO-stimulated MAPK/ERK activation and subsequently inhibit megakaryopoiesis. Finally, we confirmed the function of PSTPIP2 in megakaryocyte differentiation using mouse bone marrow progenitor cells. Ectopic expression of PSTPIP2 significantly downregulated CD41 expression, reduced CFU-GM and CFU-Mk numbers, and reduced polyploidy, while knockdown of PSTPIP2 exhibited the opposite effects. Taken together, we have identified a new negative regulator of THPO-MPL signaling that is inhibited by GATA-1 in normal megakaryocyte development. Upregulation of PSTPIP2 may contribute to abnormal megakaryopoiesis observed in GATA-1-deficient megakaryocytes. Disclosures:No relevant conflicts of interest to declare.

Blockade of Mir-150 Maturation by MLL-Fusion/MYC/Lin-28 Is Required for MLL-Associated Leukemia

AbstractAbstract 3499MicroRNAs (miRNAs), a class of small, non-coding RNAs, are important for posttranscriptional gene regulation in both health and disease. Expression of miRNAs is under stringent regulation at both transcriptional and post-transcriptional levels. Disturbance at either level could cause dysregulation of miRNAs. However, though altered expression of many miRNAs has been reported in various cancers, including acute myeloid leukemia (AML), their dysregulational mechanisms and pathologic functions remain less well understood.Here we report that mature miR-150 level is significantly downregulated in most AML samples, including those with rearrangements of the mixed lineage leukemia (MLL) gene. Strikingly, we found that despite the over 75% decrease of mature miR-150, its primary and precursor transcript abundance is increased to 2∼4 fold in human MLL-associated AML, relative to normal controls. Interestingly, we show that while MLL fusion proteins can bind to the promotor region of miR-150 and promote its primary transcription, they also negatively regulate the maturation process of miR-150 through the MYC/LIN28 functional axis.MiR-150 has been implicated as either an oncogene or a tumor suppressor in various types of solid tumors. However, its function in the pathogenesis of AML is unknown. Here we showed that ectopic expression of miR-150 dramatically inhibited cell growth and promoted apoptosis of human MLL-associated leukemic cells. Furthermore, using colony-forming/replating assays, we found that co-transduction of miR-150 and MLL-AF9 (a fusion gene resulting from t(9;11)) into mouse bone marrow (BM) progenitor cells, caused a significant reduction in colonies (down to 1∼10%; p<0.001, t-test) compared to transduction of MLL-AF9 alone. More importantly, we performed primary BM transplantation (BMT) assays and found that forced expression of miR-150 significantly delayed leukemogenesis mediated by MLL-AF9 (median overall survival, 110 days versus 56 days; p<0.001, log-rank test). We then performed secondary BMT and showed that miR-150+MLL-AF9 leukemic cells developed AML in secondary recipient mice remarkably slower than MLL-AF9 leukemic cells (median overall survival, 70 days vs. 42 days; p<0.001). These findings suggest that miR-150 plays a critical tumor suppressor role in preventing MLL-associated leukemogenesis. Moreover, through a series of studies, we identified Myb and Flt3 as critical direct targets of miR-150 in cell transformation and leukemogenesis.Previous studies have shown that there is an autoregulatory feedback loop between FLT3/MYB and HOXA9/MEIS1, and the latter two are critical downstream targets of MLL fusion proteins. In addition, FLT3 has been identified as an upstream regulator of MYC, while MYC is also a downstream target of MLL fusion proteins and an upstream regulator of Lin28. These previous findings together with the data we reported above suggest that there is a critical MLL-fusion/MYC/LIN28-miR-150-FLT3/MYB/HOXA9/MEIS1 regulatory circuit in MLL-associated leukemia (see Fig. 1). In this circuit, MLL fusion proteins function as the driver, and their presence leads to the significant up-regulation of all six downstream genes, MYC, LIN28, FLT3, MYB, HOXA9, and MEIS1, as well as the primary transcription of miR-150. The up-regulation of MYC/LIN28 results in the blockade of the miR-150 maturation process. This in turn leads to the release of miR-150 inhibition on FLT3 and MYB expression, which would enhance the expression of HOXA9, MEIS1, MYC, and LIN28, and further enhance/maintain the blockade of miR-150 maturation. As a result, the cells reach and maintain high levels of MYC/LIN28/FLT3/MYB/HOXA9/MEIS1, and thereby transform the cells and lead to leukemogenesis. Our further systematic studies confirmed the existence/fidelity of this regulatory circuit in MLL-associated leukemia.Taken together, we revealed a previously unappreciated regulatory circuit. Our findings may advance our understanding of the complex molecular mechanisms underlying the development and maintenance of MLL-associated leukemia, and may also provide new strategies to treat MLL-associated leukemia, a disease that is presently treatment resistant, and likely also other subtypes of AML (as miR-150 is down-regulated in all subtypes of AML), or even other types of cancer that also utilize at least part of the signaling circuit we have described herein. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Differentiation of mast cell subpopulations from mouse embryonic stem cells

Mast cells can generally be divided into two major groups, connective tissue mast cells and mucosal mast cells. We and others have previously shown that these mast cell populations can be developed in vitro from mouse bone marrow stem cells using a combination of specific growth factors and cytokines. Mast cell differentiation from mouse embryonic stem (ES) cells is an important alternative method when developing mast cells from an embryonic lethal genetic deficiency or to reduce the use and handling of experimental animals. In this study, we have used protocols prior known to induce connective tissue like mast cells (CTLMC) (SCF and IL-4) and mucosal like mast cells (MLMC) (SCF, IL-3, IL-9 and TGF-β) from mouse bone marrow progenitor cells and employed these protocols to study if phenotype specific mast cells can be developed from ES cells. We here demonstrate that mast cells of the different phenotypes, CTLMC and MLMC, can be derived from mouse ES cells. The mast cell populations were characterized by chymase expression, receptor expression and their difference in activation pattern and in activation-induced survival.

Repression of Mir-495, a Microrna Associated with Favorable Outcome of Acute Myeloid Leukemia Patients, Is Required for the MLL-Associated Leukemogenesis,

Abstract 3462Acute myeloid leukemia (AML) bearing MLL (mixed lineage leukemia) translocations are associated with poor survival, and only fewer than 50% of the patients survive longer than 5 years. Thus, an improved strategy leading to a higher cure rate is urgently needed to treat MLL-associated AML. MicroRNAs (miRNAs), a class of small non-coding RNAs, have been postulated to be important gene expression regulators in all biology including human leukemia. Through large-scale, genome-wide miRNA expression profiling assays, we determined that miR-495 is significantly down-regulated in the majority of human AML samples, particularly, in those with MLL rearrangements. More interestingly, through correlating the expression signature of miR-495 with clinical outcome of AML patients, we revealed that a low expression level of miR-495 is a predictor of poor prognosis in most AML patients. Our further qPCR assays confirmed that the expression of miR-495 is even more significantly downregulated in MLL-rearranged AML primary patient samples and cell lines. Through in vitro colony-forming/replating assays and in vivo bone marrow transplantation studies, we found that forced expression of miR-495 significantly inhibits the capacity of the MLL-AF9 fusion gene to support colony formation in mouse bone marrow progenitor cells in vitro and to induce leukemia in vivo. In leukemia cell lines, overexpression of miR-495 greatly inhibits the viability of the cells, while increasing apoptosis. Furthermore, by using 3 algorithms for miR-495 3'UTR binding sites, we identified several well-known MLL leukemia-related genes, e. g. BMI1, MEF2C, BID and MEIS1, as potential targets of miR-495. Results of qPCR revealed that forced expression of miR-495 significantly inhibits the expression levels of these genes in leukemia cell lines, mouse bone marrow progenitor cells, as well as mouse peripheral blood cells with MLL fusion genes. Therefore we hypothesize that miR-495 may function as a tumor suppressor in AML with MLL rearrangements by targeting essential tumor-related genes. Further studies will focus on: 1) effects of miR-495 on the functions of target genes studied in vitro and in vivo; 2) the epigenetic mechanisms and the signaling pathways involved in regulating the expression level of miR-495 in human leukemia. Disclosures:No relevant conflicts of interest to declare.