Search

Progressive destruction of pancreatic islet ��-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional ��-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide hormone that is produced by intestinal cells and foetal islets and can increase ��-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycaemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycaemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with the protection of, and/or higher frequencies of, insulin-producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycaemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin-producing ��-cells and ameliorating the disease progression in T1D.

Progressive destruction of pancreatic islet β-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional β-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide-hormone which is produced by intestinal cells and fetal islets and can increase β-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with protection of, and/or higher frequencies of, insulin producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin producing β-cells and ameliorating the disease progression in T1D.

Bacteroides fragilis (BF) is an integral component of the human colonic commensal microbiota. BF is also the most commonly isolated organism from clinical cases of intra-abdominal abscesses, suggesting its potential to induce proinflammatory responses upon accessing the systemic compartment. Hence, we examined the impact of mucosal and systemic exposures to BF on type 1 diabetes (T1D) incidence in NOD mice. The impact of intestinal exposure to BF under a chemically induced enhanced gut permeability condition, which permits microbial translocation, in T1D was also examined. While oral administration of heat-killed (HK) BF to prediabetic mice caused enhanced immune regulation and suppression of autoimmunity, resulting in delayed hyperglycemia, mice that received HK BF by intravenous injection showed rapid disease progression. Importantly, polysaccharide A–deficient BF failed to produce these opposing effects upon oral and systemic deliveries. Furthermore, BF-induced modulation of disease progression was observed in wild-type, but not TLR2-deficient, NOD mice. Interestingly, oral administration of BF under enhanced gut permeability conditions resulted in accelerated disease progression and rapid onset of hyperglycemia in NOD mice. Overall, these observations suggest that BF-like gut commensals can cause proinflammatory responses upon gaining access to the systemic compartment and contribute to T1D in at-risk subjects.

Bacteroides fragilis (BF), a Gram-negative bacterium, is an integral component of the human colonic commensal microbiota. BF is also the most commonly isolated organism from clinical cases of intra-abdominal abscesses suggesting its potential to induce pro-inflammatory responses, upon accessing the systemic compartment. Hence, we examined the impact of intestinal and systemic exposure to BF on type 1 diabetes (T1D) incidence in non-obese diabetic (NOD) mice. The impact of intestinal exposure to BF under chemically-induced enhanced gut permeability condition, which permits microbial translocation to systemic compartment, in T1D was also examined. While oral administration of pre-diabetic mice with heat-killed (HK) BF caused enhanced immune regulation and significant suppression of autoimmunity resulting in delayed hyperglycemia, mice that received small amounts of HK BF by i.v. injection showed pro-inflammatory immune response and rapid disease progression. Importantly, polysaccharide-A deficient (ΔPSA) BF failed to produce these opposing effects upon oral and systemic deliveries. Further, BF induced modulation of disease progression was observed in WT, but not TLR2-deficient, NOD mice. Interestingly, oral administration of BF under chemically-induced enhanced gut permeability condition resulted in accelerated disease progression and rapid onset of hyperglycemia in NOD mice. Overall, these observations suggest that BF-like gut commensals can cause pro-inflammatory responses upon gaining access to systemic compartment and trigger/accelerate autoimmunity and contribute to T1D in at-risk subjects.

Summary Environmental factors, including microbes and diet, play a key role in initiating autoimmunity in genetically predisposed individuals. However, the influence of gut microflora in the initiation and progression of systemic lupus erythematosus (SLE) is not well understood. In this study, we have examined the impact of drinking water pH on immune response, disease incidence and gut microbiome in a spontaneous mouse model of SLE. Our results show that (SWR × NZB) F1 (SNF1) mice that were given acidic pH water (AW) developed nephritis at a slower pace compared to those on neutral pH water (NW). Immunological analyses revealed that the NW-recipient mice carry relatively higher levels of circulating autoantibodies against nuclear antigen (nAg) as well as plasma cells. Importantly, 16S rRNA gene-targeted sequencing revealed that the composition of gut microbiome is significantly different between NW and AW groups of mice. In addition, analysis of cytokine and transcription factor expression revealed that immune response in the gut mucosa of NW recipient mice is dominated by T helper type 17 (Th17) and Th9-associated factors. Segmented filamentous bacteria (SFB) promote a Th17 response and autoimmunity in mouse models of arthritis and multiple sclerosis. Interestingly, however, not only was SFB colonization unaffected by the pH of drinking water, but also SFB failed to cause a profound increase in Th17 response and had no significant effect on lupus incidence. Overall, these observations show that simple dietary deviations such as the pH of drinking water can influence lupus incidence and affect the composition of gut microbiome.

SummaryMost patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

Systemic sclerosis (SSc) is a debilitating inflammatory and fibrotic disease that affects the skin and internal organs. Although the pathophysiology of SSc remains poorly characterized, mononuclear cells, mainly macrophages and T cells, have been implicated in inflammation and fibrosis. Inducible costimulator (ICOS), which is expressed on a subset of memory T helper (TH) and T follicular helper (TFH) cells, has been shown to be increased in SSc and associated with disease pathology. However, the identity of the relevant ICOS+ T cells and their contribution to inflammation and fibrosis in SSc are still unknown. We show that CD4+ ICOS-expressing T cells with a TFH-like phenotype infiltrate the skin of patients with SSc and are correlated with dermal fibrosis and clinical disease status. ICOS+ TFH-like cells were found to be increased in the skin of graft-versus-host disease (GVHD)-SSc mice and contributed to dermal fibrosis via an interleukin-21- and matrix metalloproteinase 12-dependent mechanism. Administration of an anti-ICOS antibody to GVHD-SSc mice prevented the expansion of ICOS+ TFH-like cells and inhibited inflammation and dermal fibrosis. Interleukin-21 neutralization in GVHD-SSc mice blocked disease pathogenesis by reducing skin fibrosis. These results identify ICOS+ TFH-like profibrotic cells as key drivers of fibrosis in a GVHD-SSc model and suggest that inhibition of these cells could offer therapeutic benefit for SSc.

The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.

The regulation of gene transcription is elementary for the function of hematopoietic stem cells (HSCs). The transcriptional repressor growth factor independence 1 (Gfi1) restricts HSC proliferation and is essential to maintain their self-renewal capacity and multipotency after transplantation. In addition, Gfi1−/− HSCs are severely compromised in their ability to compete with wild-type (wt) HSCs after transplantation. We now report that Gfi1 protects HSCs against stress-induced apoptosis, probably, by repressing the proapoptotic target gene Bax, since irradiated Gfi1−/− HSCs display higher expression of Bax and show a higher rate of apoptosis than wt HSCs. This protective function of Gfi1 appears to be functionally relevant since Gfi1−/− HSCs that express Bcl-2, which antagonizes the effects of Bax, regain their ability to self renew and to initiate multilineage differentiation after transplantation. Surprisingly, Gfi1−/−xBcl-2 transgenic mice also show a strong, systemic expansion of Mac-1+Gr-1− myeloid cells in bone marrow and peripheral lymphoid organs. These cells express high levels of the proleukemogenic transcription factor Hoxa9 and, in older mice, appear as atypical monocytoid-blastoid cells in the peripheral blood. As a result of this massive expansion of myeloid cells, all Gfi1−/−xBcl-2 mice eventually succumb to a myeloproliferative-like disease resembling a preleukemic state. In summary, our data demonstrate that Gfi1's ability to protect against apoptosis is essential for HSC function. In addition, our finding show that Gfi1 prevents the development of myeloproliferative diseases and provides evidence how Gfi1 deficiency could be linked to myeloid leukemia.

Endotoxin (bacterial lipopolysaccharide [LPS]) causes fatal septic shock via the Toll-like receptor 4 (TLR-4) protein present on innate immunity effector cells, which activates nuclear factor kappa B (NF-kappaB), inducing proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha). An early step in this process involves nuclear sequestration of the p65-RelA NF-kappaB subunit, enabling transcriptional activation of target inflammatory cytokine genes. Here, we analyzed the role of the nuclear zinc finger protein Gfi1 in the TLR response using primary bone marrow-derived macrophages. We show that upon LPS stimulation, expression of Gfi1 is induced with kinetics similar to those of nuclear translocation of p65 and that Gfi1 interacts with p65 and inhibits p65-mediated transcriptional transactivation by interfering with p65 binding to target gene promoter DNA. Gfi1-deficient macrophages show abnormally high mRNA levels of the TNF-alpha gene and many other p65 target genes and a higher rate of TNF promoter occupancy by p65 than wild-type cells after LPS stimulation, suggesting that Gfi1 functions as an antagonist of NF-kappaB activity at the level of promoter binding. Our findings identify a new function of Gfi1 as a general negative regulator of the endotoxin-initiated innate immune responses, including septic shock and possibly other severe inflammatory diseases.

Background: Following the clinical success of checkpoint blockade with CTLA-4 and PD-1 pathway-targeted agents, the field of cancer immunotherapy is rapidly expanding. Costimulatory molecules from the tumor necrosis factor receptor superfamily (TNFRSF), including OX40, may be promising targets to enhance the benefits of immunotherapy. While the functional consequences of signaling through OX40, either via OX40 ligand or agonistic antibodies, have been well documented, little attention has been paid to the temporal regulation of the OX40 receptor itself following T-cell activation and receptor agonism. Here we present functional characterization of BMS-986178, a fully human IgG1 monoclonal OX40- agonistic antibody. Methods: We developed in vitro assays using healthy donor human CD4+ effector T (Teff) and regulatory T (Treg) cells to (1) characterize the functional activity of BMS-986178 across a wide range of concentrations (0.0001-200 nM), (2) address how OX40 receptor expression is modulated following T-cell activation, and (3) determine whether antibody treatment affects receptor regulation. Analysis of murine tumor-infiltrating lymphocytes (TILs) allowed for the comparison of receptor regulation across species. Results: BMS-986178-mediated enhancement of human CD4+ Teff responses and reversal of Treg suppression was dependent on antibody cross-linking, as activity was lost in the absence of FcγR- or secondary antibody-mediated cross-linking in the assays. Maximal increases in activation markers (CD25, ICOS), proliferation, and cytokine production mediated by BMS-986178 treatment occurred at ≈20% receptor occupancy (RO). This functional enhancement was lost at relatively high antibody concentrations (>1 nM) associated with full RO. Lower concentrations of BMS-986178 (0.001-0.3 nM) were sufficient to drive upregulation of OX40, both on the cell surface and the soluble receptor; however, as RO approached 100%, a loss in surface and soluble OX40 was observed. Loss of receptor at the point of full RO was also observed in murine TILs. The downregulation of OX40 was specific to treatment with BMS-981678, as it was not observed with anti-CD28 or antibodies against other TNFRSF members. Internalization of the receptor:antibody complex as well as epigenetic regulation of the OX40 locus were examined for their role in receptor downregulation. Conclusions: These results demonstrate a clear relationship between RO and the ability of BMS-986178, an agonist OX40 antibody, to enhance T-cell responses. Furthermore, these findings provide insight into antibody-mediated receptor modulation in vitro, with potential implications for defining the optimal dose and schedule of agonist OX40 antibodies and, perhaps more broadly, for agonists targeting other costimulatory molecules. Citation Format: Marie-Claude Gaudreau, Christina Milburn, Chan Gao, Alla Pritsker, Mark Fereshteh, Zheng Yang, Bryan Barnhart, Alan Korman, Michael Quigley. Examining the dynamic regulation of OX40 following receptor agonism and T-cell activation: Implications for antibody-mediated enhancement of T-cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2782.

To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus , a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-γ and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent “Newman” strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus .

Summary The risk of developing systemic lupus erythematosus (SLE) is approximately nine times higher among women compared to men. However, very little is understood concerning the underlying mechanisms that contribute to this gender bias. Further, whether there is a link between immune response initiated in the gut mucosa, the progression of SLE and the associated gender bias has never been investigated. In this report, we show a potential link between the immune response of the gut mucosa and SLE and the gender bias of lupus for the first time, to our knowledge. Both plasma cell- and gut-imprinted- α4β7 T cell frequencies were significantly higher in the spleen and gut mucosa of female (SWR × NZB)F1 (SNF1) mice compared to that of their male counterparts. Importantly, female SNF1 mice not only showed profoundly higher CD45+ immune cell densities, but also carried large numbers of interleukin (IL)-17-, IL-22- and IL-9-producing cells in the lamina propria (LP) compared to their male counterparts. Intestinal mucosa of female SNF1 mice expressed higher levels of a large array of proinflammatory molecules, including type 1 interferons and Toll-like receptors 7 and 8 (TLR-7 and TLR-8), even before puberty. Our work, therefore, indicates that the gut immune system may play a role in the initiation and progression of disease in SLE and the associated gender bias.

The regulation of posttranscriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development, and immunity. The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. CD45 is known to be alternatively spliced, a process that is partially regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) L. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice and found that LckCre-mediated deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between double-negative 4 and double-positive cells. In addition, hnRNP L−/− thymocytes express aberrant levels of the CD45RA splice isoforms and show high levels of phosphorylated Lck at the activator tyrosine Y394, but lack phosphorylation of the inhibitory tyrosine Y505. This indicated an increased basal Lck activity and correlated with higher proliferation rates of double-negative 4 cells in hnRNP L−/− mice. Deletion of hnRNP L also blocked the migration and egress of single-positive thymocytes to peripheral lymphoid organs in response to sphingosine-1-phosphate and the chemokines CCL21 and CXCL12 very likely as a result of aberrant splicing of genes encoding GTPase regulators and proteins affecting cytoskeletal organization. Our results indicate that hnRNP L regulates T cell differentiation and migration by regulating pre-TCR and chemokine receptor signaling.

The coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes.

Hematopoiesis is sustained by a pool of multipotent hematopoietic stem cells (HSCs) that have the capacity to differentiate into cells of all blood cell lineages. The pool of long-lived HSCs is maintained throughout life by the self-renewal ability of HSCs. New evidence suggests the process of alternative splicing is an important regulator of the maturation and activation of blood and immune effector cells. It is presently estimated that almost all multi-exon genes in human genome undergo alternative pre-mRNA splicing, and aberrant splicing has been linked to a variety of human pathologies. However, the role that pre-mRNA splicing may have for HSCs behaviour remains largely unexplored. Heterogeneous nuclear ribonucleoprotein L (hnRNPL) is an RNA-binding protein that regulates alternative splicing by binding exonic splicing silencers elements (ESS) resulting in exon exclusion from the mature mRNA. RT-PCR analyses showed that hnRNPL is expressed in early stages of hematopoiesis including HSCs and lineage restricted hematopoietic progenitors. To test the role of hnRNPL in hematopoietic differentiation, we have generated conditional deficient mice, since a constitutive deletion of hnRNPL results in early embryonic lethality. Animals carrying two hnRNPL-floxed alleles (hnRNPLfl/fl) can be deleted at adult stage by the pIpC inducible MxCre transgene or by the VavCre transgene, which is expressed in all hematopoietic cells starting at embryonic stage E14. VavCre+hnRNPLfl/fl mice were not viable and did not progress further in their development than embryonic stage E17.5 and ablation of hnRNPL by pIpC injection caused a high rate of mortality in adult MxCre+hnRNPLfl/fl mice compared to control animals. Both the fetal liver (FL) of VavCre+hnRNPLfl/fl mice and the bone marrow (BM) of adult MxCre+hnRNPLfl/fl mice had a significantly reduced cellularity. Furthermore, flow cytometric analysis revealed in both FL and BM a significant reduction in frequency and absolute numbers of all mature blood cells, the lymphoid and myeloid precursors, CLPS, CMPs and GMPs and to a lesser extent the erythroid/megakaryocytic precursors (MEPs). Methylcellulose and both competitive and non-competitive transplantation assays demonstrated that HSCs lacking hnRNPL cannot generate lineage-committed progenitors and have lost their self-renewal capacity and reconstitution potential. A genome-wide analysis of mRNA expression and splicing through next-generation RNA sequencing of wild-type (WT) or VavCre+hnRNPLfl/fl E14.5 Lin- c-kit+ fetal liver cells (FLCs) revealed that hnRNPL deficiency affects not only alternative splicing but also gene expression levels in hematopoietic progenitors. In the absence of hnRNPL, genes implicated in regulating apoptosis, DNA damage response and cell division where found up-regulated in Lin- c-kit+ FLCs. Among those genes, many were p53 effector genes such as Cdkn1a, Ccng1, Trp53inp1, TrailR2, Bax and Zmat3. In addition genes that are known to be required for normal hematopoiesis and HSCs functions such as Gfi1, CD34, Csfr1, Egr1 and Runx1 were found down-regulated in those cells. Further analyses by qPCR and Western blots confirmed those findings and also showed that the level of p53 protein expression was upregulated in VavCre+hnRNPLfl/fl FLCs although the mRNA level is the same as in the WT cells suggesting that hnRNPL affects p53 mRNA translation efficiency. Similarly, several genes found differentially spliced are implicated in cell cycle progression or required for normal hematopoiesis in FL such as Bcl11a, Cdk4, Ccnd2 and TRP53bp1. These results together with an increased level of Reactive Oxygen Species (ROS) and elevated levels of phosphorylated histone H2AX (γ-H2AX, a sensor for double strand DNA breaks) suggest that hnRNPL regulates the activation of a p53 dependent DNA damage response pathway in hematopoietic stem cells. As a consequence loss of hnRNPL results in a loss of hematopoietic stem and progenitor cells. Our data also suggest that hnRNPL does not only regulate alternative splicing but also expression levels of a set of specific effector genes involved in HSC survival, proliferation, ultimately affecting self-renewal. Disclosures: No relevant conflicts of interest to declare.

Abstract 560 More than 50% of patients diagnosed with B or T-cell leukemia and lymphoma will fail current treatment protocols. This highlights the urgent need for new and improved therapies. Since the transcription factor Growth factor independent-1 (Gfi1) plays an important role in lymphoid differentiation, we explored whether it might be a suitable target for therapy. Using mouse models in which T-cell leukemia can be induced by transgenic expression of mutated forms of Notch1, by injection of the carcinogen N-Ethyl–nitrosourea (ENU) or infection with a Murine Moloney Leukemia Virus, we found that Gfi1 knockout mice had a significantly lower incidence and a longer latency period of T-ALL. To verify whether targeting Gfi1 would be a novel approach to treat B- or T-cell lymphoma, we used Mx1Cre Gfi1fl/fl mice. In these mice, injection of polyinosinic-polycytidylic acid (pIpC) activates the Mx1 promoter driven Cre expression, which ultimately leads to the deletion of the floxed Gfi1 alleles. As controls, we used Gfi1fl/fl mice, which lack the Cre recombinase and thus still express Gfi1 after pIpC injection. To elicit a T- or B-cell lymphoma, we used ENU injection combined with expression of a mutated Notch1 transgene for T-ALL, or transgenic over-expression of c-Myc for B-cell leukemia (Eμ-Myc). Using in-vivo ultrasound supported imaging, we observed complete regression of tumour masses when Gfi1 was eliminated in Mx1Cre Gfi1fl/fl mice, curing the mice of the either the T or B-cell malignancies. Strikingly, this effect was observed in the absence of any other treatment regimen. To explore the mechanisms underlying this phenomenon, we explanted tumor samples from mice, in which Gfi1 expression was either maintained or deleted and performed gene expression arrays. A comparative analysis of the array data demonstrated that loss of Gfi1 affects pathways of key importance for leukemia such as metabolism, cell cycle progression, basal transcription and apoptosis but also the response to DNA damage. It has been shown previously for non hematological malignancies that oncogenic transformation in conjuction with dysregulated cell cycle induces DNA strand breaks (DSBs), which leads to an increased p53 dependent apoptotic response in tumours compared to non-transformed cells. This forces the tumor cells to counteract this effect – for instance by selection for the loss of p53. Consistent with this concept, we noted that leukemic cells from our tumor models displayed a greater amount of DSBs and also higher rates of spontaneous apoptosis than normal cells. Interestingly, the number of apoptotic cells was further increased in those tumors where Gfi1 had been deleted. We hypothesized that Gfi1 protects leukemia cells against DSB induced apoptosis. To test this hypothesis, we irradiated in Gfi1+/+ and Gfi1−/− thymocytes, which induces DSB in thymocytes. In line with our hypothesis we found that Gfi1−/− thymocytes showed increased rates of apoptosis compared to irradiated Gfi1+/+ thymocytes and that loss of Gfi1 led to an increased induction of pro-apoptotic genes such as Bax, Noxa and Puma after irradiation. Since Bax, Noxa and Puma are all p53 target genes, we investigated a possible link between Gfi1 and p53 and found that (I) Gfi1 binds to p53, (II) that Gfi1 inhibits the transcription of p53 target genes and (III) that Gfi1 occupies p53 target gene at the same sites as p53. In summary, Gfi1 antagonizes p53 function and loss of Gfi1 sensitizes cells to p53-mediated apoptosis. Next, we used different human T-ALL cell lines and treated these cells either with sh-RNA lentivirus or morpholinos to abrogate GFI1 expression. In all cases, down-regulation of GFI1 expression led to increased apoptosis and impeded growth of human leukemia cells. Finally, we transplanted leukemic cells of T-ALL patients into NOD-Scid, IL2Rnull (NSG) mice, waited for the leukemic cells to engraft, and then injected GFI1 specific- or control morpholinos. While mice treated with control morpholino died of leukemia, the animals treated with GFI1-specific morpholinos survived showing a significant reduction of human leukemic cells in the blood, bone marrow and spleen, even with samples from patients who did not respond to first line therapy. Since morpholinos have received approval for use in humans, our data suggest that targeting GFI1 in human T-ALL patients may be a promising therapeutic target and a feasible way to complement current T-ALL treatment regimens. Disclosures: No relevant conflicts of interest to declare.

Abstract 223 A coding variant form of GFI1 (GFI136N) increases the risk to develop AML by 60% and is present in about 10–15 % of all Caucasian AML patients. To determine the underlying molecular mechanism and potentially develop new therapeutic approaches, we generated “knockin” mouse strains wherein the endogenous murine Gfi1 gene was replaced either by the human GFI1 variant (GFI136N, the form predisposing to AML) or by the more common form of GFI1 (GFI136S). In most hematopoietic compartments no difference was observable between GFI136N and GFI136S expressing mice; however, there was a 3–5 fold increase in the number of granulocytic monocytic progenitors (GMPs) and common myeloid progenitors (CMPs) in Gfi136N expressing (either homozygous or heterozygous) mice compared to wild-type or Gfi136S expressing mice(p≤0.01). Interestingly, both human and murine AML leukemic cells are thought to originate from GMPs and CMPs. To assess functional differences, we seeded GMPs from GFI136N or GFI136S knockin mice on methylcelluose or transplanted them into into syngenic animals. We found that GFI136N expressing GMPs proliferate faster and have an increased self-renewal capacity both in-vitro and in-vivo compared to GMPs carrying Gfi136S alleles. A gene expression array analysis showed that GFI136N GMPs have a stem cell-like gene signature with elevated levels of Hoxa9 expression and a deregulation of a number of oncogenes involved in the development of human AML such as Trib2, Tet2 or Idh2. It is of particular interest that Hoxa9, a known GFI1 target gene, was up-regulated 3–4 fold in GFI136N GMPs compared to in GFI136S GMPs (p≤0.01). It is known that high levels of Hoxa9 accelerate AML development in mice and are associated with a poor prognosis in AML patients. GFI1 is a transcriptional repressor and exerts its function by recruiting different histone modifying enzymes, in particular LSD1, which de-methylates histone 3 (H3) at lysine 4 (K4), or histone deacetylases (HDACs), which remove acetyl groups from H3K9 residues and G9a, which initiates dimethylation of H3K9. Both H3K4 methylation and H3K9 acetylation correlate with actived gene expression, whereas H3K9dimethyl correlates with repession. Chromatin-immuno-precipitation (ChIP) of Gfi1-bound chromatin from Lin−Sca1−c-Kit+ cells, which contains the GMP population, showed that GFI136N binds to a lesser degree to the Hoxa9 locus than GFI136S. This diminished binding of Gfi136N correlated with an increased H3K4 dimethylation and H3K9 acetylation as well as diminished H3K9 dimethylation across the Hoxa9 locus in GFI136N cells. It is likely that these epigenetic changes lead to the increased Hoxa9 expression observed in GFI136N GMPs. A more exhaustive ChIP-Seq analysis with antibodies recognizing H3K4dimethyl in Lin−Sca1−c-Kit+ cells from Gfi136N or Gfi136S mice showed significant epigenetic alterations throughout the Hoxa9 locus genome and at other GFI1 target genes. It is conceivable that these epigenetic alterations explain, at least in part, the changed gene expression signatures in GFI136N GMPs. To investigate the role of GFI136N in myeloid leukemogenesis, we induced the expression of a mutated form of KRAS (K12D) in both GFI136N and GFI136S mice. All mice developed a deadly myelo-proliferative disorder, but animals carrying the GFI136N allele succumbed to the disease within a significantly shorter latency period (17 against 31 days, p≤0.01) than GFI136S mice. We also transduced GFI136N and GFI136S GMPs with retroviral vectors directing the expression of either the AML1-Eto9a or the MLL-AF9 onco-fusion proteins typically found in human AML. We observed that GFI136N GMPs expressing MLL-AF9 or AML1-Eto9a generated 5–10 fold more colonies (p≤0.01) on methylcellulose and exhibited a higher replating efficiency than the respective GFI136S GMPs. Finally, AML blast cells from GFI136N heterozygous patients expressed higher levels of HOXA9 compared to AML blasts from GFI136S homozygous patients, suggesting that our mouse model reflects the disease predisposition in human patients. Our knockin mice are, to our knowledge, the first animal model for a human genetic variation that predisposes to leukemia. Based on the findings with this model, we propose that the human GFI136N variant predisposes to AML by inducing epigenetic changes affecting the expression of important regulators with oncogenic potential such as Hoxa9. Disclosures: No relevant conflicts of interest to declare.

Abstract 997 Growth Factor Independence 1 (GFI1) is a transcriptional repressor, which plays an important role in myeloid differentiation. We have recently described a Single Nucleotide Polymorphism (SNP) in the GFI1 gene that is found in 15% of all AML patients and increases risk of development of acute myeloid leukemia (AML) in homozygous or heterozygous carriers of this variant. The SNP generates a GFI1 variant that carries an Asparagine residue instead of a Serine at amino acid position 36 (GFI136N). To better understand how this variant might cause leukmemia, we replaced the murine Gfi1 locus with the common form of human GFI1 (denominated GFI36S) or the variant form (GFI136N) using a previously described knockin strategy. Replacement of murine Gfi1 by human GFI136S did not affect lymphoid or myeloid differentiation demonstrating that “knocked-in” human GFI1 (Gfi1hu36S/hu36S) can fully substitute the endogenous murine gene. However, mice homozygous for the variant GFI136N allele (Gfi1hu36N/hu36N) featured a 3-fold increase in the number of both Common myeloid progenitors (CMPs) and Granulocytic monocytic progenitors (GMPs) compared with wt or Gfi1hu36S/hu36S mice (p’0.001). In addition, in the GMP fraction from Gfi1hu36N/hu36N mice more dividing cells (20 %) were found compared to the GMP fraction originating from wt or Gfi1hu36S/hu36S mice (p≤0.01). Moreover, we observed a 3-fold increased expression of the proleukemogenic transcription factors Hoxa9 and Pbx1 in GMPs from Gfi1hu36N/hu36N compared to wt or Gfi1hu36S/hu36S animals (p≤0.01). This increased level of Hoxa9 would be consistent with recent publications showing that Gfi1 is required for the repression of Hoxa9 expression in GMP/CMP subsets. Interestingly, AML patients heterozygous for the GFI136N variant showed a tendency toward a 2-fold higher expression of the Hoxa9 gene when compared with GFI136S homozygous patients. To explain how the presence of the variant might predispose to leukemia, we performed a gene expression array of GMPs from Gfi1hu36N/hu36N, wt mice, Gfi1ko/ko or Gfi1hu36S/hu36S mice. Whereas wt and Gfi1hu36S/hu36S mice showed a similar gene expression pattern, the presence of the GFI136N variant led to a reprogramming of the GMP population with dysregulation of genes known to be involved in AML such as Idh1, Tet2 and Trib1. The gene expression pattern was also distinct from the pattern of sorted Gfi1ko/ko GMPs, pointing to a specific role of GFI136N in reprogramming the progenitor fraction and thus predisposing to AML. Interestingly, some of the genes differentially regulated between Gfi1hu36N/hu36N GMPs and the other GMPs were also differentially expressed between GFI36S patients and GFI136N heterozygous AML patients. We examined the biochemical features of the two Gfi1 variants in order to explain how the variant might reprogramm the GMPs and predispose to AML. Both GFI136N and GFI136S are still able to bind to previously published GFI1interaction partners such as PU.1 or CEBPe. We observed that GFI136N exhibited a different intranuclear localization than GFI136S. Whereas murine Gfi1 and human GFI136S localize in dots within the nucleus, GFI136N is mainly found along the nuclear border suggesting that nuclear localization is important for Gfi1 function. This was in accordance with a previously reported AML patient homozygous for GFI136N who also exhibited a similar aberrant nuclear localization. Finally to study a potential impact of GFI136N on myeloid leukemogenesis, we used an established mouse model in which expression of a mutated form of KRAS (K12D) can be induced causing a 100% penetrance of an AML like disease. GFI136N significantly accelerated the onset of KRAS driven leukemia in these mice which was associated with a higher number of blast cells in the bone marrow, blood and spleen (p’0.05 for blast number and latency). These data suggest that the GFI136N variant predisposes to the development of AML since it is inefficient in repressing Hoxa9 owing to an aberrant subnuclear localization. To our knowledge this is the first knock-in of a human protein variant predisposing to AML and this mouse model could help developing a targeted therapy for these patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3156 T cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers associated with mutations in NOTCH1. The Growth factor independent-1 (Gfi1) transcriptional repressor gene was originally discovered as a common target of Moloney murine leukemia virus (MMLV) proviral insertion in murine T-ALL. In fact, the Gfi1 locus is the most frequently activated gene in MMLV-induced T cell leukemia. Therefore, we investigated whether the most commonly activated gene in MMLV-induced murine T-ALL, Gfi1, could collaborate with the most commonly activated gene in human T-ALL, NOTCH1. Here, we show that GFI1 expression is associated with Notch signaling in human T-ALL (p'0.0003). Functionally, Gfi1 collaborates with Notch-induced murine T-ALL by accelerating an already rapid disease model (p=0.03) without altering the lymphoblastic nature of the disease. Furthermore, inducible deletion of Gfi1 is counter-selected in both Notch-driven retroviral and transgenic mouse models of T-ALL; whereas, constitutive absence of Gfi1 completely prevents transgenic Notch-induced T-ALL (p≤0.04). However, T-ALL tumors can form in Gfi1-/- animals using either ENU-mutagenesis or MMLV-infection, yet tumor formation is delayed (p≤0.02, p≤0.03 respectively). This suggests that Gfi1 deletion does not prevent the formation of the T-ALL initiating cell and that Gfi1 might be absolutely required for Notch-induced T-ALL. Most striking is that Gfi1 is required for T-ALL maintenance in vitro and in vivo. Using three separate Tal1-initiated murine T-ALL cell lines, the overexpression of the Gfi1 dominant-negative, Gfi1N382S, was quickly and completely counter-selected. As Gfi1 has previously been found to regulate pro-apoptotic genes in T cells, we attempted to rescue the above loss of function phenotype by overexpressing the anti-apoptotic factor Bcl2. Notably, counter-selection of Gfi1N382 is not observed or is significantly delayed in all three cell lines. In vivo, inducible deletion of Gfi1 leads to both mutagen- or Notch-induced tumor regression as measured by ultrasound. In fact, levels of Gfi1 expression directly correlate to tumor regression and disease free survival of T-ALL. Finally, targeting Gfi1 enhances the efficacy of radiation therapy and bone marrow transplantation. Deletion of Gfi1 sensitizes T-ALL tumors and T cells to p53-dependent apoptosis after exposure to DNA-damaging agents such as radiation, Etoposide or Daunorubicin by de-repression of the pro-apoptotic Gfi1 target gene Bax. These data extend the role of Gfi1 to human T-ALL and suggest that T-ALL is dependent upon Gfi1 to repress pro-apoptotic genes for tumor survival, ultimately highlighting a new therapeutic target in the fight against lymphoid malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 1486 Poster Board I-509 Hematopoietic differentiation has to be tightly regulated since uncontrolled or exaggerated development of blood cells may lead to the development of leukemia or autoimmune diseases. Many mechanisms exist to control hematopoiesis on a molecular level, including the regulation of transcription, which has been intensely studied. However, new evidence suggests the process of alternative splicing to be an important regulator of the maturation and activation of blood- and immune effector cells. One of the factors that has been identified as a potential regulator of the immune response and controls alternative splicing is “heterogenous nuclear ribonucleoprotein L” (hnRNP L). This factor affects among others the alternative splicing of the CD45 gene, which encodes the major tyrosine phosphatase expressed on all hematopoietic cells. To investigate the biological role of hnRNP L as a regulator of alternative splicing in hematopoiesis, we have generated conditional hnRNP L knockout (KO) mice carrying floxed alleles that can be deleted by co expression of Cre recombinase. Both the inducible MxCre transgene or Vav-Cre transgene, which is active in all hematopoietic cells were introduced into hnRNP Lfl/fl mice. We found that the conditional deletion of hnRNP L by the Vav Cre transgene led to early mortality before birth (at stage E17.5) and flow cytometric analysis of fetal liver of hnRNP Lfl/fl, Vav-Cre mice or bone marrow from pIpC induced hnRNP Lfl/fl Mx-Cre mice showed a deficit in erythrocyte maturation. In addition, fetal thymi from hnRNP Lfl/fl X Vav-Cre mice were severely reduced in cellularity and showed disturbed T cell maturation. Moreover, the deletion of hnRNP L results in reduced numbers of Lin−Sca1+ckit+ (LSK) cells, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs). Strikingly, while most of the progenitors and the short-term hematopoietic stem cells (HSCs) were affected by the deletion of hnRNP L, the population of long term HSCs was not reduced. We found a high percentage of Annexin V positive cells in the LSK population suggesting that the loss of progenitors and short term HSCs in hnRNP L deficient mice is due to an accelerated cell death. To test whether stem cells lacking hnRNP L were still functional, we sorted Lin−Sca1+ckit+ (LSK) cells and cultured them on either methylcellulose or the feeder cell lines OP9 and OP9-DL1. The co-culture with OP9 or OP9-DL1 cells demonstrated that hnRNP L−/− LSK cells had lost their potential to differentiate into B and T lymphocytes. Similarly, hnRNP L deficient LSK cells were unable to give rise to lymphoid, myeloid or erythroid colonies on methylcellulose. This suggests that hnRNP L is required to maintain not only the numbers of hematopoietic stem cells, but also their ability for multilineage differentiation. We conclude that the regulation of alternative splicing is an essential component of the regulatory network required to maintain hematopoietic differentiation and the functional integrity of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.