SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo

Background Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche.

CD5 Surface Expression Marks Intravascular Human Innate Lymphoid Cells That Have a Distinct Ontogeny and Migrate to the Lung

Innate lymphoid cells (ILCs) contribute to immune defense, yet it is poorly understood how ILCs develop and are strategically positioned in the lung. This applies especially to human ILCs due to the difficulty of studying them in vivo. Here we investigated the ontogeny and migration of human ILCs in vivo with a humanized mouse model (“MISTRG”) expressing human cytokines. In addition to known tissue-resident ILC subsets, we discovered CD5-expressing ILCs that predominantly resided within the lung vasculature and in the circulation. CD5+ ILCs contained IFNγ-producing mature ILC1s as well as immature ILCs that produced ILC effector cytokines under polarizing conditions in vitro. CD5+ ILCs had a distinct ontogeny compared to conventional CD5- ILCs because they first appeared in the thymus, spleen and liver rather than in the bone marrow after transplantation of MISTRG mice with human CD34+ hematopoietic stem and progenitor cells. Due to their strategic location, human CD5+ ILCs could serve as blood-borne sentinels, ready to be recruited into the lung to respond to environmental challenges. This work emphasizes the uniqueness of human CD5+ ILCs in terms of their anatomical localization and developmental origin compared to well-studied CD5- ILCs.

IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

Background: Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal. Objective: To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α. Design/Methods: Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 5 CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO 2. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P <0.05, ** P <0.01 and *** P<0.001. Results: The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p<0.0006) and 15% ± 6 (p<0.02), respectively, than that of the control (Fig 1). Importantly, the myelosuppressive effect of IFN-γ and TNF-α was significantly rescued by the addition of Ruxolitinib (at day 14, p<0.05) or IFN-γ neutralizing antibodies (B27 and B133.5, respectively) (day 7 and 14, p<0.01). Improvement in HSPC survival ranged between 1.5-3.3 fold compared to our negative control. Based on the CFU analysis, the CD34+ cells cultured in the presence of B27, B133.5 or Ruxolitinib, were able to produce more CFU at day 7 (p<0.01, p<0.01 and p<0.05 respectively)(Fig 2A). Additionally, Both B27 (p < 0.01) and Ruxolitinb (p ≤ 0.001) were found to produce more CFU-GM on day 14 (Fig 2B). Phospho flow-cytometry demonstrated a significant decrease in STAT1 phosphorylation of CD34+ cells in the presence of B27 and B133.5 (p<0.05, p<0.001, respectively). Conclusions: Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA. Figure 1 Figure 1. Disclosures Cairo: Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau. OffLabel Disclosure: Ruxolitinib was used to inhibit JAK-STAT signaling pathway in an Ex-Vivo model of Aplastic Anemia. Drug wasn't supplied by drug company.

Uncoupling p53 from an Embryonic Regulome Exhausts Quiescent CML Stem Cells through Inhibition of a HIF1alpha Molecular Program

Although it has been recognized for many years that cancer stem cells and embryonic stem cells (ESC) share molecular features, identifying ways to exploit this therapeutically has proved challenging. To date, these shared features have not been examined in the leukemic stem cells (LSC) found in patients with chronic myeloid leukemia (CML). By integrating known ES regulatory circuitry with transcriptomics datasets, including deep single-cell RNA-seq profiling of 15,670 LSC from five patients with CML, we identified a core ESC regulome in the LSC containing 1243 genes. The significant majority of this regulome (1102 genes) was up-regulated in cycling LSC, whilst quiescent LSC showed up-regulation of a characteristic set of 101 genes, unique to cells with high ESC identity and with regulatory circuitry enriched for c-Myc and Nanog modules. Membership of the ESC regulome included the TP53 gene which was transcriptionally repressed and detected at a lower frequency in quiescent LSC compared to cycling ones (11.8% vs 43.6%). We also demonstrated that tyrosine kinase inhibitors (TKI) repress the ESC regulome and TP53 expression in LSC, suggesting that the regulome safeguards against high levels of TP53 expression, thus promoting survival of quiescent LSC in the presence of TKI. We hypothesized that overcoming the influence of the regulome on TP53 expression would provide an opportunity to eradicate quiescent LSC. To this end, we used an MDM2 inhibitor (MDM2i), RG7388 (idasanutlin) or RG7112, to stabilize the p53 protein, examining its potential in combination with nilotinib (NIL) to eradicate CML LSC in vitro and in vivo, with RG7388 being the most optimized and furthest in development. The combination of NIL plus MDM2i in vitro was more effective at targeted LSC from primary patient samples than NIL treatment alone, as evidenced by reduced CFC and LTC-IC outputs (p<0.05, 0.01 respectively). Intriguingly, the combination of NIL plus MDM2i did not result in significant reductions in the number of LSC compared to NIL only, when we quantified them at the end of drug treatments in pre-clinical mouse models. Instead, we observed a functional decline of the LSC as evidenced by diminished engraftment potential in 2 o recipient mice (p<0.05; SCLtTA x BCR-ABL1 transgenic model) or diminished colony-plating potential (p<0.05). This was followed by near complete depletion of the LSC population (p<0.05) 28 days after cessation of combination drug treatment (patient-derived xenografts/PDX in immunocompromised mice). In order to understand the molecular events underpinning these drug effects on LSC, we performed RNA-seq analysis of drug-treated CD34 + cells in vitro (bulk cells), or of human CD34 + cells obtained from PDX (single cell RNA-seq). CD34 + cells treated with NIL plus MDM2i in vitro showed evidence of increased p53 stabilization and activation of p53 target genes, and this was accompanied by repression of the ESC regulome beyond that normally observed with NIL only. Similarly, in PDX we observed increased repression of the ESC regulome in human CD34 + cells exposed to the combination of NIL plus MDM2i that included repression of HIF1alpha and a signature of genes required for cellular adaptations to hypoxia, and growth factor-mediated resistance to TKI therapy. Further, single cell analysis of differentiated human CD45 + cells from our PDX model, provided compelling evidence that acquisition of this repressive signature in the LSC, through combined NIL plus MDM2i treatment, re-wires them towards a basophilic fate, consistent with functional exhaustion of the LSC compartment. In conclusion, we have identified an ESC regulome in CML LSC and demonstrate that a combination of a TKI plus an MDM2i leads to p53 upregulation which antagonizes this regulome, providing a highly effective strategy to target near complete loss of functional LSC in pre-clinical models. Our study has revealed a new therapeutic paradigm to examine in other cancer stem cell populations that utilize ESC regulatory programs. Disclosures Higgins: Roche/Genentech: Current Employment, Current equity holder in publicly-traded company. Copland: Astellas: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Jazz: Honoraria, Speakers Bureau.

An Inducible Leukemia-Associated Transcription Factor Facilitates Large-Scale Ex Vivo Generation of Functional Human Macrophages

Long-term ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) proves to be unfeasible as cellular differentiation occurs when HSPCs are detached from their supporting bone marrow stem cell niche. This issue renders it difficult to make use of the proliferation capacity of HSPCs to subsequently produce functional blood cells in relevant numbers, e.g. for cell therapy approaches. To circumvent this challenge, leukemia-associated chimeric transcription factors, including MLL fusion proteins, can be exploited for their pronounced ability to propel cell proliferation while preserving cell immaturity. By designing the protein's activity controllable, the immature state can be abolished at an arbitrary point in time enabling terminal differentiation. In this study, we employed the fusion gene mixed lineage leukemia/eleven nineteen leukemia (MLL-ENL) for engineering an inducible protein switch. For this purpose, we fused the coding sequence of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the transcription factor MLL-ENL and subsequently expressed the protein switch (DD-MLL-ENL) in human CD34+ HSPCs derived from adult healthy donors. In the presence of the specific ligand Shield1, DD-mediated protein degradation is prevented leading to massive and long-term expansion of HSPC-derived late monocytic precursors in the presence of IL-3, IL-6, SCF, FLT3-L, TPO and GM-CSF. The cells do not exhibit additional driver mutations, feature a normal karyotype and telomere length, and sustain immaturity that is strictly dependent on Shield1 supplementation every other day even after two years of ex vivo culture. Upon Shield1 deprivation, the cells completely lost self-renewal and colony-forming properties and spontaneously differentiated. By changing the cytokines to GM-CSF in combination with IFN-γ and LPS we differentiated the progenitor cells into macrophages (MΦ) (Fig. 1 A, B). Immunophenotypic characterization revealed upregulation of the monocyte/macrophage-associated surface markers CD14, CD80, CD86, CD163 and MHC class I and II, concordant with monocytic morphology as judged by cytospin preparations. Analysis of the transcription of selected inflammatory genes, including IL-6 and IL-10, revealed overlapping M1 and M2 macrophage characteristics. Furthermore, mRNA expression profiles using nCounter Systems technology covering a total of 770 myeloid innate immunity-related genes proves the cells' identity as differentiated phagocytes shown by upregulation of gene clusters involved in Fc receptor signaling, TLR signaling, antigen presentation and T cell activation. In functional assays, we demonstrated the ability of the obtained cells to migrate towards the chemokine CCL2 in a 3D chemotaxis assay, attach to VCAM-1 under flow and shear stress and produce reactive oxygen species. Regarding the cells' phagocytic capability, we could verify the uptake of bacterial particles as well as apoptotic cells in efferocytosis assays. Finally, we demonstrated IgG Fc region recognition and binding by the expressed Fcγ receptors enabling phagocytosis of lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner using rituximab (RTX), daratumumab (Dara) and trastuzumab (Trast) as a negative control (Fig. 1C). Overall, we could demonstrate the conversion of a harmful leukemic transcription factor into a useful molecular tool for large-scale ex vivo production of functional blood cells. Such engineered controllable protein switches might have the potential to be employed as molecular tools to produce functional immune cells for cell-based immunotherapeutic approaches. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Beier: Alexion: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Other: Travel reembursement. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Greif: AstraZeneca: Honoraria.

Gene Delivery Using Baculovirus in Human Hematopoietic Stem and Progenitor Cells Requires Inhibition of Cellular Innate Immune Pathways

Homology-directed gene editing of hematopoietic stem and progenitor cells (HSPCs) has the potential to treat inherited blood disorders not amendable to CRISPR-Cas9 gene inactivation or single base editing. For many diseases, one of the major hurdles is viral delivery of large DNA templates needed for gene correction. Due to limited adeno-associated virus (AAV) packaging capacity other delivery approaches are needed. Baculovirus (BV), specifically Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is a large double-stranded DNA (dsDNA) virus widely used for protein expression and AAV production. In addition, BV has been proposed as a potential therapeutic vector (Ono, Viruses 2018). BV does not replicate in mammalian cells, can deliver large quantities of DNA with virtually unlimited packaging capacity, and can express genes under the control of mammalian promoters. While capable of transducing human hepatic cells and some cell lines (Chen, Biotechnol Adv. 2011), to our knowledge BV transduction efficiency has not been tested in human CD34+ HSPCs or shown in any hematopoietic cell line. Here we show for the first time that BV can be used as a gene delivery vector for primary human CD34+ cells mobilized from healthy donors. We constructed VSV-G pseudotyped BV with a copGFP reporter flanked by 4kb homology arms (HAs) to ITGB2, a locus mutated in leukocyte adhesion deficiency type I (LAD-1) (Fig. A, top). As measured by qPCR, viral DNA was detected in CD34+ cells after transduction at a multiplicity of infection (MOI) of 50, suggesting vector binding and entry in these cells. However, although toxicity was not observed, GFP expression as assessed by flow cytometry was mostly undetectable (less than 0.1%). In contrast, robust (>70%) GFP expression was measured in 293A cells using the same BV vector, suggesting that an inhibitory cellular process was uniquely triggered in primary CD34+ cells following transduction with BV. Recent work has shown that BV can activate cellular innate immune pathways including toll-like receptors (TLRs) (Abe, J Virol 2009) and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) (Amalfi S, JVI 2020) resulting in viral clearance and attenuation of gene expression (Ono, JVI 2014). We hypothesized that inhibition of activated cellular innate immune pathways may allow for more efficient BV gene expression in human CD34+ HSPCs. To examine this possibility, we tested over a dozen small molecule inhibitors at multiple doses targeting the major dsDNA sensing innate immune pathways including cGAS-STING (Fig. A, bottom). We found that a 45-minute pre-treatment with the STING inhibitor, H-151, while slightly toxic, enhanced GFP expression several fold, from less than 0.1% to an average of 1.5% in multiple independent donors (Fig. B-C). To improve viability, we also targeted cell death pathways. We tested the pan-caspase inhibitor, zVAD-FMK, which can inhibit both innate activation of gasdermin D (GSDMD), a major dsDNA sensing pathway, as well as apoptotic cell death. We additionally tested the necroptosis inhibitor Nec-1, as necroptosis can be activated in settings of apoptotic inhibition and inflammation. Notably, the combination of both inhibitors with H-151 improved not only cell viability, but also substantially enhanced GFP expression (8%), suggesting a synergistic benefit by inhibiting both innate immune activation and cell death pathways (Fig. D-E). To assess whether BV can efficiently transduce HSPCs with long-term repopulating activity, we pre-stimulated CD34+ cells for 48 hours in culture followed by transduction with BV at an MOI of 25 with our optimized drug cocktail. We examined GFP positivity in both CD34+CD38+ progenitors and CD34+CD38- HSC enriched populations by flow cytometry. After 24 hours, we found an average of 28% GFP+ CD34+CD38- cells and 8% GFP+ CD34+CD38+ progenitors (Fig. F-G). These data suggest that using our optimized approach, BV can target more primitive HSPCs. Collectively, our results lay the groundwork for future studies characterizing innate immune responses to dsDNA viruses in CD34+ cells, and highlight the potential use of BV as a delivery system for homology-directed gene editing in HSPCs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Age-Acquired Downregulation of Lmna Leads to Epigenetic Deregulation and Altered HSPC Function

Hematopoietic stem cells (HSCs) exhibit epigenetic reprogramming and decline in function with aging, and these changes may be predisposing mechanisms for development of clonal hematopoiesis and myeloid malignancies. Our recent study characterizing the epigenetic and transcriptional landscape of young (18-30 years) and aged (65-75 years) human CD34 +CD38 - bone marrow (BM) cells identified Lamin-A/C (LMNA) as one of the most downregulated genes with aging (7.9-fold, p=1.9x10 -13). LMNA encodes the nuclear lamina protein Lamin-A/C and is mutated in the premature aging disorder Hutchinson-Gilford Progeria Syndrome. To determine whether downregulation of LMNA contributes to the phenotype of human HSC aging, we knocked down LMNA (LMNA KD) using shRNA in young, mobilized peripheral blood CD34 + cells. With an average knockdown of 60%, we observed ~33% increase in myeloid colony forming potential (p<0.05). LMNA KD also impaired differentiation in liquid culture as determined by persistence of CD34 expression in twice as many cells as controls (p<0.0001) while induction of myeloid CD11b expression was reduced by ~29% (p<0.05), as well as a trend to reduced erythroid (CD71 +GYPA +) differentiation. To investigate the effects of Lmna loss-of-function in vivo, we conditionally deleted Lmna in mice using Vav-Cre. Loss of Lmna (Lmna KO) had no effect on steady state hematopoiesis in young (10 weeks) or middle-aged (14 months) knockout mice. However, Lmna KO BM cells from middle-aged mice generated 2-fold more total colonies than wild-type, floxed controls (WT=126 vs KO=292, p<0.01). Given that nuclear architecture provides a layer of epigenetic regulation through chromatin-lamina interactions, we investigated how the epigenome changes in the context of LMNA deficiency. By super-resolution microscopy, we observed loss of LMNA localization from the nuclear periphery in LMNA KD human CD34 +cells (p<0.05). Since lamina-associated domains generally contain regions of repressed chromatin, we stained for histone H3K9me2 and observed that LMNA KD cells displayed increased scattering throughout the nucleus without a change in its density. Next, we performed ATAC-seq and identified 603 open chromatin sites showing changes in accessibility (FDR<0.1). Over 73% of these sites show reduced accessibility and were associated with genes showing reduced expression by RNA-seq. Gene ontology analysis of these sites identified genes associated with heme biosynthetic and cell differentiation processes (FDR<0.005). Moreover, gene set enrichment analysis of the RNA-seq confirmed that downregulated genes were enriched for genes downregulated in our previously reported CD34 + aging signature (NES=-1.46, FDR=0.001). To determine whether LMNA KD alters the epigenome, we performed ChIP-seq for active (H3K4me3, H3K27ac) and repressive (H3K9me2) histone marks. LMNA KD resulted in significant reduction of 36% of H3K27ac peaks, which mimics our previous observation of marked age-related losses of H3K27ac. Most differential peaks localized to gene promoters (50%) and introns (34%) and include genes involved in nuclear pore organization and gene silencing (FDR<0.001). Notably, for peaks located at intergenic regions, >35% overlapped with active enhancers we reported as lost with aging and were associated with genes involved in regulation of p38 MAPK cascade (enrichment=2.34E -5), an important signaling pathway regulating proliferation and differentiation of HSCs and leukemic cells. Taken together, LMNA deficiency recapitulates features of aging at the functional and epigenetic level. LMNA KD in young, human CD34 + cells impaired their differentiation while increasing their colony forming potential. Similarly in middle-aged mice, LMNA KO BM cells showed increased colony forming potential. Genomic changes induced by LMNA deficiency include reduced accessibility and gene transcription, accompanied by changes in localization and occupancy of histone H3K9me2 and H3K27ac, respectively. These epigenetic changes affect genes regulating differentiation and signaling pathways. Thus, we have demonstrated that in addition to its structural role, LMNA also contributes to chromatin regulation of hematopoietic pathways important for normal CD34 + function. Disclosures No relevant conflicts of interest to declare.

The Loss of Liver Kinase B1 Accelerates Maturation Time in the Developing Erythrocyte

In the US, iron-restricted anemia contributes greatly to morbidity. The erythroid iron deprivation response, characterized by a pathway in which erythropoiesis is suppressed during iron restriction, underlies this anemia. In preliminary studies, liver kinase B1 (LKB1) was implicated as a potential key component in the erythroid iron deprivation response. Normal human CD34+ hematopoietic progenitor cells cultured for 3 days in erythroid medium with 100% or 10% transferrin saturations underwent immunoblot of whole cell lysates. Reproducibly, the levels of LKB1 did not change based on the transferrin saturation; however, immunofluorescence imaging showed a shift in subcellular localization when cells were subjected to low iron conditions. To assess the effects of LKB1 loss in the erythroid compartment, we used control and LKB1 conditional knockout (STK11 F/F; EpoR-Cre+) mice. In steady-state conditions, the loss of LKB1 does not confer a change in RBC count, though, there is a baseline increase in the number of reticulocytes, and a large increase in the level of serum Erythropoietin (Epo). In a model to precipitate anemia, these mice were challenged with intraperitoneal injection of phenylhydrazine (PHZ). It was found that LKB1 is dispensable for an appropriate response to this type of stress erythropoiesis. To assess the impact of LKB1 on maturation, we performed flow cytometric analysis using an ex vivo culture system of splenic erythroblasts. LKB1-deficient erythroid progenitors show increased percentages of more advanced cells as evidenced by the surface markers CD71 and Ter119 (the mouse analogue of human glycophorin A). Current studies are underway to assess if this change is due to signaling through the AMPK pathway. These studies will provide mechanistic detail of LKB1 function and activity on erythropoiesis, improving our understanding of programs involved in maturation of differentiation and lineage selection which may ultimately help to improve health outcomes and advance treatment for various types of anemias. Disclosures No relevant conflicts of interest to declare.