Wild-type Hematopoietic Stem/progenitor Cell Research Articles

Clonal hematopoiesis related TET2 loss-of-function impedes IL1β-mediated epigenetic reprogramming in hematopoietic stem and progenitor cells

Clonal hematopoiesis (CH) is defined as a single hematopoietic stem/progenitor cell (HSPC) gaining selective advantage over a broader range of HSPCs. When linked to somatic mutations in myeloid malignancy-associated genes, such as TET2-mediated clonal hematopoiesis of indeterminate potential or CHIP, it represents increased risk for hematological malignancies and cardiovascular disease. IL1β is elevated in patients with CHIP, however, its effect is not well understood. Here we show that IL1β promotes expansion of pro-inflammatory monocytes/macrophages, coinciding with a failure in the demethylation of lymphoid and erythroid lineage associated enhancers and transcription factor binding sites, in a mouse model of CHIP with hematopoietic-cell-specific deletion of Tet2. DNA-methylation is significantly lost in wild type HSPCs upon IL1β administration, which is resisted by Tet2-deficient HSPCs, and thus IL1β enhances the self-renewing ability of Tet2-deficient HSPCs by upregulating genes associated with self-renewal and by resisting demethylation of transcription factor binding sites related to terminal differentiation. Using aged mouse models and human progenitors, we demonstrate that targeting IL1 signaling could represent an early intervention strategy in preleukemic disorders. In summary, our results show that Tet2 is an important mediator of an IL1β-promoted epigenetic program to maintain the fine balance between self-renewal and lineage differentiation during hematopoiesis.

Single-Cell Mutation Profiling and Alternative Splicing Analysis Reveals Distinct Aberrant Splicing of U2AF1S34 and U2AF1Q157 in Myeloid Malignancies

Introduction: In myeloid neoplasms, spliceosome mutations are found in approximately 50% of patients and are often associated with a poor prognosis. The mutations occur in recurrent hotspot regions, although whether different point mutations affecting the same gene have the same impact remains uncertain. Furthermore, the mechanism through which spliceosome mutations exert a clonal advantage over wild-type cells in the hematopoietic stem/progenitor cell (HSPC) compartment remains poorly understood. Single-cell multiomic techniques combining mutation analysis with RNA-sequencing are a powerful approach to study the effect of specific mutations through an intra-patient comparison of mutant and wild-type HSPCs. Up to now, this approach has not been used to study the effects of specific spliceosome mutations. To this end, we developed experimental and computational pipelines to enable joint genotyping and alternative splicing analysis from the same single cell. We further demonstrated the utility of our approach to identify specific altered splicing events in HSPCs derived from myeloproliferative neoplasm (MPN) patients in association with specific U2AF1 hotspot mutations (S34 and Q157). Methods: >20,000 lineage-CD34+ HSPCs were derived from healthy controls and MPN patients. Among those included for analysis, six patients had JAK2V617F (n=5) or CALR mutations (n=1), of which, two patients had additional U2AF1S34 mutations and three of them, U2AF1Q157 mutations. Parallel single-cell genotyping and short-read RNA-sequencing were performed using TARGET-seq (Rodriguez-Meira et al., 2019). Subsequent alternative splicing analysis was performed using the MARVEL software (https://github.com/wenweixiong/MARVEL). On average, 10,600 splice junctions were detected per cell. Confirmation of candidate alternative splicing events was performed using Nanopore and PacBio long-read sequencing. Results: Single-cell genotyping revealed U2AF1S34 and U2AF1Q157 cells were highly clonal and constituted 85.4% (on average) of the entire HSPC compartment. In contrast, single JAK2V617F mutant patients showed a much more variable clonal burden in the HSPC compartment. We performed differential splicing analysis to identify aberrant splicing events in U2AF1S34 and U2AF1Q157 cells. Differential splicing analysis identified 423 of 2,979 (14.2%) and 1,041 of 6,857 (15.2%) splicing events that were differentially spliced between U2AF1S34 and U2AF1Q157 vs. U2AF1WT cells, respectively (Fig. A). Notably, using this single-cell approach, we successfully recapitulated previously identified U2AF1S34-mediated spliced genes including GNAS and H2AFY, as well as a number of novel aberrantly spliced genes in HSPCs. The repertoire of differentially spliced events was distinct for U2AF1S34 vs. U2AF1Q157 cells. Specifically, only 14 overlapping differentially spliced events were associated with both U2AF1S34 and U2AF1Q157 genotypes. In addition, GSEA analysis identified dysregulation of ribonucleoprotein processes exclusively in U2AF1S34 cells and dysregulation of cell cycle exclusively in U2AF1Q157 cells. This suggests that U2AF1S34 and U2AF1Q157 mutations regulate different molecular pathways in HSPCs and might give rise to clonal expansions through different mechanisms. We next performed long-read sequencing with Nanopore and PacBio to confirm the full isoform sequence of candidate splicing events (Fig. B). Long-read sequencing revealed GNAS to be differentially spliced in U2AF1 S34cells due to a combination of skipped-exon and variable 5‘ and 3‘ untranslated regions. The inclusion of the skipped-exon has been experimentally shown to hyperactivate Gαs protein and downstream ERK/MAPK signalling pathway (Wheeler et al., 2021). Each cell on average expressed two GNAS isoforms (range: 0 to 5). Notably, short-read RNA-sequencing would have limited capacity to detect these isoforms, thus highlighting the advantage of single-cell long-read RNA-sequencing in deconvoluting complex splicing events in the same cell. Conclusion: In this study we demonstrate the proof-of-principle of joint genotyping and alternative splicing analysis at single-cell resolution to identify disease-relevant splicing alterations. We anticipate our approach will enable and accelerate novel biomarker discovery with therapeutic implications in myeloid neoplasms and beyond.

Single-cell multi-omics identifies chronic inflammation as a driver of TP53-mutant leukemic evolution

Understanding the genetic and nongenetic determinants of tumor protein 53 (TP53)-mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53-mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 ‘multihit’ HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53-mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53-heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53-mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53-mutant leukemia, and are of broad relevance to other cancer types.

Exendin-4 attenuates atherosclerosis progression via controlling hematopoietic stem/progenitor cell proliferation.

Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammation and plaque development in murine atherosclerotic models. However, whether they modulate hematopoietic stem/progenitor cells (HSPCs) to prohibit skewed myelopoiesis in hypercholesteremia remains unknown. In this study, GLP-1r expression in fluorescence-activated cell sorting (FACS)-sorted wild-type HSPCs was determined by capillary western blotting. Bone marrow cells (BMCs) of wild-type or GLP-1r-/- mice were transplanted into lethally irradiated low-density lipoprotein receptor deficient (LDLr-/-) recipients followed by high-fat diet (HFD) for chimerism analysis by FACS. In parallel, LDLr-/- mice were placed on HFD for 6 weeks and then treated with saline or Exendin-4 (Ex-4) for another 6 weeks. HSPC frequency and cell cycle were analyzed by FACS, and intracellular metabolite levels were assessed by targeted metabolomics. The results demonstrated that HSPCs expressed GLP-1r and transplantation of GLP-1r-/- BMCs resulted in skewed myelopoiesis in hypercholesterolemic LDLr-/- recipients. In vitro, Ex-4 treatment of FACS-purified HSPCs suppressed cell expansion and granulocyte production induced by LDL. In vivo, Ex-4 treatment inhibited plaque progression, suppressed HSPC proliferation, and modified glycolytic and lipid metabolism in HSPCs of hypercholesteremic LDLr-/- mice. In conclusion, Ex-4 could directly inhibit HSPC proliferation induced by hypercholesteremia.

Single-Cell Multi-Omics Reveals That Pegylated Interferon-Alfa Treatment Differentially Redirects Mutated and Wildtype Hematopoietic Cell Differentiation Trajectories in CALR-mutated Essential Thrombocythemia (ET) Patients

Interferon-alpha (IFN), the first approved immunotherapy for cancer, remains an effective therapy for patients with myeloproliferative neoplasms (MPN). The mechanisms of action of IFN on MPN cells are poorly understood, particularly in patients with CALR mutated (MUT) MPNs, who often exhibit clinical but not molecular responses. Previously, by developing Genotyping of Transcriptomes (GoT) that captures mutation status and single-cell RNA-seq (scRNA-seq) in high-throughput, we observed that CALR mutations led to cell identity-dependent effects on CD34 + cells, including a strong megakaryocytic progenitor (MkP) differentiation bias and fitness. We hypothesized that the IFN effects may be cell identity and mutation status dependent; thus we applied GoT to serial bone marrow aspirates (BM) from 5 patients with CALRmutated ET treated with pegylated-IFN-alfa2a who participated in MPD-RC-111/112 clinical trials.To capture the transcriptional impact of IFN, we removed experimental batch effects with Cell Hashing, in which CD34 + cells from serial BM were uniquely labeled and combined for the same GoT experiment (Fig. 1A). Cell clustering based on transcriptomic data alone revealed that the cells on active treatment clustered based on cell identity and IFN effects (Fig. 1B). When off therapy for 3 weeks, the strong transcriptional effects of IFN were largely lost (Fig. 1B).Next, we batch corrected and integrated across time points for each BM sample (Fig. 1C). We observed that IFN caused large shifts in the composition of wildtype (WT) and MUT cell subsets (Fig. 1D). IFN resulted in a dramatic expansion of WT lymphoid progenitors with a corresponding diminution of other progenitors (Fig. 1E). MUT cells at baseline were enriched for MkPs, compared to WT cells; after treatment, we observed an expansion of the immature myeloid (IMP) and neutrophil progenitors, with a less striking expansion of lymphoid progenitors (Fig. 1E).As IFN has been reported to induce cell cycling of murine hematopoietic progenitor cells, we examined whether a differential increase in proliferation by IFN underlies the differentiation shifts in WT and MUT cells. Cell cycle gene expression of ProB cells increased after treatment similarly in MUT and WT cells, while cell cycle expression of IMPs was increased to a greater extent in MUT cells (Fig. 1F), consistent with the differential shifts in populations.Next, we performed differential expression analysis between baseline and treated WT and MUT cells, respectively. We observed enrichment of the IFN pathways post-therapy, whereas TNF-a signaling was downregulated (Fig. 1G). Uniquely in the MUT cells, TGF-b signaling was downregulated, which may underlie improvements in marrow fibrosis following IFN therapy (Fig. 1G).Finally, as the differentiation biases of IFN persisted after discontinuation, we hypothesized that IFN results in chromatin remodeling of the earliest hematopoietic stem progenitor cells (HSPCs), with respect to transcription factor (TF) accessibility. We leveraged single nuclei chromatin accessibility (snATAC-seq) as a powerful measure of TF regulatory activities. We developed GoT-ATAC, an adaptation of the Multiome platform (10x Genomics), to capture snRNA-seq, snATAC-seq and somatic genotyping within the same cells in high-throughput (Fig. 1H). We applied GoT-ATAC to CD34 + cells from the same clinical trial cohorts (Fig. 1I, n = 3 patients: 3 baseline, 2 treated) and identified the expected enrichment of IRFs and STAT2 in treated HSPCs (Fig. 1J). Accessibility of BCL11A, critical for early lymphoid development, was increased in treated MUT and WT HSPCs. We also identified enhanced motif accessibility of PU.1 which can associate with IRF and is essential for myeloid and lymphoid differentiation. Uniquely within the treated MUT cells, we observed enhanced CEBPA motif enrichment, which regulates myeloid differentiation, together with PU.1.In conclusion, GoT revealed that IFN reshapes the differentiation landscape by promoting early lymphoid development and, uniquely in MUT cells, myeloid differentiation, providing a novel mechanism of actions underlying the effects of IFN in MPN patients. Downregulations of TNF-a and TGF-b signaling were other key molecular consequences of IFN. Lastly, GoT-ATAC demonstrated that IFN governs master regulators of hematopoietic differentiation as a function of the underlying mutational status. [Display omitted] DisclosuresMimitou: Immunai: Current Employment. Smibert: Immunai: Current Employment. Hoffman: AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Kartos Therapeutics, Inc.: Research Funding.

Introduction of STAG2 Mutation in an iPSC Model of Transient Abnormal Myelopoiesis Mimics Down Syndrome Myeloid Leukemia

BackgroundChildren with Down syndrome (DS) have a high risk for acute myeloid leukemia (DS-ML). Genomic characterization of DS-ML blasts showed the presence of unique mutations in GATA1, an essential hematopoietic transcription factor, leading to the production of a truncated from of GATA1 (GATA1s). GATA1s together with trisomy 21 is sufficient to develop a pre-leukemic condition called transient abnormal myelopoiesis (TAM). Approximately thirty percent of these cases progress into DS-ML by acquisition of additional somatic mutations in a step-wise manner. We previously developed a model for TAM by introducing disease-specific GATA1 mutation in trisomy 21 induced pluripotent stem cells (iPSCs) leading to the production of N-terminally truncated short form of GATA1 (GATA1s) (Barwe et al., 2021). In this study, we introduced co-operating mutation in STAG2, a member of the cohesin complex recurrently mutated in DS-ML but not in TAM, and evaluated its effect on hematopoietic differentiation.MethodsTwo different iPSC lines with trisomy 21 with or without GATA1 mutation as described in Barwe et al., 2021, were used. CRISPR/Cas9 gene editing was performed to introduce STAG2 mutation to generate a knockout of STAG2. Hematopoietic differentiation of these iPSC lines was performed using STEMdiff Differentiation kit. ProteinSimple Wes system was used for western blot analysis. Multi-dimensional flow cytometry was used for immunophenotypic analysis of megakaryoblasts cultured in lineage expansion media for 5 days. Multi-lineage colony forming potential was assessed by Methocult colony forming assay using day 10 hematopoietic stem progenitor cells (HSPCs).ResultsHematopoietic differentiation of GATA1 and STAG2 double mutants in two independent trisomy 21 iPSC lines confirmed GATA1s expression and the loss of functional STAG2 protein (Fig. 1A). GATA1s expressing HSPCs collected on day 12 post differentiation showed reduced erythroid (CD71+CD235+) and increased megakaryoid (CD34+CD41+ within CD41+ compartment) and myeloid (CD18+CD45+) population compared to disomy 21 HSPCs with wild-type GATA1, consistent with our previous study (Fig. 2B). STAG2 knockout HSPCs showed higher erythroid population (P=0.033 and 0.016 in T21-1S and T21-2S respectively) and reduced myeloid population while it had no significant effect on the megakaryoid population in both iPSC lines. The GATA1s/STAG2 knockout HSPCs showed reduced erythroid, but higher megakaryoid and myeloid population compared to wild-type HSPCs. Strikingly, the immature megakaryoid population was significantly higher in the double mutant HSPCs compared to single mutant alone in both iPSC lines (P=0.005 and 0.004 for T21-1GS and T21-2GS respectively), indicating that the STAG2knockout co-operated with GATA1s for increasing megakaryoid population.The trisomy 21 iPSC line with wild-type GATA1 developed CFU-GEMM (colony-forming unit granulocyte erythroid macrophage megakaryocyte), CFU-GM (CUF granulocyte-macrophage) and BFU-E (burst-forming unit erythroid) colonies in Methocult. GATA1 mutation, unlike STAG2 mutation, inhibited the formation of CUF-GEMM and BFU-E colonies. The number of CFU-GM colonies in T21-2GS was significantly reduced compared to T21-2G (Fig. 1C, p=0.002). Lineage expansion and immunophenotyping of these HSPCs in megakaryocyte-specific media showed that these cells expressed markers closely resembling DS-ML immunophenotype. Of note, the myeloid markers, CD13 and CD11b are the only two markers expressed on majority of DS-ML blasts compared to TAM blasts (Karandikar et al., 2001) (Yumura-Yagi et al., 1992). The percentage of CD13 and CD11b expressing cells was higher in megakaryoblasts expanded from iPSC lines with STAG2 GATA1 double mutant (Fig. 1D). The number of cells expressing CD117, a stem cell marker shown recently to be involved in DS-ML progression, were highest in T21-1GS and T21-2GS lines when compared to their respective isogenic family of GATA1 mutant lines.ConclusionGATA1s and STAG2 knockout co-operated to increase the megakaryoid population and the percentage of cells expressing DS-ML markers. We have developed a model system representing DS-ML, which can be used for understanding the individual and synergistic contribution of these gene mutations in disease initiation and progression. [Display omitted] DisclosuresBarwe: Prelude Therapeutics: Research Funding. Gopalakrishnapillai: Geron: Research Funding.

JAK2V617F mutant endothelial cells promote neoplastic hematopoiesis in a mixed vascular microenvironment

The chronic myeloproliferative neoplasms (MPNs) are clonal stem cell disorders. The hematopoietic stem/progenitor cell (HSPC) compartment in patients with MPNs is heterogeneous with the presence of both wild-type and JAK2V617F mutant cells. Mechanisms responsible for mutant stem cell expansion in MPNs are not fully understood. Vascular endothelial cells (ECs) are an essential component of the hematopoietic microenvironment. ECs carrying the JAK2V617F mutation can be detected in patients with MPNs. Utilizing an ex vivo EC-HSPC co-culture system with mixed wild-type and JAK2V617F mutant ECs, we show that even small numbers of JAK2V617F mutant ECs can promote the expansion of JAK2V617F mutant HSPCs in preference to wild-type HSPCs during irradiation or cytotoxic chemotherapy, the two treatments commonly used in the conditioning regimen for stem cell transplantation, the only curative treatment for patients with MPNs. Mechanistically, we found that both cell-cell interactions and secreted factors are important for JAK2V617F mutant EC-mediated neoplastic hematopoiesis. Further understanding of how the JAK2V617F mutation alters vascular niche function will help identify new strategies to not only control neoplastic cell expansion but also prevent disease relapse in patients with MPNs.

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X‐linked hyper‐IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one‐size‐fits‐all editing strategy for effective T‐cell correction, selection, and depletion and investigated the therapeutic potential of T‐cell and HSPC therapies in the HIGM1 mouse model. Edited patients’ derived CD4 T cells restored physiologically regulated CD40L expression and contact‐dependent B‐cell helper function. Adoptive transfer of wild‐type T cells into conditioned HIGM1 mice rescued antigen‐specific IgG responses and protected mice from a disease‐relevant pathogen. We then obtained ~ 25% CD40LG editing in long‐term repopulating human HSPC. Transplanting such proportion of wild‐type HSPC in HIGM1 mice rescued immune functions similarly to T‐cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T‐cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.

TGF-β Signaling Contributes to the Clonal Dominance of Jak2V617F Hematopoietic Stem/Progenitor Cells

Increased TGF-β expression in the bone marrow microenvironment is present in most cases of myeloproliferative neoplasms (MPNs), however, its contribution to disease pathogenesis is not well understood. Multiple prior studies show that TGF-β suppresses the proliferation of hematopoietic stem/progenitor cells (HSPCs) in vitro. However, its impact on HSCs in vivo is less clear, with conflicting data showing that TGF-β suppresses or induces HSPC proliferation, depending upon the dose of TGF-β and cell context. Here, we explore the hypothesis that HSPCs carrying a Jak2V617F mutation are resistant to the suppressive effects of TGF-β, conferring a fitness advantage that contributes to their expansion in MPNs. To test this hypothesis, we first assessed TGF-β signaling in wildtype (WT) and Jak2V617F HSPCs. Whereas TGF-β1 induced robust Smad2/3 phosphorylation in WT phenotypic hematopoietic stem cells (HSCs), it was barely detectable in Jak2V617F HSCs. Interestingly, this phenotype was lost in lineage-restricted progenitors, where TGF-β1-induced Smad2/3 phosphorylation was comparable in WT and Jak2V617F cells. These data suggest that TGF-β signaling, at least Smad2/3 phosphorylation, is selectively loss in Jak2V617F HSCs but not lineage-restricted progenitors. Competitive repopulation assays (CRAs) were performed using WT and Jak2V617F HSPCs in which Tgfbr2 was deleted to abrogate TGF-β signaling in HSCs. We first established bone marrow chimeras containing equal amounts of WT and UBC-CreERT2; Tgfbr2f/f HSPCs (hereafter referred to as TR2KO cells). Tamoxifen was given 6 weeks after transplantation to activate Cre expression and delete Tgfbr2. Multilineage donor chimerism of the Tgfbr2-deleted cells increased over time, suggesting that TGF-β signaling negatively regulates HSPC repopulating activity. However, when competing Jak2V617F cells with Jak2V617F TR2KO cells, the TR2KO compartment no longer had competitive advantage, suggesting that Jak2 mutated HSPC are insensitive to the suppressive effect of TGF-β. To extend these findings, CRAs were established that included the following 4 cohorts: 1) WT vs. Jak2V617F; 2) TR2KO vs. Jak2V617F; 3) TR2KO vs. Jak2V617FTR2KO; and 4) WT vs. Jak2V617FTR2KO. In each case, a 5:1 ratio of competitor to Jak2-mutated cells was used and tamoxifen was given 6 weeks after transplantation to induce both Cre-mediated deletion of Tgfbr2 and activation of the Jak2V617F allele. In cohort 1, as expected, donor chimerism with Jak2 mutated cells increased over time (Figure 1A, black). Consistent with this finding, Jak2 mutant chimerism in HSPCs was increased at 12-14 weeks (Figure 1B). In cohort 2 (orange), the loss of TGF-β signaling in WT HSPCs completely abrogated the competitive advantage of Jak2V617F HSPCs. In fact, the TR2KO cells outcompeted Jak2V617F HSPCs. In cohort 3 (red), we asked whether loss of TGF-β signaling in both WT and Jak2V617F HSPCs "leveled the playing field". Indeed, no expansion of Jak2 mutant peripheral blood leukocytes or HSPCs was observed in these mice. Finally, in cohort 4 (blue), we observed that loss of TGF-β signaling in Jak2 mutant cells had little impact on their repopulating activity. To assess the impact of TGF-β signaling on WT and Jak2V617F HSPC proliferation, we generated bone marrow chimeras containing either WT vs. Jak2V617F HSPCs or TR2KO vs. Jak2V617FTR2KO HSPCs. Of note, in both types of bone marrow chimeras, the presence of Jak2 mutant cells is expected to increase the local level of endogenous TGF-β. As expected, in the WT vs. Jak2V617F chimeras, the Jak2V617F HSPCs were less quiescent than WT cells. Surprisingly, loss of TGF-β signaling resulted in an increase in HSC quiescence in both TR2KO and Jak2V617F TR2KO HSCs. Of note, the loss of TGF-β signaling again "leveled the playing field" with a similar percentage of quiescent TR2KO vs. Jak2V617FTR2KO HSCs. Collectively, these data suggest that attenuated TGF-β signaling in Jak2V617F HSPCs plays a key role in the pathogenesis of MPN. Specifically, the data support a model in which increased local production of TGF-β by Jak2V617F hematopoietic cells selectively inhibits WT HSPCs, thereby promoting the expansion of Jak2V617F HSPCs. Targeting TGF-β signaling early in the course of MPN or in persons with Jak2V617F clonal hematopoiesis may prevent or delay clonal progression. Disclosures No relevant conflicts of interest to declare.

Cell Competition and Microenvironment in the Development of Myeloproliferative Neoplasms

Introduction The myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion. The acquired kinase mutation JAK2V617F plays a central role in MPNs. Both JAK2 wild-type (WT) clones and JAK2V617F-mutant clones co-exist in many MPN patients. Little is known about earliest steps of MPN disease initiation and how the mutant cells expand over WT cells. In this work, we tested the hypothesis that competition between WT and JAK2V617F-mutant cells acts to prevent mutant cell expansion in MPNs, but that a JAK2V617F-bearing microenvironment can overcome this competition, leading to the development of a MPN. Methods JAK2V617F Flip-Flop (FF1) mice and Tie2-Cre mice were crossed to generate a strain in which human JAK2V617F is expressed specifically in hematopoietic cells and vascular endothelial cells (Tie2FF1). Results First, we isolated JAK2WT and JAK2V617F-mutant CD45+CD201+CD150+CD48- (here termed HSPCs) from either WT mice or Tie2FF1 mice and performed a series of in vitroco-culture assays. We found that JAK2V617F HSPCs displayed a higher proliferation rate than JAK2WT HSPCs when cultured separately. In contrast,when placed in direct co-culture, proliferation of JAK2WT HSPCs was greatly increased that no significant difference between WT and mutant HSPCs was detected. Similar results were also obtained when using a transwell co-culture system, suggesting that cell-cell contact was not required for the competitive co-existence we observed in mixed JAK2WT / JAK2V617F HSPC cultures in vitro. (Figure 1) Next, we performed marrow transplantation assays to assess the competition between JAK2WT and JAK2V617F HSPCs in vivo. We found that, when 50% JAK2V617F marrow cells and 50% JAK2WT marrow cells were transplanted together into lethally irradiated WT mice, the JAK2V617F donor cells had no engraftment advantage over JAK2WT donor cells and the recipient mice had normal blood cell counts during more than 4-month follow up. This result contrasts our and other reports that a JAK2V617F-positive MPN develops following transplantation of 100% JAK2V617F-mutant donor cells into WT recipients. These observations suggest that the presence of WT cells can alterthe behavior of JAK2V617F-mutant stem cells in vivo. This conclusion was further supported by in vivoBrdU labeling experiments, which demonstrated that the proliferation rate of JAK2V617F-mutant Lin-cKit+Sca1+CD150+CD48-(here termed HSCs)was 2-fold higher than JAK2WT HSCs when transplanted separatelyinto lethally irradiated WT recipients; however, when transplanted together, WT HSCs displayed a significantly higher proliferation rate (3-fold) than mutant HSCs. (Figure 2) Endothelial cells (ECs) are an essential component of the hematopoietic niche and many studies have established that a significant number of ECs in MPN patients carry the JAK2V617F mutation. Using the same Tie2FF1 murine model, we previously reported that a JAK2V617F-bearing vascular niche promotes the expansion of JAK2V617F HSPCs over JAK2WT HSPCs, and the level of Chemokine (C-X-C motif) ligand 12 (CXCL12) was upregulated in JAK2V617F-mutant ECs compared to WT ECs. Cell cycle analysis using Hoechst33342 and Pyronin Y staining demonstrated that, while there were no differences in WT and mutant HSPC cell cycle status when transplanted into a WT vascular niche, JAK2V617F-mutant HSPCs were less quiescent (more cycling) than WT HSPCs when transplanted into a JAK2V617F-mutant vascular niche. (Figure 3) To test the hypothesis that JAK2V617F-mutant vascular niche overcomes the competition between WT and mutant cells to promote mutant HSPC expansion through increased CXCL12 level, we used the same competitive repopulation assay as in Figure 2 where both JAK2WT and JAK2V617F marrow cells were injected together into lethally irradiated WT recipient. At 12wks post transplantation, the recipient mice were treated with a single dose of CXCL12 (400ng, intravenously) before BrdU labeling. We found that CXCL12 treatment stimulated JAK2V617F-mutant HSC proliferation to a greater extent than JAK2WT HSPCs. (Figure 4) Conclusions Cell competition between WT and JAK2V617F-mutant cells prevents mutant stem cells from expansion, but a mutant microenvironment (e.g. JAK2V617F-mutant vascular niche) can overcome this competition to promote mutant stem cell expansion and the development of a murine MPN. Disclosures No relevant conflicts of interest to declare.

Relatively Low Sensitivity of CD109(-) Hematopoietic Stem/Progenitor Cells (HSPCs) to TGF-β: A Possible Mechanism Responsible for the Preferential Commitment of Piga Mutant HSPCs in Immune-Mediated Bone Marrow Failure

[Background] An increase in the numbers of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] blood cells is often detected in patients with acquired aplastic anemia (AA) and low-risk myelodysplastic syndrome (MDS), and is associated with good response of their bone marrow (BM) failure to immunosuppressive therapy. Although some immune mechanisms are thought to play a role in the preferential commitment of hematopoietic stem/progenitor cells (HSPCs) with PIGA mutations in such BM failure patients, the exact mechanisms are unknown. Our previous studies suggested that GPI(-)T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) were less susceptible to TGF-ƒÀ-mediated inhibition of proliferation triggered by anti-CD3 and anti-CD28 antibodies than GPI(+)T cells of the same patient. The lower sensitivity of PIGA mutant HSPCs to TGF-ƒÀ, a cytokine capable of inhibiting the cell cycling of dormant HSPCs, than GPI(+) HSPCs may also explain the preferential commitment of GPI(-) HSPCs in immune-mediated BM failure. However, little is known about the GPI-APs that affect the sensitivity of HSPCs to TGF-ƒÀ.[Objectives/Methods] We assessed the roles of GPI-APs in the signal transduction of CD34(+) cells of a PNH patient and the myeloid leukemia cell line TF-1 in response to TGF-ƒÀ. We also assessed the TGF-ƒÀ-mediated inhibition of cell proliferation in TF-1 cells with or without a PIGA mutation. CD109, a GPI-AP that serves as a TGF-ƒÀ co-receptor in human keratinocytes, of TF-1 cells, was knocked out from TF-1 cells using a CRISPR-Cas 9 system, and the sensitivity to TGF-ƒÀ was compared between CD109(+) and CD109(-) TF-1 cells.[Results] The treatment of BM mononuclear cells from a florid PNH patient with TGF-ƒÀ induced SMAD2 phosphorylation in GPI(-) CD34(+) cells to a lesser degree than in GPI(+) CD34(+) cells (fold increase in pSMAD2 MFI, 1.0 vs. 2.6, Figure 1). TGF inhibited PIGA-mutant TF-1 (PNH-TF-1) proliferation to a lesser degree (percentage of inhibition, 19%}13%) than wild-type TF-1 cells (67%}3%) in an MTT-based proliferation assay. Transfection of PIGA into PNH-TF-1 cells restored GPI-AP expression as well as sensitivity to TGF-ƒÀ (53%}10% vs. 19%}13% in PNH-TF-1 cells). CD109 coimmunoprecipitated with TGF-ƒÀ in TF-1 cells, and its expression was confirmed on BM CD34+ cells of healthy individuals, particularly CD34+CD38+CD123-CD45RA- megakaryocyte-erythroid progenitor cells, as well as on TF-1 cells. The pSMAD2 induction in CD109(-) TF-1 cells by TGF-ƒÀ was less pronounced (relative increase in pSMAD2 MFI, 7.65}2.15 vs. 10.74}2.28) than that in CD109(+) TF-1 cells (Figure 2).[Conclusions] CD109 deficiency is involved in the lower sensitivity of GPI(-) HSPCs to TGF-ƒÀ than GPI(+) HSPCs. This deficiency may account for the preferential activation of PIGA mutant HSPCs in immune-mediated BM failure, in which TGF-ƒÀ suppresses activation of wild-type HSPCs. [Display omitted] [Display omitted] DisclosuresHosokawa:Aplastic Anemia and MDS International Foundation: Research Funding. Nakao:Alexion Pharmaceuticals: Honoraria, Research Funding.

Overexpression and Knockout of Mir-126 Both Promote Leukemogenesis through Targeting Distinct Gene Signaling

MicroRNAs (miRNAs) have been implicated in the pathogenesis of various types of cancers. We reported previously that miR-126 likely functioned as an oncogene in acute myeloid leukemia (AML) (Li Z, et al. PNAS. 2008 Oct 7; 105:15535-40), which was confirmed by others (de Leeuw DC, et al. Cancer Research. 2014 Apr 1,74:2094-105; Dorrance AM, et al. Leukemia. 2015). However, it was also reported that miR-126 knockdown in normal hematopoietic stem/progenitor cells (HSPCs) resulted in expansion of long-term HSCs (Lechman ER, et al. Cell Stem Cell. 2012 Dec 7,11:799-811). Thus, the role of miR-126 in normal and malignant hematopoiesis warrants further investigation.Here we show that miR-126 is particularly overexpressed in t(8;21) AML and increased expression of miR-126 is associated with poor prognosis in patients with t(8;21) AML. To determine the function of miR-126 in leukemogenesis, we conducted both gain- and loss-of-function in vivo studies of miR-126 in t(8;21) AML models via mouse bone marrow transplantation (BMT). Surprisingly, we found that both overexpression and knockout of miR-126 accelerated leukemogenesis by the t(8;21) fusion genes, AML1-ETO (AE) or AML1-ETO9a (AE9a; a potent oncogenic isoform of AE). For example, forced expression of miR-126 (by retroviral transduction?) significantly accelerated AE9a-mediated leukemogenesis (median survival of AE9a+miR-126 vs. AE9a: 234 days vs. 317 days, p <0.01), and AE9a -transduced miR-126-/- HSPCs (miR-126KO+AE9a) caused leukemia significantly faster than did AE9a -transduced wild-type HSPCs (AE9a) in recipients (median survival: 130 days vs. 317 days, p <0.0001).To assess the impact of miR-126 overexpression and depletion on long-term self-renewal of t(8;21) leukemia stem/initiating cells (LSCs/LICs), we performed serial mouse BMT assays with leukemic BM cells collected from the prior generation of BMT recipients as donor cells. In the secondary mouse BMT assay, we found that the AE9a+miR-126 group showed similar leukemia development to the miR-126KO+AE9a group (median survival: 79 days vs. 81 days, p =0.22), and both groups had significantly shorter survival (p <0.01) than did the AE9a group (median survival: 116 days). Strikingly, the AE9a +miR-126 group developed leukemia significantly faster than did the miR-126KO+AE9a group in both tertiary and quaternary BMT (median survival: 28 days vs. 43 days, p =0.0001 in tertiary; 35 days vs. 49 days, p <0.0001 in quaternary), and both groups developed leukemia significantly faster (p <0.0001) than did the AE9a group. Our limiting dilution assays with mouse BM leukemic cells collected from secondary BMT recipients as donor cells showed that the estimated LSCs/LICs frequency of the AE9a+miR-126 group is significantly greater (p <0.001) than that of the miR-126KO+AE9a group (1/2,476 vs. 1/27,399; >10 fold), and both were significantly higher (p <0.01) than that (1/166,619) of the AE9a group. Furthermore, consistent with the association of increased expression of miR-126 with poor survival of t(8;21) AML patients, our in vivo and in vitro studies demonstrated that depletion or inhibition of miR-126 significantly sensitized t(8;21)-fusion-induced mouse leukemia or primary human leukemia cells to standard chemotherapy.Our mechanistic studies indicate that miR-126 overexpression activates genes that are highly expressed in LSCs/LICs and/or primitive HSPCs through directly targeting ERRFI1 and SPRED 1, which in turn activate the MAPK signaling pathway. On the other hand, miR-126 knockout activates genes that are highly expressed in committed, more differentiated hematopoietic cells and triggers the WNT/β-catenin signaling pathway by inducing FZD7 expression. We show that ERRFI1, SPRED 1 and FZD7 are bona fide targets of miR-126, and their increased expression is associated favorable survival of t(8;21) AML patients. Overall, our data show that both gain- and loss-of-function of a single miRNA (e.g., miR-126) can promote tumorigenesis and enhance long-term self-renewal/progression of LSCs/LICs, through targeting distinct gene signaling and thus being associated with different biological consequences. As miR-126 depletion can sensitize AML cells to standard chemotherapy, our data also suggest that miR-126 represents a promising therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation.

Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis. We investigated the potential for HSPC transplantation to treat corneal defects in cystinosis. We isolated HSPCs from transgenic DsRed mice and systemically transplanted irradiated Ctns-/- mice. A year posttransplantation, we investigated the fate and function of HSPCs by in vivo confocal and fluorescence microscopy (IVCM), quantitative RT-PCR (RT-qPCR), mass spectrometry, histology, and by measuring the IOP. To determine the mechanism by which HSPCs may rescue disease cells, we transplanted Ctns-/- mice with Ctns-/- DsRed HSPCs virally transduced to express functional CTNS-eGFP fusion protein. We found that a single systemic transplantation of wild-type HSPCs prevented ocular pathology in the Ctns-/- mice. Engraftment-derived HSPCs were detected within the cornea, and also in the sclera, ciliary body, retina, choroid, and lens. Transplantation of HSPC led to substantial decreases in corneal cystine crystals, restoration of normal corneal thickness, and lowered IOP in mice with high levels of donor-derived cell engraftment. Finally, we found that HSPC-derived progeny differentiated into macrophages, which displayed tunneling nanotubes capable of transferring cystinosin-bearing lysosomes to diseased cells. To our knowledge, this is the first demonstration that HSPCs can rescue hereditary corneal defects, and supports a new potential therapeutic strategy for treating ocular pathologies.

Deconvoluting MLL1-dependent pathways in hematopoiesis and leukemogenesis

The Mixed Lineage Leukemia gene (MLL1, previously termed HRX, ALL-1, MLL) was identified in humans as a gene at the site of common chromosomal translocations in infant leukemia.1 It is now clear that MLL1 represents a family of histone methyltransferases (HMTs) including its closest relative MLL2 and MLL3, MLL4, SETD1A, SETD1B and possibly MLL5.2 Most of these proteins possess histone H3, lysine 4 (H3K4) HMT activity encoded by the SET domain (for Suvar 3–9, Enhancer of Zeste, Trithorax, epigenetic regulators). Studies of the Drosophila homeotic regulator and MLL1 ortholog, Trithorax, established the paradigm for mechanisms by which these proteins function in vivo.3 For example, the fact that Trithorax regulates clustered homeodomain (Hox) genes prompted this line of investigation in the first loss-of-function mouse Mll1 mutants, which were demonstrated to mis-express multiple Hox genes.4, 5, 6 Since the discovery that the SET domain encodes HMT activity,7, 8 great attention has been paid to this particular enzymatic activity in regulating downstream target genes. To determine the function of MLL1 in hematopoietic stem/progenitor cells (HSPCs), we previously analyzed conditional knockout animals and determined that deletion of Mll1 rapidly reduced HSPCs through proliferation and cell fate defects.9 Using this model system, we identified genes that were deregulated upon Mll1 deletion in the hematopoietic stem cell (HSC)-enriched LSK/CD48neg population. Small-scale chromatin immunoprecipitation experiments identified a series of transcriptional regulators that were direct MLL1 target genes including Mecom, Pbx1 and Prdm16, which are known to control HSC self-renewal and quiescence.10 To determine whether maintenance of these target genes required the HMT activity of MLL1, a mouse strain in which homozygous germline deletion of the SET domain of MLL1 (ΔSET) was used for the analysis of the hematopoietic system.6 Surprisingly, we found that H3K4 methylation at target genes such as Hoxa9, Mecom and Prdm16 was absolutely normal in hematopoietic cells of ΔSET-mutant animals, as was the expression level of these genes. We also found that retroviral MLL-AF9 expression generated leukemia in ΔSET HSPCs that was indistinguishable in latency or phenotype from leukemia generated in wild-type HSPCs. Collectively, none of the known biological functions of MLL1 in the hematopoietic system required the HMT activity. To understand how MLL1 maintains the expression of its target genes, a survey of histone modifications that undergo changes upon deletion of Mll1 was performed. We identified only histone H4 lysine 16 acetylation (H4K16Ac) as immediately corresponding to the decrease in transcriptional initiation upon MLL1 removal from the target gene promoter regions. This modification is performed by the histone acetyl transferase MOF (also called Myst1 or KAT8), previously discovered as part of the MLL1 protein complex.11 Furthermore, inhibition of SIRT1, a deacetylase that removes H4K16Ac, was sufficient to prevent the loss of transcription at MLL1 targets. These observations together with our data from the MLL1▵SET knockout mouse provide a clear but unexpected mechanism for the maintenance of target gene expression by MLL1 in the hematopoietic cells.12 These studies highlight differences between endogenous MLL1 and MLL-fusion oncoprotein gene regulation mechanisms, suggesting that differential targeting in leukemia may be feasible.

Glypican-3–mediated inhibition of CD26 by TFPI: a novel mechanism in hematopoietic stem cell homing and maintenance

AbstractDirectional migration determines hematopoietic stem/progenitor cell (HSPC) homing, which depends upon the interaction between the chemokine CXCL12 and its receptor CXCR4. CD26 is a widely expressed membrane-bound ectopeptidase that cleaves CXCL12 thereby depleting its chemokine activity. We identified tissue-factor pathway inhibitor (TFPI) as a biological inhibitor of CD26 in murine and human HSPCs. We observed low-level TFPI expression in endothelial cells in the bone marrow (BM), which did not increase following radiation injury. Treatment of HSPCs with TFPI in vitro led to enhanced HSPC migration toward CXCL12, as well as homing and engraftment in the BM upon transplantation. We found that Glypican-3 (GPC3), a heparan sulfate proteoglycan expressed on murine as well as human HSPCs, mediated this effect. TFPI did not affect CD26 activity, migration, or homing of GPC3−/− HSPCs, while it affected GPC1−/− HSPCs similar to wild-type HSPCs. Moreover, proliferation of GPC3−/− but not GPC1−/− BM HSPCs was significantly increased, which was associated with a decrease in the primitive HSC pool in BM and an increase in proportion of the circulating HSPCs in the peripheral blood. Hence, we present a novel role for TFPI and GPC3 in regulating HSC homing as well as retention in the BM.

Stress Hematopoiesis Reveals Abnormal Control of Self-Renewal, Lineage-Bias and Myeloid Differentiation in Mll Partial Tandem Duplication (Mll-PTD) Hematopoietic Stem/Progenitor Cells

AbstractAbstract 3501Rearrangements of the Mixed-Lineage Leukemia (MLL) gene occur in a variety of aggressive human leukemias. The most common ones are balanced translocations in which the genomic sequences encoding the N-terminal portion of MLL are fused to sequences encoding the C-terminus of another translocation partner in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL). Another mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and AML, but rarely seen in ALL, is partial tandem duplication (MLL-PTD). The MLL–PTD was first identified in de novo AML with a normal karyotype or trisomy 11. Cloning of this region revealed partial duplications within the 5′ region of the MLL gene. These duplications consist of an in-frame repetition of MLL exons in a 5′–3′ direction and produce an elongated protein. The incidence of MLL–PTD was 8% in unselected adult and childhood AML and 5% in MDS. However, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Mll-PTD knock-in mouse model, its expression is regulated by endogenous promoter, has been generated to study the function of Mll-PTD in vitro and in vivo and to identify its downstream targets. This mouse model provides a powerful genetic tool to identify disruptions in normal cellular regulation as a result of this mutation, as well as a model to characterize the contribution of the Mll-PTD in leukemogenesis. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state due to increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT–derived phenotypic short-term (ST)-HSCs/multipotent progenitors (MPPs) and granulocyte/macrophage progenitors (GMPs) have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. In conclusion, the MllPTD/WT mouse model provides unique genetic and biochemical tool to identify new targets and pathways responsible for the altered differentiation/repopulating properties, self-renewal activity, lineage bias and myeloid differentiation blockade relevant to MLL-PTD MDS and AML. This model should also help us to understand the underlying mechanism(s) for each of the phenotypes we found in this study and facilitate improved therapies and patient outcomes in the future. Disclosures:No relevant conflicts of interest to declare.

Modeling MLL-PTD Related Myelodysplastic Syndromes in Mouse.

AbstractAbstract 2811MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we’ve recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML.We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn’t develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures:No relevant conflicts of interest to declare.

Rho Gtpase Cdc42 Is Essential for Endothelial Niche Maintenance in the Bone Marrow and Hematopoiesis

AbstractAbstract 508Adult hematopoietic stem/progenitor cells (HSPCs) are maintained in the unique microenvironment of bone marrow (BM) niche to sustain life-long blood production of mammals. Several types of cells in the BM, including endothelial cells (ECs), have been shown to contribute to the niche components in regulating the homeostasis of HSPCs as well as leukemia stem cells in the BM. However, because of the current technical limitations in studying BM ECs in mice, particularly the lack of an EC-specific inducible gene targeting method in adult mice and controversies surrounding the suitable murine EC culture conditions in vitro, the signaling pathways that regulate the putative BMEC niche remain poorly understood. Here we have utilized several complementary mouse models to achieve an inducible, BMEC-specific deletion of Cdc42 to unveil an essential role of this Rho family GTPase in regulating BMEC morphogenesis and proliferation, and in maintaining hematopoietic homeostasis of adult mice. First, by using the interferon-inducible Mx1-Cre;Cdc42lox/lox and the tamoxifen inducible Rosa26-CreER;Cdc42lox/lox mice engrafted with syngeneic WT HSPCs, we found that both conditions of inducible deletion of Cdc42 in the BM microenvironment, including BMECs, caused sinusoidal dilation, a drastic decline of endothelial capillary density, and a loss of endothelial progenitor activity and mature endothelial cells, in the bone marrow. Significantly, the BMEC phenotypes of these non-specific Cre-driver genetic models could be recapitulated by using a mouse model of Tie2-CreER;Cdc42lox/lox that allows inducible endothelial lineage-specific deletion of Cdc42 in the BM in response to tamoxifen, which we have shown rigorously by lineage tracing to be a suitable model for BMEC characterizations (Feng et al., ASH abstract, 2012). Second, we have established an in vitro culture system that expands and maintains primary BMECs in culture, allowing a direct assessment of the proliferation, survival, adhesion, migration, tube formation, as well as capillary assembly in 3D, activities of the BMECs. Deletion of Cdc42 by in vivo tamoxifen induction or in vitro Cre transduction of the BM cells of Tie2-CreER; Cdc42lox/lox genotype resulted in a complete loss of angiogenesis activity of BMECs that was associated with a drastic reduction of adhesion, tube-formation, and 3D capillary assembly capabilities of BMECs. In addition, endothelial cell proliferation was also compromised, as seen by a decrease of CyclinD1 expression and a diminished BrdU incorporation. These genetic results were phenocopied by findings using a Cdc42 specific inhibitor, CASIN, as CASIN treatment inhibited BMEC adhesion, diminished BM endothelial colony-forming activity, and suppressed BMEC tube-formation. A rescue experiment using retroviral expression of a panel of effector-coupling defective Cdc42 mutants and/or constitutively active PAK or AKT mutant in the Cdc42 knockout BMECs identified AKT and p21 PAK as the major downstream effectors of Cdc42 in BMEC maintenance. Third, using the three inducible mouse models, we found that Cdc42 in BMECs and microenvironment is required for maintaining hematopoietic homeostasis. Deletion of Cdc42 in BMECs caused a mobilization of HSPCs to the peripheral organs and a subsequent reduction of wild-type HSPCs in the BM, as assayed by genotypic FACS analysis, colony-forming activity and competitive transplantation of BM cells from endothelial Cdc42-null or control mice. Taken together, our results demonstrate that the Rho GTPase Cdc42 in BMECs mediates critical signaling cascade through PAK and Akt in maintaining the BMEC angiogenesis and morphogenesis. Cdc42 in BMECs could serve as a potential therapeutic target for bone marrow diseases including leukemia and lymphoma. Disclosures:No relevant conflicts of interest to declare.

Stress hematopoiesis reveals abnormal control of self-renewal, lineage bias, and myeloid differentiation in Mll partial tandem duplication (Mll-PTD) hematopoietic stem/progenitor cells

AbstractOne mechanism for disrupting the MLL gene in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is through partial tandem duplication (MLL-PTD); however, the mechanism by which MLL-PTD contributes to MDS and AML development and maintenance is currently unknown. Herein, we investigated hematopoietic stem/progenitor cell (HSPC) phenotypes of Mll-PTD knock-in mice. Although HSPCs (Lin−Sca1+Kit+ (LSK)/SLAM+ and LSK) in MllPTD/WT mice are reduced in absolute number in steady state because of increased apoptosis, they have a proliferative advantage in colony replating assays, CFU-spleen assays, and competitive transplantation assays over wild-type HSPCs. The MllPTD/WT-derived phenotypic short-term (ST)–HSCs/multipotent progenitors and granulocyte/macrophage progenitors have self-renewal capability, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice with an unexpected myeloid differentiation blockade and lymphoid-lineage bias. However, MllPTD/WT HSPCs never develop leukemia in primary or recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for full leukemogenic transformation. Thus, the Mll-PTD aberrantly alters HSPCs, enhances self-renewal, causes lineage bias, and blocks myeloid differentiation. These findings provide a framework by which we can ascertain the underlying pathogenic role of MLL-PTD in the clonal evolution of human leukemia, which should facilitate improved therapies and patient outcomes.

Noncanonical NF-κB Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

RelB and nuclear factor κB (NF-κB2) are the main effectors of NF-κB noncanonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-κB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Furthermore, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-κB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.