Primary Bone Marrow Transplantation Research Articles

Abstract A013: Sex-dependent hematopoietic stem cell aging and leukemogenic potential

Abstract Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders. But how HSC aging and sex co-influence leukemogenesis is poorly understood. By examining HSC aging in young, aging (75% lifespan) and longevity (110% lifespan) mice in males vs females, we have discovered that aging males have a significantly higher HSC frequency than females, and that male HSC aging is characterized by an expansion of Lin-CD150+SP HSCs and a decrease of Lin-CD150-SP HSCs, whereas females show opposite HSC changes towards aging and longevity. In line with HSC changes, aging males exhibit an increase of white blood cell counts that is primarily driven by increased myeloid cells, whereas aging females shows a decrease of white blood cell counts that is primarily driven by reduced lymphoid cells. To understand leukemogenic potential of aging HSCs in the true setting of organismal aging, we generated the first aging mouse model of chronic myeloid leukemia (CML) induced by BCR-ABL1 (Oncogene, 2021, 40: 3152-3163). We found that aging male HSCs are significantly more susceptible to BCR-ABL1 transformation and CML induction in aging male mice than aging female HSCs for CML induction in aging female mice. However, in the absence of oncogenic translocation genes, longevity female but not male mice rapidly develop B/myeloid mixed phenotype acute leukemia, a disease of HSC origin, in primary bone marrow transplantation recipients. Leukemias in our aging mouse models phenocopy those of humans in that the incidence of myeloid leukemia is higher in aging men than in women overall, but longevity women have a higher incidence of myeloid leukemia. Using single cell RNA sequencing analysis of HSCs, we found distinct cell clustering patterns and signaling pathways of aging HSCs between male and female mice. The top pathways seen in male HSC aging involve TGFβ and TNFα signaling that have been linked to poor drug response and persistence of human CML stem cells, while those for female HSC aging involve IFNα/γ signaling pathways that have been associated with good drug response of human CML stem cells. Together, our results demonstrate sex-dependent HSC aging and leukemogenic potential and that molecular changes in aging HSCs can inform drug response of leukemia stem cells. Our findings provide novel insight into the etiology of leukemia in males vs females and suggest potential benefit to use sex-based prevention and therapeutic strategies for leukemia in men vs women. Citation Format: WenYong Chen. Sex-dependent hematopoietic stem cell aging and leukemogenic potential [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A013.

Diaphanous-Related Formin Protein mDia2 Regulates Hematopoietic Stem and Progenitor Cell Functions through Srf-Integrins Pathway

Distinct mature cell types in mammalian blood system are derived from the Hematopoietic Stem/Progenitor cells (HSPCs), which are multipotent progenitor cells with self-renewing and engraftment ability. Various intrinsic and extrinsic molecules modulate HSPCs' fates to either undergo self-renewal or differentiation. The Diaphanous-related formin protein mDia2, is an effector of Rho GTPases. Previously, we reported that hematopoietic-specific mDia2 knock out mice developed anemia due to ineffective erythropoiesis caused by bi-nucleated erythroblasts. However, how mDia2 is involved in HSPC functions are unknown. Here we show that mDia2 expression is enriched in HSPCs. Although mDia2 was largely dispensable for HSPCs' function at steady-state, hematopoietic-specific inactivation of mDia2 severely impaired the competitive repopulation capacity of HSPCs. After primary bone marrow transplantation, the absolute number of bone marrow HSPCs from mDia2 knockout (KO) transplants was significantly decreased. mDia2 deficient LSK cells also showed skewed differentiation with increased percentage of more differentiated multi-potent progenitor cells (MPP), which was further supported by loss of stem cell quiescence. Under serial transplantation, mice transplanted with mDia2 knockout (KO) bone marrow showed rapid lethality. Importantly, these defects can be rescued by the constitutively active mDia2 mutant mDia2-ΔDAD. Furthermore, mDia2 KO HSPCs showed a marked reduction in the motility ability from bone marrow to the peripheral blood upon G-CSF stimulation. Loss of mDia2 also severely compromised the recovery of the mice from 5-FU-induced myelosuppression.mDia2 functions to nucleate linear actin filaments. We found that loss of mDia2 expression in HSPCs reduced the F-actin levels. This further attenuated the adhesion of HSPCs that was confirmed by adhesion assay and decreased protrusion formation in vitro . We demonstrated that the defect in F-actin formation caused by mDia2 deficiency repressed the transcriptional activity of serum response factor (SRF), which caused down-regulation of SRF target genes such as Acta2, Actb and SRF itself. Ectopic expression of SRF rescued HSPC phenotypes with loss of mDia2. We further identified that the expression levels of several integrins, including Itga2, Itgam (encodes CD11b) , Itagal (encodes CD11a)and Itgb2 (encodes CD18) , were significantly attenuated in mDia2 KO c-Kit+ HSPCs. Chromatin immunoprecipitation (ChIP) sequencing data further revealed intronic SRF binding sites in the genomic loci of Itgam and Itgb2, therefore suggesting Itgam and Itgb2 as new downstream targets of SRF pathway. Our data revealed a novel mDia2-SRF-integrin axis that regulates HSPCs adhesion and engraftment. DisclosuresNo relevant conflicts of interest to declare.

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies

Total marrow irradiation(TMI) has significantly advanced radiation conditioning for hematopoietic cell transplantation inhematologicmalignancies by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. However, the relapse rate remains high, and the lack of a preclinical TMI model has hindered scientific advancements. To accelerate TMI translation to the clinic, we developed a TMI delivery system in preclinical models. A Precision X-RAD SmART irradiator was used for TMI model development. Images acquired with whole-body contrast-enhanced computed tomography (CT) were used to reconstruct and delineate targets and vital organs for each mouse. Multiple beam and CT-guided Monte Carlo-based plans were performed to optimize doses to the targets and to vary doses to the vital organs. Long-term engraftment and reconstitution potential were evaluated by a congenic bone marrow transplantation (BMT) model and serial secondary BMT, respectively. Donor cell engraftment was measured using noninvasive bioluminescence imaging and flow cytometry. Multimodal imaging enabled identification of targets (skeleton and spleen) and vital organs (eg, lungs, gut, liver). In contrast to total body irradiation (TBI), TMI treatment allowed variation of radiation dose exposure to organs relative to the target dose. Dose reduction mirrored that in clinical TMI studies. Similar to TBI, mice treated with different TMI regimens showed full long-term donor engraftment in primary BMT and second serial BMT. The TBI-treated mice showed acute gut damage, which was minimized in mice treated with TMI. A novel multimodal image guided preclinical TMI model is reported here. TMI conditioning maintained long-term engraftment with reconstitution potential and reduced organ damage. Therefore, this TMI model provides a unique opportunity to study the therapeutic benefit of reduced organ damage and BM dose escalation to optimize treatment regimens in BMT and hematologic malignancies.

Reduction in Bone Marrow Insulin-like Growth Factor-1 Levels Enhances Long-Term Hematopoietic Stem Cell Engraftment Following Bone Marrow Transplantation

Efficient donor hematopoietic stem cell (HSC) engraftment is critical for successful bone marrow transplantation (BMT) and requires interactions between donor HSC and host-derived HSC niche elements. Due to disruption of perivascular stem cell niches induced by myeloablative BMT conditioning, initial engraftment of HSC following BMT occurs in niches located near the endosteum. We have previously shown that myeloablative BMT conditioning with total body irradiation (TBI) induces expansion of endosteal niche osteolineage cells in a time course correlating with increases in bone marrow (BM) levels of insulin-like growth factor (IGF)-1. We also have shown that inhibition of IGF-1 Receptor (IGF1R) signaling blocks osteolineage cell expansion after TBI, resulting in poor engraftment of long-term (LT)-HSC after BMT and suggesting a critical role for IGF-1/IGF1R interactions in regulating engraftment after BMT. We now present data in which we have defined the cellular sources of BM IGF-1 following TBI and have surprisingly discovered that reduction in BM IGF-1 levels promotes LT-HSC retention at baseline and donor LT-HSC engraftment following BMT.Using mice with floxed IGF-1 alleles (IGF1L/L) and with tissue-specific expression of Cre-recombinase, we generated models with deletion of IGF-1 derived from mature osteolineage cells (Col1A1CreIGF1Exc), hematopoietic cells (CD45CreIGF1Exc), or liver (AlbCreIGF1Exc). We additionally generated a conditional IGF-1 deletion model (MX1CreIGF1Exc) in which polyI:polyC (pI:pC) treatment disrupts IGF-1 production from all 3 of the above sources.By qPCR analysis, CD45CreIGF1Exc BM showed marked reduction (>95%) in BM IGF-1 mRNA at baseline and following TBI, suggesting that hematopoietic cells produce most of the locally derived IGF-1 in BM. Unexpectedly however, neither deletion of osteoblast-derived (Col1A1CreIGF1Exc) nor hematopoietic cell-derived (CD45CreIGF1Exc) IGF-1 significantly reduced BM extracellular or intracellular IGF-1 protein levels at baseline, or extracellular BM IGF-1 levels following TBI. In contrast, elimination of liver-derived IGF-1 in AlbCreIGF1Exc mice not only reduced plasma IGF-1 levels by 80%, but surprisingly reduced BM extracellular IGF-1 protein levels by ≥80% both at baseline and 48 hours post-TBI, and reduced BM intracellular IGF-1 protein levels by >80% and >60% at baseline and post-TBI, respectively. These data suggest that most IGF-1 protein in BM during BMT is derived from liver. Conditional IGF-1 deletion in MX1CreIGF1Exc mice resulted in severe reduction of both BM IGF-1 mRNA and protein levels, combining effects seen in CD45CreIGF1Exc and AlbCreIGF1Exc mice. None of these models, however, showed deficits in TBI-induced endosteal cell expansion, suggesting that other ligands or perhaps only low levels of IGF-1 are required to induce IGFR1 signaling-dependent endosteal cell expansion.Unexpectedly, reduction of BM IGF-1 in ALBCreIGF1Exc and MX1CreIGF1Exc mice caused a 3-4 fold increase (% of total BM) in BM Lin-cKit+Sca1+CD48-CD150+ or Lin-cKit+Sca1+CD34-CD135- HSC. ALBCreIGF1Exc BM displayed enhanced multi-lineage long-term repopulating capacity in competitive transplant assays, proving that these cells are functional LT-HSC. Given this baseline LT-HSC enhancement, we asked if reduction in BM IGF-1 levels impacted capacity of ALBCreIGF1Exc BM niches to engraft donor HSC after BMT. Using competitive secondary transplantation assays to assess numbers of engrafted WT GFP+ LT-HSC 3 weeks after primary BMT into ALBCreIGF1Exc or control mice, we found that reduced IGF-1 in the ALBCreIGF1Exc BM microenvironment resulted in >2.5 fold enhancement of donor LT-HSC engraftment. In an initial search of downstream mediators of this effect, we discovered that reduction of IGF-1 levels in MX1CreIGF1Exc BM results in a 2.5-fold increase in post-TBI CXCL12 mRNA expression, suggesting that reducing BM IGF-1 may enhance donor LT-HSC engraftment by promoting LT-HSC homing and maintenance within HSC niches.Taken together our data define a critical role for IGF-1/IGF1R interactions in regulating the efficiency of donor HSC engraftment following BMT. Therapeutic strategies to reduce BM IGF-1 may prove to be a valuable approach to improving engraftment following clinical BMT. DisclosuresNo relevant conflicts of interest to declare.

Alox5 Functions As Both Tumor Suppressor and Drug Sensitizer in AML

IntroductionAcute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant expansion of dysfunctional myeloid progenitors and the suppression of normal hematopoiesis. Despite of the intensive efforts in developing novel therapeutic strategies in the past decades, the standard chemotherapy, i.e., the "5+3" regimen, is still the main approach for treating AML, and resistance to the standard chemotherapy remains the major cause of relapse and therapeutic failure. Therefore, it is crucial to understand the mechanisms underlying AML pathogenesis and chemotherapy resistance, and thus identify new therapeutic targets.The arachidonate5-lipoxygenase gene (Alox5) is known to be involved in various physiological and pathological processes, including oxidative stress response, inflammation and cancer. Previous reports show that loss of Alox5 impairs the function of leukemic stem cells in BCR-ABL-induced chronic myelogenous leukemia (CML), as well as the long-term self-renewal of hematopoietic cells of AML with RUNX1-ETO9a, MLL-AF9 and PML-RARA. To our surprise, our Affymetrix microarray-based, genome-wide gene expression profiling of primary AML patient samples reveals that Alox5 is expressed at a particularly lower level in certain AML subtypes (e.g., MLL-rearranged AML) that are associated with poor prognosis than in AML subtypes (e.g., t(8;21) and inv(16) AMLs) that are associated with favorable prognosis. The aim of this study is to identify the influence and mechanism of Alox5 overexpression in AML pathogenesis and therapeutic response.MethodsTo assess the potential effect of Aloxon leukemic cell viability and drug response/resistance, AML cell lines with stable ectopic expression of Alox5 or control plasmids induced by lentivirus were treated with DMSO control, doxorubicin (DOX) or cytarabine (Ara-C). Cell viability and proliferation assays, i.e., MTT assays, were performed. To determine the influence of Alox5 on the transformation capacity, viability and drug response of mouse BM progenitor cells bearing MLL-AF9, colony-forming/replating assay (CFA) and MTT assay were conducted. To evaluate the effect of Alox5 on AML progression and chemotherapeutic response in vivo, we first carried out primary bone marrow transplantation (BMT) by use of hematopoietic progenitor cells retrovirally co-infected with MLL-AF9 and Alox5 or empty vector as primary donors, and then applied the leukemic blast cells isolated from the primary leukemic recipients as donor cells in a secondary BMT followed by "5+3" chemotherapy.Gene Set Enrichment Analysis (GSEA) was used to analyze the signal pathway enrichments in the BMT samples based on RNA sequencing results.ResultsIn AML cell lines, lentivirus induced overexpression of Alox5 resulted in a moderate repression of cell viability, while significantly sensitizing the cells to DOX and Ara-C treatment. In mouse BM progenitor cells with MLL-AF9, forced expression of Alox5 remarkably enhanced the inhibitory effect of DOX and Ara-C on the colony forming capacity and cell viability.Secondary BMT assay results showed that overexpression of Alox5 inhibited MLL-AF9-induced AML progression (MLL-AF9+Alox5+Ctrl, with medium survival of 44.5 days; MLL-AF9+Ctrl, with medium survival of 39.5 days; P=0.001) in vivo. The "5+3" chemotherapy alone indeed improved survival as compared with MLL-AF9+Ctrl (MLL-AF9+DOX/Ara-C, with medium survival of 50 days; P<0.001). The therapeutic effect of DOX+Ara-C treatment was remarkably enhanced by Alox5 overexpression, and the medium survival was prolonged to >200 days (MLL-AF9+Alox5+DOX/Ara-C) (P<0.001) (Fig. 1A).RNA-seqand GSEA suggested that overexpressing Alox5 activates the E2F signaling and oxidative stress response, while repressing the K-ras and Stat pathways (Fig. 1B).ConclusionsOur results suggest that while forced expression of Alox5 shows a moderate tumor suppressor role in the progression of MLL-AF9-mediated AML, it significantly enhanced the effect of the standard "5+3" chemotherapy both in vitro and in vivo. The potential mechanism is through targeting E2F, oxidative stress response, K-ras and Stat signaling pathways, which were all known to be closely associated with tumorigenesis and drug response/resistance. Our study indicates the restoration of Alox5 in combination with the classic chemotherapy as a potential novel therapeutic strategy in treating AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Osteoblastic VEGF Coordinates Remodeling of the Hematopoietic Stem Cell Niche

Osteoblasts (OBs) and the bone marrow (BM) vasculature constitute hematopoietic stem and progenitor cell (HSC/HSPC) niches. Immature perivascular osteolineage cells enforce HSC quiescence while signals from mature OBs are capable of bone and vascular remodeling. We previously demonstrated that bone anabolic parathyroid hormone (PTH) stimulation increases HSPCs with short and long-term repopulating activity. Based upon the coupled nature of osteogenesis and angiogenesis as well as the multifaceted role blood vessels play in HSC regulation, we investigated effects of PTH on the BM vasculature. Both genetic PTH receptor activation in maturing OBs (Col1caPTH1R) and systemic PTH treatment increased functional blood vessel branching imaged intravitally in the calvaria (14±2 vs 34±3 and 14±2 vs 27±4 branchpoints, n=3-4 mice/group, 3-7 regions analyzed per mouse). These changes were confirmed histologically in long bones, where PTH-induced tortuosity was accompanied by the emergence of α smooth muscle actin (αSMA)+ bone-associated vascular networks, likely small-caliber arterioles, containing red blood cell rouleaux, while control marrow displayed sparse, non-bone-associated αSMA+ vessels. Further, PTH increased morphologically-heterogeneous BM microvessels (167±18 vs 348±39 per section, n=5 mice/group) and endothelial cell (EC) abundance (0.09±0.02% vs 0.2±0.02%, n=4-5 mice/group). VEGF-A and FGF2, known to be important proangiogenic signals in bone, were increased after PTH treatment of osteoblastic cell lines and primary osteolineage cells in vitro, and in bone-associated cells of mice treated in vivo. Because the perivascular milieu is heterogeneous and can support HSC quiescence or activation depending on vessel type and stimuli, we tested whether tuning BM angiogenic responses modulated effects of PTH on HSCs. Treatment with the anti-VEGF-A monoclonal antibody bevacizumab (αVEGF) precluded PTH-induced vascular branching in calvarial BM and reduced pre-established vascular branching in Col1caPTH1R mice (34±3 vs 21±1 branchpoints, n=4 mice/group, 3-5 regions analyzed per mouse), while PTH-induced bone anabolism, EC abundance and bone-associated αSMA+ small-caliber vessels were sustained. Moreover, αVEGF did not change the frequency of CD51+ PDGFRα+ or PDGFRα+ Sca1+ immature mesenchymal cells reported to regulate HSCs. The altered balance of marrow sinusoidal vs arteriolar structures we quantified in PTH + αVEGF-stimulated BM would be expected to improve HSC support based on niche remodeling, therefore we tested the hematopoietic consequences. αVEGF did not alter frequencies of phenotypically-defined HSCs in the BM or mature hematopoietic cells and platelets in the peripheral blood (PB). Remarkably, αVEGF augmented PTH-induced repopulation of the PB in primary BM transplantation (p = 0.0013, n=10 recipients/group) and sustained the repopulating ability (p < 0.0001, n=10 recipients/group) and BM engraftment (~70 fold) of cells in secondary transplantation. Competitive transplantation of HSPCs sorted from PTH + αVEGF-treated mice showed enhanced repopulating ability, confirming niche-mediated improvement of HSPC function. Unbiased analysis of sorted stem and progenitor cells demonstrated that PTH, alone or in combination with αVEGF, broadly reduced multipotent progenitor (MPP) gene expression, including markers of cell proliferation. Notably, microenvironmental activation with PTH alone uniquely decreased a cluster of transcripts associated with hematopoietic differentiation in MPPs, suggesting progenitor cell reeducation by PTH activation versus niche reapportioning by combined PTH and VEGF antagonism. Because PTH also increases FGF2, reported to expand HSCs and stabilize blood vessels, we tested whether FGF signaling is necessary for PTH to establish long-term HSC niches. In vivo FGF receptor 1 inhibition significantly reduced long-term hematopoietic repopulating ability of PTH + αVEGF-treated BM cells in secondary transplantation (p = 0.0046, n = 16-17 mice/group), suggesting VEGF-A and FGF2 have opposing HSC effects in the PTH-activated microenvironment. These data define the HSC niche as a regulatory network, and identify mature OBs as the cellular source of signals that serve to coordinate HSC-supportive niches, demonstrating functional cooperation of the different constituents of the bone marrow microenvironment. DisclosuresOff Label Use: Bevacizumab (trade name Avastin, Genentech/Roche) is used by the authors as a strategy to block VEGF-A in the bone marrow microenvironment.. Calvi:Fate Therapeutics: Patents & Royalties.

Role Of Sf3b1 On Hematopoiesis

Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown.The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice.In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance.To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

PU.1 Is Essential For MLL Leukemia Via Activation Of The Meis/HOX Pathway and A Monocytic Cytokine Mediated Anti-Apoptotic Inflammatory Program

Aberrant transcriptional programs play a critical role in the development of acute myeloid leukemias (AMLs). Although persistent over-expression of MEIS1 and HOXA9 has been shown to be essential for the initiation and maintenance of MLL-associated leukemia, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion-driven MEIS/HOX pathway, dictate the development of MLL leukemia. Considering that AMLs with MLL translocation are typically associated with the monocytic lineage (FAB M4 and M5), we explored the potential role of the monocytic lineage-specific transcriptional program in MLL leukemia.Using 97 genome-wide expression profiles of human MLL leukemias, we constructed an MLL distinctive transcriptional regulatory network. In addition to well-known transcriptional factors in leukemia development such as MEIS1 and HOXA family genes, we identified a highly active monocyte-specific gene signature that includes transcription factor PU.1. In our effort to determine the functional role of PU.1 in MLL leukemia, we found that lower PU.1 expression significantly delayed the onset of MLL- AF9 induced leukemia in primary bone marrow transplantation assay. MLL leukemia failed to maintain in vivo upon induced deletion of the PU.1 gene. To examine the clinical relevance of the PU.1 in AML patients, we further performed multivariate Cox proportional-hazards regression analysis in four published datasets of patients with AML, for whom gene expression and time-to-event data were available. We found that a PU.1-regulated 40-gene signature showed profound concordance with prognosis in segregating high-risk and low-risk AML patients. When specific subgroups of AMLs were examined, the PU.1 expression signature could predict patient outcome for MLL patients, but not in other major AMLs, such as t(8;21), t(15;17) and inv(16).We further explored the molecular mechanisms underlying the critical role of the PU.1 program in MLL leukemia. Functional annotation of this PU.1 expression signature identified the MEIS/HOX pathway (MEIS1, FLT3, KIT), as well as key genes in the inflammatory response (AIF1, NF-KB1 and CD180). We showed that PU.1 is required to maintain high expression of Meis1 and Pbx3 and also important downstream genes in the MEIS/HOX pathway that includes known MEIS/HOX targets c-Kit and Flt3. Using ChIP-sequencing, we demonstrated that PU.1 interacts with the MEIS/HOX regulatory program through co-binding with MEIS1 at the target genomic regions in a MLL-ENL cell line. In our effort to determine the role of PU.1-controlled inflammatory response genes, we found that the growth inhibition in PU.1 knockdown MLL leukemic cells was partially rescued by addition of the monocytic inflammatory cytokine AIF1. AIF1 provides an anti-apoptotic effect through activation of the NF-ƒÛB pathway and additional known apoptosis regulators. Interestingly, AML patients with higher expression of both AIF1 and MEIS1 had a significantly shorter overall survival time than those with lower expression of both genes. Patients with high expression of either MEIS1 or AIF1 had medium survival possibilities. Notably, the prognostic value of AIF1 and MEIS1 remained in those with monocytic AMLs (P=0.00079), but not in the non- monocytic group of patients (p=0.105). Collectively, these results strongly suggest that the monocyte-specific inflammatory cytokine AIF1 is an MEIS/HOX independent essential regulator in monocytic AMLs such as MLL leukemia.Loss of function PU.1 is leukemogenic in mouse models. Suppression of PU.1 activity is also required for the development of human myelocytic M2/M3 leukemia. Here we reveal a converse role for PU.1 as an essential positive regulator in the development of MLL myeloid leukemia, mostly M4/M5 monocytic AMLs. Our study demonstrats that the monocyte-specific PU.1-driven transcriptional program independently contributes to the development of myeloid MLL leukemia, in parallel with the MLL fusion pathway. PU.1 and downstream macrophage specific inflammatory cytokine AIF1 have important prognostic value and may serve as novel therapeutic targets for MLL leukemias. Disclosures:No relevant conflicts of interest to declare.

MiR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia

HOXA9 and MEIS1 have essential oncogenic roles in mixed lineage leukaemia (MLL)-rearranged leukaemia. Here we show that they are direct targets of miRNA-196b, a microRNA (miRNA) located adjacent to and co-expressed with HOXA9, in MLL-rearranged leukaemic cells. Forced expression of miR-196b significantly delays MLL-fusion-mediated leukemogenesis in primary bone marrow transplantation through suppressing Hoxa9/Meis1 expression. However, ectopic expression of miR-196b results in more aggressive leukaemic phenotypes and causes much faster leukemogenesis in secondary transplantation than MLL fusion alone, likely through the further repression of Fas expression, a proapoptotic gene downregulated in MLL-rearranged leukaemia. Overexpression of FAS significantly inhibits leukemogenesis and reverses miR-196b-mediated phenotypes. Targeting Hoxa9/Meis1 and Fas by miR-196b is probably also important for normal haematopoiesis. Thus, our results uncover a previously unappreciated miRNA-regulation mechanism by which a single miRNA may target both oncogenes and tumour suppressors, simultaneously, or, sequentially, in tumourigenesis and normal development per cell differentiation, indicating that miRNA regulation is much more complex than previously thought.

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

AbstractAbstract 2617Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs.On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004).In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT.These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures:No relevant conflicts of interest to declare.

Strategic Nonmyeloablative Conditioning: CD154:CD40 Costimulatory Blockade at Primary Bone Marrow Transplantation Promotes Engraftment for Secondary Bone Marrow Transplantation after Engraftment Failure

There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 costimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T cell depletion inhibits generation of the humoral immune response after primary BMT, as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, because almost all CFSE-labeled donor cells were killed at 0.5 and 3 h in sensitized recipients in in vivo cytotoxicity assay, reflecting Ab-mediated cytotoxicity. CD154:CD40 costimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.

Id1, but not Id3, directs long-term repopulating hematopoietic stem-cell maintenance

E-proteins are widely expressed basic helix-loop-helix (HLH) transcription factors that regulate differentiation in many cell lineages, including lymphoid, muscle, and neuronal cells. E-protein function is controlled by HLH inhibitors such as Id and SCL/TAL1 proteins, which recently have been suggested to play a role in hematopoietic stem cell (HSC) differentiation. However, the precise stages when these proteins are expressed and their specific functions are not entirely clear. Using a knock-in mouse model where the sequence for the enhanced green fluorescent protein (GFP) was inserted downstream of the Id1 promoter, we were able to track Id1 expression on an individual cell basis and detected Id1 expression in long-term repopulating HSCs (LT-HSCs). Functional assays showed that the Id1/GFP(+)Lin(-)Sca1(+)c-kit(Hi) population was highly enriched for LT-HSCs. Consistent with this expression pattern, Id1 deficiency led to a 2-fold reduction in the number of LT-HSCs defined as Lin(-)Sca1(+)c-kit(Hi)CD48(-)CD150(+). Primary bone marrow transplantation studies revealed that Id1 is dispensable for short-term engraftment. In contrast, both Id1(-/-) whole bone marrow and Lin(-)Sca1(+)c-kit(Hi)Thy1.1(Lo)-enriched HSCs, but not Id3(-/-) marrow, displayed impaired engraftment relative to wild-type controls in secondary transplantation assays. These findings suggest a unique role for Id1 in LT-HSC maintenance and hematopoietic development.

The Membrane Proximal Portion of the Thrombopoietin Receptor (Mpl) Elicits Novel Pivotal Signals for Hematopoietic Stem Cell Self-Renewal.

Thrombopoietin (Tpo) and its receptor, mpl, are important for hematopoietic stem cell (HSC) self-renewal in addition to their roles in megakaryocyte and platelet development. In this study we dissected the signaling pathways that mpl transduces to support megakaryocytopoiesis as well as stem cell production and function using knockin mice bearing a truncated mpl receptor lacking the distal 60 amino acids (delta-60). This 60 amino acid deletion removes three major signaling tyrosines on the mpl cytoplasmic domain, but retains the membrane proximal Box 1 and Box 2 domains that are required for JAK2 activation. We show that delta-60 bone marrow (BM) cells cultured in Tpo generated normal numbers of megakaryocytes but with greatly reduced ploidy. Freshly-isolated delta-60 megakaryocytes showed marked reduction in most, if not all, known Tpo-stimulated signaling pathways, including Stat5, Stat3, Akt, and p42/44MAPK. Using competitive bone marrow transplantation (BMT) to analyze HSC activities and serial BMT to assess HSC self-renewal, we found that delta-60 mice displayed normal short-term (ST-HSC) activities and marginally compromised long-term (LT-HSC) stem cell activities in primary BM transplantation. In addition, delta-60 mice supported HSC self-renewal for at least two serial BMTs. Thus, our results reveal a pivotal role for the membrane proximal region of the mpl receptor in maintaining stem cell activity and self-renewal by eliciting novel intracellular signaling pathways.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer

Many mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem cells by gene transfer

AbstractMany mutations of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD) cause G6PD deficiency in humans. Some underlie severe forms of chronic nonspherocytic hemolytic anemia (CNSHA) for which there is no definitive treatment. By using retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein that harbor the human G6PD (hG6PD) complementary DNA, stable and lifelong expression of hG6PD was obtained in all the hematopoietic tissues of 16 primary bone marrow transplant (BMT) recipient mice and 14 secondary BMT recipients. These findings demonstrate the integration of a functional gene in totipotent stem cells. The average total G6PD in peripheral blood cells of these transplanted mice, measured as enzyme activity, was twice that of untransplanted control mice. This allowed the inference that the amount of G6PD produced by the transduced gene must be therapeutically effective. With the same vectors both the cloning efficiency and the ability to form embryoid bodies were restored in embryonic stem cells, in which the G6PD gene had been inactivated by targeted homologous recombination, thus effectively rescuing their defective phenotype. Finally, expression of normal human G6PD in hG6PD-deficient primary hematopoietic cells and in human hematopoietic cells engrafted in nonobese diabetic/severe combined immunodeficient mice was obtained. This approach could cure severe CNSHA caused by G6PD deficiency.

Improved Expression in Hematopoietic and Lymphoid Cells in Mice After Transplantation of Bone Marrow Transduced With a Modified Retroviral Vector

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.

Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion

Whether hematopoietic stem cells can proliferate without limit, or whether their regenerative capacity declines with repeated division, has been debated for decades. Prevailing opinion favours an intrinsic ‘decline’, a view based on the finite degree to which murine bone marrow can be serially transplanted [1,2], the diminished self-renewal of spleen colony-forming cells (CFU-s) subjected to repeated passage [1], and the failure of stem cells to regenerate to normal levels after even a single transplantation [3,4]. However, serial transfer experiments did not specifically monitor input and output of long-lived stem cells (long-term reconstituting cells, LTRCs), leaving competing interpretations unresolved. We have re-examined the issue by quantitating 7–12 month LTRCs during sequential transplantations. Although these cells recovered to only 4% of normal levels after primary bone marrow transplantation, at each passage they increased around 10-fold relative to the amount transplanted, attaining an estimated cumulative expansion of 8400-fold over the original input after four transfers. Expansion was limited by transfer of increasing numbers of marrow cells and specifically of LRTCs, suggesting an extrinsically determined ceiling to stem cell growth. Conversely, expansion was enhanced in vivo by administration of stem cell factor (SCF, c-kit ligand) and interleukin-11. The results challenge the view that expansion of passaged stem cells is limited by exhaustion, and indicate that augmentation after transplant is limited by extrinsic mechanisms whose effects are reversible either by further transfer of the stem cells into irradiated hosts or by administration of exogenous cytokines.