Murine Hematopoietic Stem Cells Research Articles

MicroRNA-127-3p controls murine hematopoietic stem cell maintenance by limiting differentiation.

The balance between self-renewal and differentiation is crucial to ensure the homeostasis of the hematopoietic system, and is a hallmark of hematopoietic stem cells. However, the underlying molecular pathways, including the role of micro-RNA, are not completely understood. To assess the contribution of micro-RNA, we performed micro-RNA profiling of hematopoietic stem cells and their immediate downstream progeny multi-potent progenitors from wild-type control and Pbx1-conditional knockout mice, whose stem cells display a profound self-renewal defect. Unsupervised hierarchical cluster analysis separated stem cells from multi-potent progenitors, suggesting that micro-RNA might regulate the first transition step in the adult hematopoietic development. Notably, Pbx1-deficient and wild-type cells clustered separately, linking micro-RNAs to self-renewal impairment. Differential expression analysis of micro-RNA in the physiological stem cell-to-multi-potent progenitor transition and in Pbx1-deficient stem cells compared to control stem cells revealed miR-127-3p as the most differentially expressed. Furthermore, miR-127-3p was strongly stem cell-specific, being quickly down-regulated upon differentiation and not re-expressed further downstream in the bone marrow hematopoietic hierarchy. Inhibition of miR-127-3p function in Lineage-negative cells, achieved through a lentiviral-sponge vector, led to severe stem cell depletion, as assessed with serial transplantation assays. miR-127-3p-sponged stem cells displayed accelerated differentiation, which was uncoupled from proliferation, accounting for the observed stem cell reduction. miR-127-3p overexpression in Lineage-negative cells did not alter stem cell pool size, but gave rise to lymphopenia, likely due to lack of miR-127-3p physiological downregulation beyond the stem cell stage. Thus, tight regulation of miR-127-3p is crucial to preserve the self-renewing stem cell pool and homeostasis of the hematopoietic system.

Long-Term Engraftment of ESC-Derived B-1 Progenitor Cells Supports HSC-Independent Lymphopoiesis.

SummaryIt is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.

High-fat diet disturbs lipid raft/TGF-\u03b2 signaling-mediated maintenance of hematopoietic stem cells in mouse bone marrow

Despite recent in vivo data demonstrating that high-fat diet (HFD)-induced obesity leads to major perturbations in murine hematopoietic stem cells (HSC), the direct role of a HFD is not yet completely understood. Here, we investigate the direct impact of a short-term HFD on HSC and hematopoiesis in C57BL/6J mice compared with standard diet-fed mice. We detect a loss of half of the most primitive HSC in the bone marrow (BM) cells of HFD-fed mice, which exhibit lower hematopoietic reconstitution potential after transplantation. Impaired maintenance of HSC is due to reduced dormancy after HFD feeding. We discover that a HFD disrupts the TGF-β receptor within lipid rafts, associated to impaired Smad2/3-dependent TGF-β signaling, as the main molecular mechanism of action. Finally, injecting HFD-fed mice with recombinant TGF-β1 avoids the loss of HSC and alteration of the BM’s ability to recover, underscoring the fact that a HFD affects TGF-β signaling on HSC.

Sequential Cyclophosphamide and Trametinib Improve Clinical Graft Versus Host Disease and Survival in Murine Hematopoietic Stem Cell Transplant

Background Ongoing acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (alloSCT) causes significant morbidity and mortality with no standard therapy except corticosteroids, which are broadly immunosuppressive and toxic. Cyclophosphamide (Cy) given day 3 and day 4 is effective for prophylaxis of aGVHD but has proven ineffective when given later as in ongoing aGVHD in both murine and human studies. We previously demonstrated that the MEK inhibitor trametinib (Tram) is effective for aGVHD prophylaxis in murine models of aGVHD through the novel mechanism of inhibition of naive T cells (CD44−CD62L+). Our hypothesis is that ongoing aGVHD could be better inhibited by sequential Cy and post-Cy Tram inhibition (to prevent naive T cells from re-initiating alloreactivity) without compromising graft-versus-malignancy (GVM) responses. Methods Mice were administered sequential Cy and Tram post SCT starting day 12. Efficacy of treatment was assessed in a murine model of MHC-mismatched alloSCT. Overall survival was assessed and clinical GVHD was scored three times weekly. Peripheral blood, spleen, and lymph node cells phenotypes were determined by flow cytometry. GVM was also assessed with A20 luciferase/YFP (A20luc/YFP) lymphoma cells imaged with in vivo imaging system (IVIS). Results Overall survival following sequential Cy and Tram was identical to that seen with T cell depleted bone marrow alone and significantly superior to treatment using either single-agent Cy or Tram (p Conclusions Combination cyclophosphamide and trametinib demonstrates superior overall survival and limits GVHD severity without affecting regulatory T cells or GVM in murine allogeneic stem cell models. Utilizing Cy and Tram are readily translatable and support the notion that clinical approaches can be developed in the future.

Origin and development of classical dendritic cells.

Classical dendritic cells (cDCs) are mononuclear phagocytes of hematopoietic origin specialized in the induction and regulation of adaptive immunity. Initially defined by their unique T cell activation potential, it became quickly apparent that cDCs would be difficult to distinguish from other phagocyte lineages, by solely relying on marker-based approaches. Today, cDCs definition increasingly embed their unique ontogenetic features. A growing consensus defines cDCs on multiple criteria including: (1) dependency on the fms-like tyrosine kinase 3 ligand hematopoietic growth factor, (2) development from the common DC bone marrow progenitor, (3) constitutive expression of the transcription factor ZBTB46 and (4) the ability to induce, after adequate stimulation, the activation of naïve T lymphocytes. cDCs are a heterogeneous cell population that contains two main subsets, named type 1 and type 2 cDCs, arising from divergent ontogenetic pathways and populating multiple lymphoid and non-lymphoid tissues. Here, we present recent knowledge on the cellular and molecular pathways controlling the specification and commitment of cDC subsets from murine and human hematopoietic stem cells.

Isolation of Murine Hematopoietic Stem Cells.

Bone marrow resident hematopoietic stem cells (HSCs) are responsible for the lifetime generation of the wide profusion of blood and immune cell types found in the body. In addition, therapeutically, in the context of bone marrow transplantation, HSCs have been successfully deployed to restore normal blood-forming capacity in patients being treated with high-dose chemotherapy for hematologic malignancies. The known ability of bone marrow transplantation to either restore or reset the immune system and to engender immune tolerance has suggested that HSCs may be applied therapeutically for a wider range of clinical conditions, including immunological/autoimmune disorders and allogeneic organ transplantation. Herein, we describe a flow-cytometry-based method to isolate mouse HSCs for continued experimental investigation into such therapeutic uses.

Depletion of murine fetal hematopoietic stem cells with c-Kit receptor and CD47 blockade improves neonatal engraftment

Key Points Fetal injection of antibodies against the c-Kit receptor and CD47 effectively depletes host HSCs in immunocompetent mice. In utero depletion of host HSCs increases long-term engraftment after neonatal hematopoietic cell transplantation.

Murine HSCs contribute actively to native hematopoiesis but with reduced differentiation capacity upon aging.

A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.

Murine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs

The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5–7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2–7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2–7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41− population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.

CD166 Engagement Augments Mouse and Human Hematopoietic Stem and Progenitor Function Via Activation of Stem Cell-Associated and Cell Cycle Pathways

Functions of hematopoietic stem and progenitor cells are regulated by cellular signaling networks and by the cellular and non-cellular elements of the hematopoietic niche. CD166 is highly expressed on both human and murine hematopoietic stem cells (HSC) and on murine osteoblasts and has been identified as a critical regulator of their functions. CD166 engages in trans-homophilic interactions with other CD166 molecules. However how homophilic or heterophilic engagement of CD166 (with CD6) improves HSC function is unknown.Since we described that CD166 is expressed on murine and human HSC and murine osteoblasts, we hypothesized that CD166-CD166 homophilic engagement is critical for the hematopoiesis enhancing activity (HEA) that is mediated by osteoblasts. CD166+LSK cells cultured for 7d on either WT osteoblasts or recombinant murine CD166 (rmCD166) showed increased colony forming units (CFU) compared to CD166-LSK cells cultured identically (p<0.05). Moreover, after 7d, the frequency of Lin-Sca1+ cells was higher in co-cultures containing rmCD166 and CD166+LSK than cultures initiated with CD166-LSK (p<0.05).Next, we determined the effect of loss of CD166 expression on progenitors and osteoblasts on hematopoietic functions using CD166 knockout mice (CD166-/-). CD166-/- LSK cells co-cultured with rmCD166 showed decreased CFU production compared to WT LSK cultured similarly (p<0.05). In addition, WT HSC cultured on CD166-/- osteoblasts for 7d showed decreased fold change in CFU relative to WT HSC cultured over WT osteoblasts (p<0.05). In vivo transplantation studies to corroborate our in vitro results are ongoing. To determine if CD166-CD166 interactions also enhance hematopoietic functions of human HSC, we cultured CD34+CD166+ and CD34+CD166- HSC isolated from cord blood with recombinant human CD166 (rhCD166). On d7, CD34+CD166+ HSC cultured with rhCD166 showed 30±4-fold increase in CFU production compared to d0, whereas CD34+CD166- HSC had only 9±0.4-fold increase. Taken together, these data suggest that CD166-CD166 homophilic interactions enhance hematopoietic functions and loss of this homophilic interaction negatively impacts HSC.To elucidate the underlying signaling mechanism of CD166-CD166 mediated HEA, we cultured WT or CD166-/- SLAM LSK cells with rmCD166 for 20hr and performed single-cell (sc) RNA seq. We analyzed for differential gene expression (DEG) using LGMT model and identified 518 upregulated and 174 downregulated genes in CD166-/- HSC compared to WT. Following pathway enrichment analysis of DEGs, we identified 148 canonical pathways enriched by the upregulated genes in CD166-/- HSC, including cell cycle, translational regulation, and mitochondria-related signaling pathways. 268 pathways were impacted by the downregulated genes in CD166-/- HSC, including oxidative stress response, and metabolism. Moreover, CXCR4 signaling, PDGF signaling and glucocorticoid receptor signaling pathways were also downregulated in CD166-/- HSC.A cell trajectory reflecting associations among cells revealed a single cluster of CD166-/- HSC. CD166-/- HSC are linked with low expression of stemness marker genes, and high expression of genes regulating cell cycle, oxidative phosphorylation, and glucose metabolism. In addition, ER-stress and oxidative stress responsive genes are overexpressed in CD166-/- HSC. In CD166-/- HSC, genes of all enriched pathways were highly connected in the co-expressed networks, which indicates that in HSC, the impact of loss of CD166 on cell cycle, metabolism, growth factors and stemness pathways is highly associated. Sixteen hub genes including Suclg1, Eif4a1, Cox4i2, Jak3, Runx3, and Cdk6 were identified in the co-expression network.We next applied bi-clustering algorithm QUBIC to identify modules of co-upregulated genes to analyze the transcriptomic variations of DEG in CD166-/- HSC and identified 39-gene co-upregulation modules forming one big block in CD166-/- HSC and 35-gene modules forming three blocks in WT HSC indicating that modules closely corresponded to CD166's impact over different cell states rather than on individual pathways.Overall, our studies suggest that homophilic CD166 interactions involving HSC are required for maintenance of essential pathways that sustain HSC function and progenitor cell production including stemness, mitochondrial function, metabolism, cell cycle and growth factor signaling. DisclosuresNo relevant conflicts of interest to declare.

Epidermal Growth Factor Receptor - Mediated DNA Repair Regulates Hematopoietic Stem Cell Regeneration

Hematopoietic myelosuppression is the principal dose-limiting toxicity of curative chemotherapy and radiotherapy in patients with cancer. Both chemotherapy and radiotherapy deplete hematopoietic stem cells (HSCs) via induction of DNA damage. DNA damage contributes to both HSC dysfunction and risk for malignant transformation over time. Extrinsic signals capable of promoting DNA repair in HSCs following injury can potentially improve HSC function and may decrease risk for dysplasia and leukemia over time. Here, we show that treatment with epidermal growth factor (EGF) decreases DNA damage in murine HSCs following irradiation via activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ) repair. Specifically, we observed that in vitro treatment of irradiated bone marrow (BM) ckit+sca-1+lin- (KSL) hematopoietic stem/progenitor cells (HSPCs) with 100 ng/ml EGF decreased HSPC DNA damage at 1 hour as measured by gamma-H2AX foci and Comet assay (p<0.0001, p<0.0001). Further, EGF treatment increased phosphorylation and nuclear localization of DNA-PKcs, which is integral to NHEJ repair, and increased phosphorylation of Akt in irradiated HSPCs. Inhibition of Akt or DNA-PKcs completely abrogated EGF - mediated DNA repair and EGF - mediated recovery of HSPCs following irradiation in vitro. In irradiated (500 cGy) C57BL/6 mice, subcutaneous administration of 10 μg/day of EGF significantly increased recovery of peripheral blood white blood cells and lymphocytes (p=0.03, p=0.04), as well as BM SLAM+KSL cells (HSCs) at day +14 (p=0.0004). Treatment with the DNA-PKcs inhibitor, NU7441, abrogated EGF - mediated recovery of peripheral blood WBCs in irradiated mice (p=0.007), suggesting that EGF - mediated mitigation of radiation injury in vivo was dependent on DNA-PKcs - mediated NHEJ repair. Importantly, EGF treatment of irradiated mice also substantially increased the survival of irradiated mice compared to irradiated control mice (p=0.009) and caused the recovery of long-term HSCs capable of 20 week competitive repopulation in congenic recipient mice (p=0.01, 20 weeks).Conversely, doxycycline inducible, cell - specific suppression of EGFR in hematopoietic cells using SCL-tTA;EGFR-DN mice caused a significant depressed regeneration of BM SLAM+KSL cells and KSL cells at day +10 after irradiation (p=0.005, p=0.02, respectively). SCL-tTA;EGFR-DN mice also displayed significantly decreased survival at day +30 compared to EGFR wild type mice (23.1% survival vs. 58.3% survival, p=0.04). Consistent with our hypothesis, BM HSPCs from EGFR-DN mice displayed increased DNA damage in response to total body irradiation, as measured by gamma H2AX foci and the Comet assay (p<0.0001, p=0.04). Coordinately, BM HSPCs from EGFR-DN mice displayed decreased phosphorylation of Akt and DNA-PKcs in response to irradiation in vitro (p<0.0001, p=0.004).These studies suggest that EGFR - mediated DNA repair is necessary for HSC and progenitor cell recovery following irradiation and that EGF treatment accelerates hematopoietic regeneration via augmentation of NHEJ repair in HSCs. EGF treatment following high dose chemotherapy and/or radiotherapy may increase HSC function and lessen near - term hematologic toxicities and morbidities. DisclosuresNo relevant conflicts of interest to declare.

The Lysine Acetyltransferase Tip60 Is Required for Hematopoietic Stem Cell Maintenance

Hematopoietic stem cells (HSCs) are maintained for their two defining properties, a self-renewal and a multi-lineage differentiation ability, under various epigenetic processes. One of the epigenetic processes essential for HSC is a lysine acetylation, which was demonstrated in several previous studies utilizing knockout mice of a gene encoding a lysine acetyltransferase (KAT), such as Cbp/p300, Moz, or Mof. The lysine acetyl transferase 5, Kat5 (also called Tip60), is a member of MYST KAT family, defined by a protein containing a C2HC-type zinc finger and an acetyl-CoA binding domain, consisting of five members: Tip60, Moz, Morf, Hbo1, and Mof. Tip60 regulates gene transcription, genome stability, cell growth and apoptosis and has been demonstrated to be essential for embryonic development.To assess the role of Tip60 in murine HSC, we generated Tip60 conditional knockout mice in two different Cre strains (Mx1-Cre and Vav-iCre), wherein Tip60 gene was successfully deleted in HSCs. Tip60 abrogation induced embryonic and adult lethality due to hematopoietic failure. Remarkably, HSCs were rapidly depleted upon Tip60 disruption, exhibiting robust apoptosis, aberrant cell cycle progression, and accumulated DNA damages. Tip60Δ/Δ fetal LSK cells transduced with retroviruses expressing wild-type Tip60 recovered a long-term hematopoiesis in transplantation experiments, whereas acetyl transferase defective Tip60 did not rescue the reconstitution to any detectable level, suggesting that the Tip60 acetyltransferase activity is essential for HSC function. Gene set enrichment analysis in RNA-seq demonstrated that Tip60 was significantly associated with genes involved in cell-cycle and DNA repair process, and Myc transcriptional factors target gene sets. A genome-wide chromatin profiling corroborated these findings, uncovering a strong similarity between Tip60 and c-Myc binding genomic regions. Considering similarities of HSC phenotype between our Tip60 conditional knockout mice and c-myc and N-myc double knockout mice (Laurenti, E. et al., Cell Stem Cell 2008), Tip60 maintains HSC by regulating Myc target genes.We next evaluated alteration in histone modifications evoked by Tip60 deletion, performing ChIP-seq analysis in Tip60f/f; Rosa26-CreERT2 and Tip60f/f fetal c-Kit+ cells that were treated with 4-OHT. Intriguingly, we found that acetylated H2A.Z was enriched at the Tip60-bound active chromatin and Tip60 deletion reduced the acetylation level of H2A.Z at Myc target genes promoters, whereas neither H3K27 acetylation level, active promoter / enhancer mark, nor H3K27 tri-methylation level, repressive mark, were altered. Collectively, our results demonstrate that Tip60 - H2A.Z could be an epigenetic axis critical for active transcription of Myc target genes to maintain murine HSC. DisclosuresNo relevant conflicts of interest to declare.

Distinct Gene Expression Patterns Drive Proliferation of Dominant-Negative RUNX1 Immortalized HSPCs

The transcription factor RUNX1 is a master regulator of normal hematopoiesis and is involved in cell fate decisions. RUNX1 mutations have been shown to contribute to the development of myeloid neoplasms, and in myelodysplastic syndromes (MDS) it is one of the most frequently mutated genes. Such mutations lead to RUNX1 proteins that lack transactivation activity or DNA-binding ability resulting in a loss of its tumor suppressor function. The dominant-negative short isoform RUNX1a resembles truncated RUNX1 mutants and inhibits the function of the full-length RUNX1 proteins. Additionally, a recently published study identified overexpression of RUNX1a but not full-length RUNX1 in CD34+-cells from patients with myelodysplastic/myeloproliferative disease, which increased with disease progression. This strongly suggests that truncated RUNX1 plays a pivotal role in myelodysplastic disease. However, the precise molecular functions of mutating RUNX1, particularly with respect to the identity of RUNX1 target genes conferring its tumor suppressor function, remain unclear.Previously, our group reported that overexpression of RUNX1a immortalized murine hematopoietic stem and progenitor cells (HSPCs) in vitro. Immunophenotyping of these cells confirmed the expansion of an immature subpopulation defined as Lin- Sca1+ Kit+ (LSK). This phenotype was reversed upon turning RUNX1a-expression off and led to a loss of Sca1 expression (Lin- Kit+, LK). To further understand the molecular consequences of RUNX1a overexpression we sorted the LK cells and LSK cells before and 36h after RUNX1a-expression was turned off. Next, we performed microarray analysis to assess differential gene expression in these different subpopulations. Gene set enrichment analysis (GSEA) identified upregulation of genes highly expressed in hematopoietic stem cells (HSC) and leukemic stem cells (LSCs) in RUNX1a-expressing LSK cells compared to those LSK cells in which RUNX1a expression was turned off. Conversely, a gene signature associated with stemness and self-renewal was lost in LK-cells when RUNX1a expression was turned off. Among the eleven leading edge genes, we found genes implicated in leukemogenesis, stem cell regulation, or both such as Erg, Meis1 and Bcl11a.To further understand the role of RUNX1a in vivo we transplanted C57Bl6 mice (n=29) with HSPCs expressing RUNX1a in a competitive reconstitution setting. Consistent with the immortalization of HSPCs in vitro, RUNX1a-overexpressing HSPCs expanded in the bone marrow of transplanted mice. We observed significantly higher frequencies of LK (2.9-fold) and LSK cells (5-fold) in the RUNX1a-expressing bone marrow cells compared to transplanted control mice. High frequencies of RUNX1a-expressing cells in the bone marrow were associated with lower frequencies of RUNX1a-expressing cells in the peripheral blood indicating a differentiation block. In addition, we found that 85% of the RUNX1a-expressing cells were committed to the myeloid lineage (CD11b+/Ly6G+) at the expense of the lymphoid lineage (B220 and CD3e). Moreover, RUNX1a expression led to an increased percentage (65%) of immature erythroblasts (Ter119-) in the bone marrow compared to control cells (55%).In summary, we have demonstrated that RUNX1a overexpression immortalized HSPCs by upregulation of genes involved in leukemogenesis, stemness and self-renewal. In vivo such HSPCs showed a competitive advantage that was associated with a block of differentiation. Our study, particularly the gene expression analysis, provides novel insights into genetic drivers contributing to the development of myeloid malignancies in patients with RUNX1 mutations. DisclosuresNo relevant conflicts of interest to declare.

BRCA1/2 Containing Complex 3 (BRCC36) Is Recurrently Mutated in AML with t(8;21) and Associated with Increased Sensitivity to Chemotherapy through Impairment of the DNA Damage Repair Pathway

IntroductionBRCA1/BRCA2-containing complex 3 (BRCC36) is a Lys63-specific deubiquitinating enzyme (DUB) involved in DNA damage repair. Mutations in BRCC36 have been identified in 2-3% of patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). The role of BRCC36 mutations in de novo AML and their impact on DNA damage-inducing cytotoxic chemotherapy sensitivity is not clear.AimWe aimed to determine the incidence of BRCC36 mutations in AML and their impact on outcome and drug sensitivity in vitro.MethodsWe analyzed the entire coding region of BRCC36 for mutations in 191 AML cases with t(8;21) (q22;q22.1) and 95 cases with inv(16) (p13.1q22) using a customized targeted sequencing panel. Data for de novo AML was derived from The Cancer Genome Atlas Research Network (TCGA) data set (NEJM 2013). Lentiviral CRISPR/Cas9 was used to inactivate BRCC36 in t(8;21)-positive AML cell lines - Kasumi-1 and SKNO-1 - and murine hematopoietic stem and progenitor cells (LSKs). Knockout was confirmed by a cleavage assay as well as Western blot. AML1-ETO-9a was expressed by a retroviral vector. Cell lines and LSK cells were treated with different concentrations of doxorubicin or cytarabine and their viability was assessed seven days post treatment. DNA damage was assessed through phospho-γH2AX staining using flow-cytometry.ResultsBRCC36 mutations were identified in 7 out of 191 patients (3.7%) with t(8;21) AML and none of 95 patients with inv(16). In the TCGA data set one out of 200 patients (0.5%) with de novo AML had a BRCC36 mutation. This patient had a complex karyotype and would be considered as secondary AML with myelodysplastic-associated changes according to the 2016 WHO classification. Six of the 7 mutations were missense or nonsense mutations that were predicted to be deleterious to BRCC36 function. One mutation affected a splice site at exon 6, resulting in an impaired splicing capability. With intensive standard chemotherapy all patients with BRCC36 mutations achieved a complete remission and had an estimated relapse-free and overall survival of 100% after a median follow up of 4.2 years. Given its role in DNA damage repair, we hypothesized that BRCC36 inactivation sensitizes AML cells to DNA-damage inducing drugs. In order to test this, we generated BRCC36 knockout Kasumi-1 and SKNO-1 cell lines using CRISPR-Cas9. BRCC36 inactivation had no impact on cell growth on either of the cell lines. However, we found that BRCC36 knockout cells were significantly more sensitive to doxorubicin as compared to the parental cells with normal BRCC36. This was accompanied by a significant increase in DNA damage as assessed by phospho-γH2AX in BRCC36 knockout vs control cells after doxorubicin treatment. In contrast, BRCC36 inactivation had no impact on cytarabine sensitivity. We next assessed drug sensitivity in primary murine leukemic cells expressing AML1-ETO-9a. Again, inactivation of BRCC36 resulted in a significant higher sensitivity to doxorubicin but not cytarabine.ConclusionWe found BRCC36 to be recurrently mutated in t(8;21)-positive AML Inactivation of BRCC36 was associated with impairment of the DNA damage repair pathway and thus higher sensitivity to DNA damage-inducing chemotherapy. This might be also reflected by the favorable clinical outcome of patients with BRCC36 mutated t(8;21)-positive AML, a finding which has to be confirmed in a large patient cohort. DisclosuresPaschka:Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Otsuka: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Other: Travel support; Janssen: Other: Travel support; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Astex: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Bullinger:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding.

Syndecan-2 Surface Expression Identifies Hematopoietic Stem Cells with Increased Repopulating Capacity

Syndecans are transmembrane glycoproteins, which can regulate cell proliferation, growth, and adhesion through interactions with neighboring proteins within the plasma membrane or at the cytoplasmic interface. Although syndecans have been described to regulate aberrant signaling in hematological malignances, the role of syndecans in regulating normal hematopoietic stem cell (HSC) proliferation, differentiation, and self-renewal is largely unknown. We demonstrate that syndecan-1 and syndecan-3 are expressed on the surface of < 10% of murine hematopoietic stem and progenitor cells, whereas, syndecan-4 is expressed on 50% of lineage negative (Lin-) progenitor cells and 98% of c-Kit+Sca-1+Lin- (KSL) hematopoietic stem/progenitor cells, KSL CD34-CD48-CD150+/- short-term HSCs, and 100% of CD34-CD48-CD150+ long-term HSCs. Interestingly, we find that syndecan-2 is expressed by 11% of KSL CD34-CD48-CD150+/- short-term HSCs and 36% of CD34-CD48-CD150+ long-term HSCs. More specifically, our data demonstrate a 15-fold increase in syndecan-2 surface expression on KSL CD34-CD48-CD150+ HSCs compared to Lin- progenitor cells (p<.0001, ****). Collectively, these data suggest that syndecan-2 may be a marker for long-term HSCs. In keeping with this hypothesis, we found that syndecan-2+ CD34- KSL cells produce two-fold more granulocyte, erythrocyte, monocyte, megakaryocyte (GEMM) colonies compared to syndecan-2- CD34- KSL cells (p=.0017, **). Cell cycle analyses revealed a significant increase in BrdU incorporation in syndecan-2+ KSL cells compared to syndecan-2- KSL cells (90% versus 40%, p<.0001, ****). Competitive repopulation assays comparing syndecan-2+ or syndecan-2- CD34- KSL bone marrow cells demonstrated that mice transplanted with syndecan-2+ CD34- KSL cells displayed threefold increased donor multilineage hematopoietic cell repopulation compared to mice transplanted with syndecan-2- CD34- KSL cells. These data suggest that syndecan-2 expression marks a highly proliferative population of HSCs with increased multilineage repopulating capacity and that syndecan-2+ HSCs can be readily isolated to enhance the efficacy of hematopoietic cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

SHIP1 Inhibition As Novel Therapeutic Approach in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the Western world. Malignant B cells are blocked from differentiating into immunoglobulin producing-plasma cells and clonally accumulate in the spleen, lymph nodes, bone marrow and peripheral blood. CLL is characterized by immunosuppression throughout all disease stages, which is mediated by increased numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Jitschin and Braun et al., Blood 2014) and direct inhibitory effects of the malignant CLL cells on T cells (Christopoulos etal., Blood 2011).Over the past decade, significant improvement in understanding the pathogenesis of CLL has highlighted the importance of active B cell receptor (BCR) signaling. This has revealed promising targeted treatment options, including the small molecule inhibitors targeting the phosphatidylinositol-3-kinase (PI3K) signaling pathway. Idelalisib and Duvelisib are under clinical investigation for CLL treatment, however, treatment-related toxicities are limiting their application and none of these approaches are curative, highlighting the importance of functional anti-tumor immune responses in CLL for prolonged treatment efficacy.Here, we are testing a novel approach that aims to selectively target CLL B cells and simultaneously restore an appropriate immune cell function. The phosphatase SH2-domain-containing inositol 5ʹ-phosphatase 1 (SHIP1) serves as negative feedback molecule and downregulates PI3K signaling in B cells by dephosphorylating the 5'phosphate of Phosphatidylinositol (3,4,5)-trisphosphate. We hypothesize that CLL cells rely on such negative regulators for optimal survival and can only tolerate a maximum signaling level. We are therefore testing whether SHIP1 inhibition induces hypersignaling and thereby CLL cell death. Furthermore, we are investigating whether SHIP1 inhibition simultanously stimulates immune responses, as it has been shown to induce expansion of murine hematopoietic and mesenchymal stem cell compartments (Brooks et al., Stem cells 2014). 3α-Aminocholestane (3AC) is a small molecule inhibitor of SHIP1 and can be used for pharmacological inhibition.First, we investigated the expression and phosphorylation levels of SHIP1 in CLL. We found SHIP1 to be expressed at various levels in CLL peripheral blood and strongly phosphorylated compared to age-matched healthy donors. Besides, SHIP1 transcription is upregulated in lymph nodes as compared to peripheral blood, which is in line with the assumption of increased BCR signaling in secondary lymphoid organs. We then set out to investigate the consequences of SHIP1 phosphatase inhibition. Similarly, to recent findings in acute lymphoblastic leukemia (Chen et al., Nature 2015), pharmacological inhibition of SHIP1 lead to rapid cell death of CLL cells. We further investigated the mode of cell death and observed specific features of apoptosis, namely caspase 3/7 activation and phosphatidylserine exposure on the outer cell membrane. This has been tested on primary CLL patient samples and T cell leukemia/lymphoma 1 (TCL1)-driven murine CLL cells and was not observed or significantly less pronounced in other lymphoma cell lines or healthy primary B cells. To confirm the specificity of the observed effects, we genetically activated AKT with a GFP reporter in the TCL1-driven mouse model in vivo and in vitro. By tracking GFP-expressing CLL cells, we observed an initial expansion followed by rapid cell death in vitro. When we induced AKT activation in vivo, GFP+ CLL cells were not detectable in the peripheral blood, total CLL count declined upon induction and we found decreased tumor burden in the secondary lymphoid organs, particularly in the lymph nodes. In addition to the direct effects on CLL cells, we sought to investigate the impact of SHIP1 inhibition on other immune cell functions. We observed that SHIP1 inhibition lowers the activity threshold of T cells: When we stimulated a reporter cell line with suboptimal doses of anti-CD3, 3AC treatment significantly enhanced the response rate.Therefore, we propose SHIP1 as a novel interesting target in CLL. In contrast to kinase inhibition and downregulation of the BCR signaling strength, phosphatase inhibition and BCR signaling overaction provides an attractive new treatment strategy for elimination of malignant CLL cells and stimulation of immune responses. DisclosuresNo relevant conflicts of interest to declare.

Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4+and CD8+ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease. DisclosuresRettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding.

CRISPR/Cas9 Generation of Npm1-Alk in Transplantable Murine Hematopoietic Stem Cells Accurately Models ALK-Positive Lymphoma in Recipients

Chromosomal rearrangements resulting in generation of novel fusion oncogenes are common in hematologic malignancies. These disease drivers are key therapeutic targets and form the basis of animal model development for preclinical studies. For example, retroviral introduction of the chronic myeloid leukemia (CML) fusion BCR-ABL to hematopoietic stem cells (HSCs) results in a myeloproliferative disease similar to accelerated-phase human CML when transplanted to recipient mice. This model and many others are established traditionally through retroviral or germline introduction of human fusion oncogenes to the murine genome. Recent advances in CRISPR/Cas9 technology now permit direct editing of the murine genome to create endogenous genotypes that more accurately reflect configurations found in human diseases. To date, these techniques have not been successfully applied to the modeling of fusion oncogene-driven hematologic malignancies.Anaplastic Large Cell lymphoma (ALCL) is a T-cell non-Hodgkin lymphoma common in adolescents and young adults and driven in ~70% of cases by chromosomal rearrangements involving Anaplastic Lymphoma Kinase (ALK). Most commonly, t(2:5; p23:q35) fuses the 3‘ ALK kinase domain to the 5‘ oligomerization domain of the constitutively expressed Nucleophosmin1 (NPM1) gene. An existing immunocompetent model of ALK+ lymphoma employs a CD4 promoter-driven NPM1-ALK transgene but results in immature T-lymphomas in about two-thirds and B-lineage plasma-cell lymphomas in the rest of animals. We employed CRISPR/Cas9 vectors containing guide strands designed to generate double-stranded DNA cleavage in mouse chromosomes 11 and 17 at breakpoints predicted to generate an in-frame Npm1-Alk fusion oncogene. Wild-type HSCs derived from fetal livers were divided and subjected to either transient CRISPR or mock transfection during a brief ex vivo passage followed by immediate transplantation to sub-lethally irradiated wild-type recipients.Mice initially transplanted with CRISPR-modified HSCs developed an ALK+ large cell lymphoma with a latency of ~9 months, while mock transfected controls sacrificed in parallel were phenotypically normal. qPCR analysis of lymphoid organs from mice that developed disease showed extremely high expression of Alk and Tnfrsf8, which encodes CD30. Pathologically, tumors contained large malignancy T cells with anaplastic morphology. Immunohistochemistry confirmed ALK protein expression, including nuclear localization classic for NPM1-ALK, and intense CD30 cell-surface staining. One recipient instead developed a CD30-negative ALK+ diffuse large B-cell lymphoma. Transplantation of primary T-lymphoma cells to secondary recipients resulted also in ALK+ T-cell lymphomas in recipients with more rapid onset infiltrating lymph nodes, spleen, liver, and other organs. T-cell receptor clonality analyses through β-chain deep sequencing show an oligoclonal T-cell disease in both primary and secondary recipients.We therefore demonstrate successful genomic editing of transplantable murine hematopoietic stem cells to generate a novel model of a fusion oncogene-driven hematologic malignancy. These methods are widely applicable to additional lymphomas and leukemias and could fuel development of improved in vivo preclinical model systems. DisclosuresNo relevant conflicts of interest to declare.

Genetic Dissection of p53 Driven Senescence of Bone Marrow Mesenchymal Cells in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by bone marrow infiltration of abnormally proliferating leukemic blasts which results in fatal anemia, bleeding and infectious complications due to compromised normal hematopoiesis. Patients with complete remission (CR) but incomplete blood cell count recovery (CRi) have significantly shorter survival compared to CR patients. Although there is a correlation between CRi and minimal residual disease (MRD), the two variables were shown to be independent risk factors for relapse development (1). The mechanism by which AML induces bone marrow failure in patients is largely unknown. Here, we demonstrate that AML derived MSCs highly express p53 and p21 proteins and are more senescent compared to their normal age-matched controls as demonstrated by high β-galactosidase staining (figure 1. A, B&C). Emerging evidence indicates that the aging of endosteal niche cells results in lower reconstitution potential of hematopoietic stem cells (2). To functionally evaluate the effects of AML on bone marrow MSCs, we utilized a murine leukemia model of the AML microenvironment. We transplanted Osx-Cre;mTmG mice with AML cells and compared the senescence of MSCs in normal bone marrow (Figure 1.D) with AML (Figure 1.E). Consistent with our initial findings in human, AML strongly induced senescence of osteoblasts. This suggests that AML suppresses normal hematopoiesis by inducing senescence in the hematopoietic niche. To address the role of p53 signaling in senescence of MSCs we generated a traceable conditional p53 gain/loss model specifically in bone marrow MSCs using Osx-Cre;mTmG; Mdm2fl/+ and Osx-Cre;mTmG;p53fl/fl mice respectively (Figure 1.F). Deletion of p53 in bone marrow MSCs resulted in an increased population of osteoblasts (GFP+) in Osx-Cre;mTmG;p53fl/fl mice in comparison to Osx-Cre;mTmG mice suggesting that p53 loss in osteoblasts inhibits senescence of osteoblasts. In order to evaluate p53 activity after recombination of p53fl alleles in the osteoblasts, we isolated MSCs from bone marrows and analyzed the expression of p21.P21 was significantly down regulated in osteoblasts (GFP+) derived from Osx-Cre;mTmG;p53fl/fl mice whereas its expression in the hematopoietic cells from same tissue (tdTomato+) remained comparable to p53 wild type suggesting that p21 as the master regulator of senescence is regulated by p53 in bone marrow mesenchymal cells. To evaluate the effect of p53 loss in osteoblasts and its impact on hematopoietic cells, we isolated the GFP+ cells (osteoblasts) and RFP + cells (hematopoietic) by FACS. Senescent cells, non-cell autonomously, modulate the bone marrow microenvironment through the senescence-associated secretory phenotype (SASP). We analyzed the expression of fifteen SASP cytokines by QPCR. Deletion of p53 in bone marrow mesenchymal cells strongly abrogated the expression of several SASP cytokines. Interestingly several Notch target genes such as Hey1 and Hey2 were highly induced in MSCs following p53 deletion suggesting a role for Notch signaling in hematopoietic failure following AML induced MSCs senescence.Our data suggest that AML induces senescence of endosteal niche resulting in hematopoietic failure. These findings contribute to our understanding of the role of p53 in leukemia MSCs and could have broad translational significance for the treatment of hematopoietic failure in patients with AML.Chen X, et al. (2015) Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol 33(11):1258-1264.Li J, et al. (2018) Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging. Sci Rep 8(1):2833. [Display omitted] DisclosuresAndreeff:Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Jazz Pharma: Consultancy; Reata: Equity Ownership.

The NAD+ Salvage Pathway Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance and Asymmetric Division

During the last 50 years, with over a million transplants, hematopoietic stem cell (HSC) transplantation has been the first and most extensively exploited stem cell therapy and the only curative regime for most acute leukemias. Nevertheless, the success of HSC transplantation and all other intensive ablative chemotherapy regimes is still overshadowed by a procedure-associated mortality of ~25%, due to both graft versus host disease and to the infectious complications associated with the severe leucopenia following bone marrow (BM) ablation, even in spite of standard support with HSC-stimulating factor G-CSF. It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response can severely compromise HSC regeneration.Here we show that the NAD+-boosting agents Nicotinamide Riboside (NR) and Nicotinamide MonoNucleotide (NMN) reduce mitochondrial activity within HSCs through increased mitochondrial clearance via autophagy and possibly the Unfolded Protein Response mitochondria (UPRmt), leading to increased asymmetric HSC divisions as measured by asymmetric mitochondrial distribution in single cell pair-daughter analysis. This process was abrogated in Nrk1-/-;Nrk2-/- double knock out mice, which cannot incorporate NR into the NAD+ salvage pathway. Contrary to controls, purified murine HSC underwent self-renewal in minimal culture conditions in presence of NR, and human CD34+ hematopoietic progenitors (hCD34+) cultured in vitro in presence of NR could repopulated NSG mice in primary and secondary transplant recipients.In vivo, NR dietary supplementation resulted in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improved survival by 80%, and accelerated blood recovery after murine lethal irradiation and limiting-HSC transplantation. In human xenotransplanted immune-deficient NSG mice, NR increased the production of human leucocyte progeny from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD+ boosting strategies on the most primitive blood stem cells, establishes a novel link between mitochondrial stress, mitophagy and stem cell fate decision, and unveils the potential of NR in vivo supplementation to improve recovery of patients suffering from hematological failure including post-chemo/radiotherapy. DisclosuresNo relevant conflicts of interest to declare.