Immature Hematopoietic Stem Cells Research Articles

Effects of Black Raspberry Supplementation on Methylation Pathways in Vav-creAsxl1fl/flTet2fl/fl Double Knockout Mice with Early-stage Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDS) are a subset of myeloid malignancies defined by clonality of immature hematopoietic stem cells that leads to faulty blood cell development. These syndromes can lead to an increased risk of infection and may transform into acute myeloid leukemia, making it critical to determine effective treatments for the condition. While hypomethylating agents such as azacitidine and decitabine, as well as stem cell transplants, have been delineated as favored treatments for MDS, not all patients are physiologically receptive to these treatments. However, black raspberries (BRBs) have been shown to exert hypomethylating effects in various malignancies, with minimal adverse effects and thus a broader range of potential candidacies. This study aimed to investigate the potential of BRBs to exert such effects on MDS using Addition of Sex Combs Like/Tet Methylcytosine Dioxygenase 2 (Asxl1/Tet2) double knockout mice (Vav-cre Asxl1fl/fl Tet2fl/fl), which typically manifest symptoms around 25 weeks of age, mirroring genetic mutations found in humans with MDS. Following a 12-week dietary supplementation of Vav-cre Asxl1fl/fl Tet2fl/fl mice with 5% BRBs, we observed both hyper- and hypomethylation at multiple transcription start sites and intragenic locations linked to critical pathways, including hematopoiesis. This methylation profile may have implications for delaying the onset of MDS, prompting a need for in-depth investigation. Our results emphasize the importance of exploring whether an extended BRB intervention can effectively alter MDS risk and elucidate the relationship between BRB-induced methylation changes, thus further unlocking the potential benefits of BRBs for MDS patients.

Early Molecular Steps in Human Bone Marrow Stem Cell Differentiation Trajectories

Understanding the molecular signatures of human hematopoietic stem/progenitor cells (HSCPs) and gene network changes leading to differentiation induction are of utmost importance to achieve therapeutic hematopoietic stem cell (HSC) expansion. The identification of surface markers has improved the prospective isolation of human bone marrow HSCs over the last decades, but the isolated population still remains very heterogeneous. Single cell sequencing approaches have the potential to unbiased assort stem cells along their differentiation stages and trajectories, and to decipher molecular markers in differentiation hierarchies. However, whole transcriptome single cell sequencing often suffers from inadequate detection of low-expressed genes with increased drop-outs. To address this limitation and to provide sensitive mRNA quantification in human HSPCs, we developed a selected targeted gene panel (600 genes) in combination with 50 protein markers to cover most relevant genes/proteins of early HSPC differentiation and cell cycle status. We employed single cell CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) based multi-omic profiling to >60.000 high-quality FACS-enriched human bone marrow HSPCs (CD34 + and CD34 +CD38 - cells) from 15 healthy donors representing young, mid and old age groups. Here, we demonstrate the most comprehensive molecular map of early human HSC differentiation and provide evidence of early branching points into lineage trajectories across different age groups using Slingshot pseudotime analysis. Our data demonstrate that the HSC branched into two main lineages, with the first branching point giving rise to megakaryocytic and erythroid progenitors (MKP/ERP), followed by lymphoid and myeloid progenitors (LMP/MDP). As expected, the most immature HSCs were defined by high expression of CD34, CD90, CRHBP, HOPX, HLF, MLLT3. However, we observed a number of hitherto undescribed HSC markers. The MKP/ERP lineage was defined by GATA1, TFRC, CD36, MPL and VWF expression. Furthermore, we found CD274, the Programmed Cell Death receptor ligand 1, to be selectively expressed in the MKP cluster. Re-clustering and trajectory analysis of the most immature CD34 +CD38 -/low cells confirmed early lineage commitment, suggesting that an MKP/ERP development does not include myeloid or lymphoid progenitors. A similar pattern was observed for all age groups. Interestingly, we observed increased expression of novel marker genes ( HLA-E, DLK1, ADGRG6, ADGRG1, MMRN1) and proteins (CD137 and CD273) in the most immature HSCs. Besides a gradual upregulation of CD38 along differentiation, differentiated cells showed lower CD45 levels. Furthermore, MKP/ERP committed cells showed a downregulation of SPINK2. TradeSeq analysis defined the continuous single cell expression changes in genes and markers upon early differentiation and lineage commitment, allowing the detection of decision-making gene networks. Our data adds further evidence to the revised model of hematopoiesis that challenges the dogma of a common myeloid progenitor giving rise to erythroid, megakaryocytic and myeloid cells. Furthermore, we see a structured hierarchical organization with a sequential loss of lineage potential and not an early commitment of uni-lineage restricted precursors. We report on the upregulation of ADGRG6, ADGRG1, DLK1, HLA-E, MMRN1 CD137 and CD273 in the most immature HSCs. Molecular and functional characterization of CD273 hi and low HSCs are presented in an adjacent report by our group at this meeting (Schmachtel et al.). In conclusion, we provide a valuable resource for the continuous regulation of genes and surface proteins along early differentiation of very immature human HSCs.

Screening for Intracellular Phosphorylation Cascades That Positively and Negatively Regulate the Self-Renewing Proliferation of Immature HPCs

For the purpose of studying hematological diseases involving complex lineages of cells, hematopoietic differentiation using patient-derived iPS cells has already proved to be powerful in the analysis of various diseases. However, in terms of sourcing for the expansion of its use to larger scale studies, the time-consuming and inefficient differentiation process still presents a significant obstacle. In this context, the development of a method that allows the proliferation of progenitor cells (HPCs) which maintains their ability to differentiate into multiple lineages is an important issue. In vivo, immature multipotent hematopoietic stem cells (HSCs) reside in the bone marrow, where intracellular signals via phosphorylation cascades co-ordinately regulate the expression pattern of transcription factors (TFs), leading to fate decisions within the microenvironment between self-renewal and one-way differentiation into HPCs. In contrast, iPS cell-derived hematopoietic differentiation does not involve HSCs and the microenvironment differs from that of bona fide cells. Therefore, the signal combinations required to amplify iPS cell-derived HPCs without further differentiation should naturally differ from those observed in HSCs in vivo. Based on this assumption, we constructed a CRISPRa and CRISPRi screen for phosphorylation-related genes using iPS cell-derived differentiation to search for factors that enable long-term maintenance of multipotent HPCs. First, Doxycycline-inducible dCas9-VPR (CRISPRa) or dCas9-KRAB-MecP2 (CRISPRi) expression cassettes were introduced into human iPS cells before, and then acted in combination with the phosphorylation-focused sgRNA libraries at the time when HPCs begin to be produced from mesodermal progenitors during their hematopoietic differentiation. Then, to identify the sgRNAs of interest with single-cell accuracy, colony formation assays were performed by isolating HPCs one by one by single-cell sorting. 2 weeks later, cells were collected only from wells that had formed CFU-Mix. Amplicon-sequences were performed using genomic DNA extracted from those cells and the sgRNAs that significantly contributed to the phenotype were identified by calculating the difference in enrichment with control sgRNAs. As a result, the CRISPRa screen identified a group of intracellular signals related with eight candidate genes known to be specifically activated in immature cells in the normal hematopoiesis. On the other hand, the results of CRISPRi showed that suppression of necroptosis-related signalling probably promotes long-term culture of HPCs in the stroma-free, artificial culture circumstances. Our results showed the potential to obtain multipotent HPCs from iPS cells over a long period of time and in large quantities, simply by adjusting the intracellular signals without artificial adding or removing of individual genes.

Tumor Necrosis Factor from Leukemic Microenvironment Provides Non-Leukemic Hematopoietic Stem Cells with Megakaryocyte Lineage Bias

Background: Acute myeloid leukemia (AML) is characterized by proliferation of malignant cells and impairment of normal hematopoiesis, leading to the severe bone marrow failure. Although genetic abnormalities and pathogenesis of AML are now investigated in detail, the precise mechanism impairing normal hematopoiesis in AML is not fully elucidated yet. Hematopoietic stem cells (HSC) retain the self-renewal capacity together with multipotent differentiation potential into all mature blood cells, playing a major role in maintaining the normal hematopoiesis. The ability of HSC is maintained by complex network of intrinsic factors and external stimulation from microenvironment called as HSC niche. According to these backgrounds, we hypothesized that the impairment of normal hematopoiesis in AML is caused by abnormal function of non-leukemic (nl) HSC. To verify this hypothesis, we isolated nl-HSC and HSC niche cells from MLL-AF9-induced AML mice and further analyzed their function and gene expression profiles. Results: The frequencies of nl-HSC/hematopoietic progenitor fraction (Lin-cKit+Sca1+ cells, HSPC) were significantly increased at the initiation phase of leukemic proliferation in AML mice (leukemic cells : 10-30% in BM)（normal: 0.76+/-0.34%, AML: 2.07 +/-0.93 %, p<0.001）. Surface expression level of CD41 was also upregulated (normal: 15.1+/- 0.526%, AML: 25.4+/- 9.28%, p<0.05). Interestingly, the results of in vitro culture of nl-HSPC demonstrated that they proliferated more efficiently than HSPC from normal mouse BM, and that they tended to differentiate into megakaryocyte (MgK) lineage. Gene set enrichment analysis revealed that MgK-related gene sets and several inflammatory cytokines signaling pathways, including tumor necrosis factor (TNF) pathway, were statistically enriched in nl-HSC. These data suggested that nl-HSC/HSPC retain the MgK-biased character. On the other hands, the gene expression profiles of perivascular stromal cells (ICAM1+CD31-CD45-TER119-cells) in AML mice demonstrated the significantly decreased expression of HSC-supporting factors (e.g., lpl, ptn, rspo2, angptl4, kitl, cxcl12), when compared with those in normal mice. These data suggested that the changes of multiple external humoral factors from leukemic environment induce functional alternation of nl-HSC. To further investigate the role of inflammatory humoral factors in HSC self-renew and differentiation, we next utilized polyvinyl alcohol (PVA)-based ex vivo HSC expansion system and focused on the role of TNF signaling. In the analysis of highly purified normal HSC (CD150+CD48-CD34-cKit+Sca1+Lin-cells) with PVA, TNF alpha increased both proliferation and MgK differentiation. UMAP analysis of single cell RNA sequence data for ex vivo-expanded HSCs with TNF alpha showed the increased frequencies of the clusters expressing MgK-related genes (e.g., itga2b and gp1ba) and relatively decreased immature HSC related genes (e.g., hlf and procr). The results of competitive repopulation assay using ex-vivo expanded normal HSC with TNF alpha stimulation in HSC promoted myeloid differentiation of HSC, rather than self-renew and expansion of HSC. Conclusion: Our data suggested that TNF alpha from leukemic microenvironment may play a key role for the driver signaling of HSCs to MgK, eventually resulting in the impairment of normal HSC maintenance.

High Throughput Multi‐omic Characterization of Clonal Hematopoietic Stem Cells During Progression of Myeloid Malignancies

Hematopoietic stem cells (HSCs) are responsible for giving rise to all other lineages of blood cells in the body. Over time, mutations in HSCs can promote the outgrowth of clonal populations that outcompete other HSCs, resulting in a phenomenon called Clonal Hematopoiesis of Indeterminate Potential (CHIP). Though not cancerous in and of itself, CHIP can progress to more serious hematologic disorders, such as the Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). The mechanisms of clonal expansion, by which certain mutant HSCs acquire a competitive advantage over other HSCs, currently remain largely undeciphered, as are the mechanisms by which clonal HSCs drive the initiation of MDS and contribute to the development of AML. Moreover, previous studies have shown that such mutant HSCs are resistant to conventional therapies and may act as reservoirs for disease relapse and progression. In the past, researchers were hindered by bulk cell analyses and the relative rarity of HSCs, but advances in single‐cell omics have now enabled us to explore the molecular heterogeneity of clonal HSCs and identify distinct clonal populations based on genotype and cell surface phenotype. We used single cell RNA sequencing to examine the transcriptional dynamics of purified HSCs from MDS and AML patients before and after treatment, as well as from age matched elderly controls. Interestingly, dimensionality reduction methods such as UMAP and tSNE revealed a reservoir of control HSCs that clustered with MDS HSCs. Upon comparison with other normal control HSCs, we found that genes associated with aging, mitochondrial function, and particular ion channels were strongly upregulated in these “MDS‐like” control HSCs, while genes involved in ribosomal and translation activity, along with certain surface markers, were substantially downregulated. Additionally, ribosomal transcripts were depleted in MDS HSCs from patients who did not respond to treatment. These results support the notion that the most immature HSCs, which impose the strictest constraints on translation, might clonally expand and initiate CHIP and/or MDS upon acquiring driver mutations, also serving as treatment resistant populations that underlie disease relapse. We also subjected purified MDS HSCs to simultaneous single cell targeted DNA sequencing and cell surface phenotyping, which allowed us to correlate the cell surface phenotype and genotype for specific clonal populations and identify cell surface markers that can be used to isolate HSCs with enhanced engraftment ability. Taken together, these results indicate that combined single cell genotyping and phenotyping can be used to track clonal populations across different stages of pathogenesis, providing further insight about the development of CHIP and its progression to MDS and AML.

Identification and immune responses of thrombocytes in bacterial and viral infections in grass carp (Ctenopharyngodon idella)

Thrombocytes are an important component in peripheral blood cells and play a crucial role in immune regulation. CD41 is one of the biomarkers of thrombocytes. In this study, grass carp (Ctenopharyngodon idella) CD41 protein was expressed in Escherichia coli and purified by affinity chromatography. Subsequently, New Zealand rabbits were immunized with this protein via subcutaneous injection. The antibody titer examined by enzyme linked immunosorbent assay was 1:12800. The concentration of rabbit polyclonal antibody purified by HiTrap-rprotein-AFF affinity chromatography column was 1.9 mg/mL. The specificity was identified by SDS-PAGE, Western blot, flow cytometry, and indirect immunofluorescence assays. The purified antibody was used to screen grass carp thrombocytes, and CD41+ cells were 14.13%. CD41+ cells were further verified by Giemsa staining, transmission electron microscopy and RT-PCR. mRNA expression of CD41 in thrombocytes was not affected by viral or bacterial challenge in vitro, while CD41 transcripts were remarkably induced post pathogenic infections in vivo, which results from the immature hematopoietic stem cells and thrombocytes. Indirect immunofluorescence assay revealed that grass carp reovirus (GCRV) could not invade thrombocytes; however, mRNA expressions of some representative innate immune genes (IFN1, IL-1β, TNFα and Mx2) were significantly up-regulated post GCRV challenge. Meanwhile, the transcripts of some innate immune genes (IL-6 and TNFα) were swiftly increased post bacterial infection. These results indicated that the rabbit anti-CD41 polyclonal antibody possesses good specificity and can effectively bind to the CD41 protein on the surface of grass carp thrombocytes. Grass carp thrombocytes participate in immune regulation in viral and bacterial infections.

3177 – NEURAL CREST CONTRIBUTION TO THE MURINE HSC SPECIFICATION NICHE

Hematopoietic stem cells (HSCs) are developmentally specified from endothelium of the dorsal aorta (DA) in the aorta-gonad-mesonephros (AGM) region of the midgestation mouse embryo. Their emergence requires signals from neighboring cell populations in the microenvironment. We previously showed that neural crest cells (NCCs), a migratory population of cells that in mouse migrates to the DA around midgestation, are required for HSC specification in zebrafish. Although NCC-derived differentiated sympathetic neurons have been proposed to promote HSC emergence in the mouse through secretion of catecholamines, an earlier role for NCCs in HSC specification prior to sympathetic neuronal differentiation has not been demonstrated. In the fish model, the NCC requirement precedes sympathetic neuronal differentiation. In our recent studies, we have found that mouse NCCs reach the dorsal aorta between E9.5 and E10 but only express catecholamine-synthesizing enzymes after E10.5, when the definitive hematopoietic program initiates. We hypothesize that NCCs play a role in HSC specification in mouse prior to neuronal differentiation and secretion of catecholamines. To test this possibility, we have used an inducible diphtheria toxin mouse model to ablate NCCs before they reach the DA. Although we find an increase in immature hematopoietic stem and progenitor cells, we note a decrease in pre-HSCs type II. Our data suggest that NCCs play a distinct role in HSC specification at a specific maturational transition point during early specification. Hematopoietic stem cells (HSCs) are developmentally specified from endothelium of the dorsal aorta (DA) in the aorta-gonad-mesonephros (AGM) region of the midgestation mouse embryo. Their emergence requires signals from neighboring cell populations in the microenvironment. We previously showed that neural crest cells (NCCs), a migratory population of cells that in mouse migrates to the DA around midgestation, are required for HSC specification in zebrafish. Although NCC-derived differentiated sympathetic neurons have been proposed to promote HSC emergence in the mouse through secretion of catecholamines, an earlier role for NCCs in HSC specification prior to sympathetic neuronal differentiation has not been demonstrated. In the fish model, the NCC requirement precedes sympathetic neuronal differentiation. In our recent studies, we have found that mouse NCCs reach the dorsal aorta between E9.5 and E10 but only express catecholamine-synthesizing enzymes after E10.5, when the definitive hematopoietic program initiates. We hypothesize that NCCs play a role in HSC specification in mouse prior to neuronal differentiation and secretion of catecholamines. To test this possibility, we have used an inducible diphtheria toxin mouse model to ablate NCCs before they reach the DA. Although we find an increase in immature hematopoietic stem and progenitor cells, we note a decrease in pre-HSCs type II. Our data suggest that NCCs play a distinct role in HSC specification at a specific maturational transition point during early specification.

Matične ćelije - opšti pregled - od razvojnih hemobioloških koncepata do "(auto)grafting"-a u kliničkoj praksi

Conventional hematopoietic stem cell transplantation is a well-known treatment method for numerous acquired and congenital hematopoietic disorders, disorders of the immune system, as well as certain metabolic disorders. Stem cells (SCs) can be defined as cells capable of self-renewal with a high proliferative capacity and the potential to differentiate into functionally competent mature cells. Stem cells can be divided into embryonic SCs (ESCs) and tissue-specific or adult SCs - such as bone marrow (BM) stem cells, peripheral blood (PB) stem cells, and SCs derived from umbilical cord blood (UCB), as well as other non-hematopoietic or somatic SCs. SCs in adults are characteristically considered to be restricted in their regenerative and differentiative potential, while embryonic stem cells are 'true' totipotent/pluripotent cells, due to their ability to develop into endoderm, ectoderm, or mesoderm - all three embryonic tissue types in the human body. They are the most promising, but also the most controversial type of potentially transplantable SCs. Immature hematopoietic SCs have the potential of differentiating, not only into all blood cells, but also into some somatic cell types (SC plasticity). In different clinical settings, the transplantation of immature stem cells leads to the repopulation of recipient bone marrow, with subsequent complete, stable, and long-term reconstitution of hematopoiesis. Given that immature stem cells are also capable of homing to different tissues, autologous stem cell implantation into a damaged and/or ischemic area induces their colonizing and consecutive transdifferentiating into cell lineages of the host organ, including neovascularization. Thus, they are clinically applicable in the field of regenerative medicine for the treatment of myocardial, brain, vascular, liver, pancreatic, and other tissue damage. The purpose of this overview is to recapitulate the key developments in the rapidly evolving area of stem cell research, as well as to review the use of SCs in conventional transplantations and in regenerative medicine. Additionally, a brief critical evaluation of our own stem cell research will be summarized.

Leukemogenic Pathway of MLL-AF4 Fusion-Positive Acute Lymphoblastic Leukemia

Background Many hematologic malignancies, including leukemia, are caused by genetic abnormalities such as chromosomal translocation. These gene defects usually perturb differentiation and proliferation of hematopoietic stem/progenitor cells. As observed in most cancers, multiple gene defects are required to develop overt leukemia, and multi-step tumorigenesis is also the rule in leukemogenesis. Leukemia-specific fusion genes are usually generated early in leukemogenesis as essential genetic defects (1st hit) producing pre-leukemic stem cells. Additional genetic defect(s) occur in pre-leukemic stem cells, finally generating leukemic stem cells that expand to present overt leukemia. Infant acute lymphoblastic leukemia (ALL) are frequently characterized by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion genes. Amongst the > 50 genes that have been identified as fusion partners of MLL, fusion with AF4 is characteristically and commonly observed in infant ALL, representing a hallmark of poor prognosis. In most cases of ALL with MLL-AF4, the fusion gene originates in utero as a primary event. Although mutations in RAS and FLT3 are frequently observed in MLL-AF4 -positive ALL, recent genetic data showed significantly lower number of accompanying mutations, which suggested that 2nd hit mutations were not necessary for developing MLL fusion-positive leukemia. The necessity of 2nd hit mutations in leukemogenesis with MLL-AF4 is still under debate. Materials and Methods Several previous studies have modeled leukemic transformation by introducing fusion genes into hematopoietic stem/progenitor cells; however, reproduction of certain forms of ALL, such as MLL-AF4 -positive infant ALL, has been challenging. We used the immature hematopoietic cells derived from mouse embryonic stem (ES) cells as targets for MLL-AF4 -mediated transformation. The MLL-AF4 fusion gene was introduced into mouse ES cells to obtain ES cell clones with stable expression of MLL-AF4 . These ES cells were conditioned to generate embryoid bodies (EBs) and subsequently differentiated into hematopoietic lineages. Tie2-positive cells, which contain immature hematopoietic stem cells, were separated and transplanted into immunodeficient non-obese diabetic/Shi-scid, IL-2Rγ null (NOG) mice to investigate leukemia development. Retroviral insertional mutagenesis was used to introduce additional genetic defect(s) as candidates for tumor-promoting 2nd hit mutations. Purified Tie2-positive cells were transduced using the murine stem cell virus (MSCV) and transplanted into NOG mice. Results Mouse ES cells with stable expression of the MLL-AF4 fusion were viable and could proliferate and differentiate to produce EBs. However, EBs derived from MLL-AF4 -positive ES cells differentiated via the mesenchymal pathway rather than into the Tie2-positive hematopoietic lineage. Mice transplanted with Tie2-positive cells with MLL-AF4 expression did not develop leukemia within the observed period. However, addition of an additional genetic change by retroviral insertional mutagenesis accelerated tumorigenesis in the recipient mice. The MLL-AF4 -expressing Tie2-positive cells with insertional mutagenesis showed clonal expansion and formed mostly extra-medullary tumors with B220 expression, but did not fully develop into tumors of hematopoietic organs. Viral integration sites of these tumors were analyzed to determine additional genetic events inducing tumor formation. In the MLL-AF4 model, viral integration often caused overexpression of gene(s) located near the integration site. Conclusion Considering the in utero formation of MLL-AF4 harboring pre-leukemic stem cells in ALL, hematopoietic progenitor cells or immature fetal cells may be potential targets of leukemic transformation. Immature hematopoietic cells such as the Tie2-positive cells derived from ES cells could be suitable candidates for transformation with MLL-AF4 . Clonal expansion of MLL-AF4 -positive cells after introduction of retroviral insertional mutagenesis suggests the necessity of additional genetic change(s) that activate certain target gene(s) in leukemogenesis with MLL-AF4 . We are currently analyzing the roles of these genes in the leukemic transformation of MLL-AF4 -positive hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

Pathophysiology of chronic lymphocytic leukemia and human B1 cell development.

Chronic lymphocytic leukemia (CLL), the most frequent type of leukemia in adults, is a lymphoproliferative disease characterized by the clonal expansion of mature CD5+ B cells in peripheral blood, bone marrow, and secondary lymphoid tissues. Over the past decade, substantial advances have been made in understanding the pathogenesis of CLL, including the identification of recurrent mutations, and clarification of clonal architectures, transcriptome analyses, and the multistep leukemogenic process. The biology of CLL is now better understood. The present review focuses on recent insights into CLL leukemogenesis, emphasizing the role of genetic lesions, and the multistep process initiating from very immature hematopoietic stem cells. Finally, we also review progress in the study of human B1 B cells, the putative normal counterparts of CLL cells.

PF337 ALRN‐6924, A DUAL INHIBITOR OF MDMX AND MDM2 THAT CAUSES MINIMAL THROMBOCYTOPENIA IN PATIENTS, DISRUPTS DIFFERENT STAGES OF THROMBOPOIESIS COMPARED TO MDM2‐ONLY INHIBITION

Background:ALRN‐6924 is a cell‐permeating α‐helical peptide that disrupts interactions of the p53 tumor suppressor protein with its endogenous inhibitors, MDMX and MDM2. ALRN‐6924 can thereby restore p53‐dependent anti‐cancer activity in p53 wild type tumors. In an ongoing ALRN‐6924 clinical trial with 96 solid tumor and lymphoma patients enrolled as of 13Aug2018 there have been 1/0 adverse events (AE) of Grade 3/4 thrombocytopenia (TCP), respectively, at doses that yield objective responses (NCT02264613; Meric‐Bernstam et al, ASCO 2017; Shustov et al, ASH 2018). In contrast, small molecule MDM2‐only inhibitors induce Grade 3–4 TCP as a common AE and dose‐limiting toxicity.Aims:To explore the differences in TCP grades and frequencies from published solid tumor trials for ALRN‐6924 and MDM2i's, we investigated the effects of dual MDM2/MDMX‐inhibition vs. MDM2‐only inhibition during different stages of megakaryocyte maturation.Methods:Clonogenicity was measured in megakaryocyte (MK), erythroid (E), and granulocyte/monocyte myeloid (GM) progenitor colony‐forming unit (CFU) assays. Human bone marrow CD34+ cells seeded in methylcellulose semi‐solid media containing IL‐3 and IL‐6; and TPO, Epo or GM‐CSF together with ALRN‐6924, RG7388 (idasanutlin), or controls. CFU‐MK, CFU‐E, and CFU‐GM colonies were measured after 14 days of culture. Megakaryocyte maturation was modeled in vitro using a 14‐day liquid culture assay. Human bone marrow CD34+ cells were cultured in liquid medium containing SCF and TPO. Megakaryocyte maturation was assessed by measuring the expression of CD41 and CD42. To assess the effects of ALRN‐6924 and RG7388 on platelet development, drugs were added to the culture at different stages during the 14‐day period of megakaryocyte maturation in vitro. DNA synthesis was measured by BrdU, and apoptosis by Annexin V/propidium iodide. Healthy C57BL/6 mice treated with ALRN‐6924 3x/week were monitored for blood counts 2x/week; hematopoietic stem and progenitor cells in bone marrow were measured at 4 weeks.Results:In vitro, ALRN‐6924 disrupted MK colony formation more potently than RG7388, with IC50's of 0.024 and 0.12 μM, respectively, while their effects were comparable in CFU‐E and CFU‐GM assays with IC50's for both of ≈ 0.2 μM. Conversely, RG7388 disrupted megakaryopoiesis by inducing apoptosis and cell cycle arrest in maturing MKs 10‐fold more potently than ALRN‐6924. Consistent with these findings, ALRN‐6924 induced minimal effects on platelet counts in mice at doses that are effective in xenotransplanted and syngeneic tumor models, despite a significant decrease in MK and erythroid progenitors, while more immature hematopoietic stem and progenitor cells numbers remained unperturbed.Summary/Conclusion:ALRN‐6924 potently inhibits the clonogenic capacity of megakaryocyte progenitors in MK‐CFU assays but it is less potent during the process of megakaryocyte maturation than the MDM2‐only inhibitor RG7388. These findings suggest that the disruption of later stages of megakaryocyte maturation more severely affects platelet production, ultimately resulting in thrombocytopenia. However, inhibition of less mature megakaryocyte progenitors, prior to entering terminal maturation, appears to be better tolerated. This may explain the differences in TCP grades and frequencies from published solid tumor trials for ALRN‐6924 vs. MDM2‐only inhibitors, and suggests that MDMX in bone marrow cells, where MDMX RNA expression is the highest of all 37 normal tissues reported in the Protein Atlas, may contribute to these differences.

Unraveling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis.

B-cell acute lymphoblastic leukemia is the commonest childhood cancer. In infants, B-cell acute lymphoblastic leukemia remains fatal, especially in patients with t(4;11), present in ~80% of cases. The pathogenesis of t(4;11)/KMT2A-AFF1+ (MLL-AF4+) infant B-cell acute lymphoblastic leukemia remains difficult to model, and the pathogenic contribution in cancer of the reciprocal fusions resulting from derivative translocated-chromosomes remains obscure. Here, “multi-layered” genome-wide analyses and validation were performed on a total of 124 de novo cases of infant B-cell acute lymphoblastic leukemia uniformly diagnosed and treated according to the Interfant 99/06 protocol. These patients showed the most silent mutational landscape reported so far for any sequenced pediatric cancer. Recurrent mutations were exclusively found in K-RAS and N-RAS, were subclonal and were frequently lost at relapse, despite a larger number of non-recurrent/non-silent mutations. Unlike non-MLL-rearranged B-cell acute lymphoblastic leukemias, B-cell receptor repertoire analysis revealed minor, non-expanded B-cell clones in t(4;11)+ infant B-cell acute lymphoblastic leukemia, and RNA-sequencing showed transcriptomic similarities between t(4;11)+ infant B-cell acute lymphoblastic leukemias and the most immature human fetal liver hematopoietic stem and progenitor cells, confirming a “pre-VDJ” fetal cellular origin for both t(4;11) and RASmut. The reciprocal fusion AF4-MLL was expressed in only 45% (19/43) of the t(4;11)+ patients, and HOXA cluster genes are exclusively expressed in AF4-MLL-expressing patients. Importantly, AF4-MLL/HOXA-expressing patients had a significantly better 4-year event-free survival (62.4% vs. 11.7%, P=0.001), and overall survival (73.7 vs. 25.2%, P=0.016). AF4-MLL expression retained its prognostic significance when analyzed in a Cox model adjusting for risk stratification according to the Interfant-06 protocol based on age at diagnosis, white blood cell count and response to prednisone. This study has clinical implications for disease outcome and diagnostic risk-stratification of t(4;11)+ infant B-cell acute lymphoblastic leukemia.

Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation.Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these.Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv.In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks.To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML.Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. DisclosuresNeri:Philochem AG: Equity Ownership.

Single-Cell Sequencing in Normal and Malignant Hematopoiesis.

Hematopoiesis is one of the best studied adult stem-cell systems, with a differentiation hierarchy progressing from immature hematopoietic stem cells to over 10 distinct mature cell types. Recent technological breakthroughs now make it possible to define transcriptional profiles in thousands of individual cells. Facilitated by the wealth of prior data on cell purification and analysis strategies, hematopoiesis has been one of the earliest experimental systems to which many of these new single-cell sequencing technologies have been applied. In this review, the authors focus on recent studies, which have shed light on heterogeneity within individual populations as well as the relationships between populations, and also attempt to characterize the differences between normal and disease/perturbed states.

Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation.Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation).Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs.We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo.Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Network of Activated Cancer Signaling Pathways in a High Risk Subgroup of Acute Myeloid Leukemia Targetable By SRC-Kinase Inhibitors

Resistance to therapy including potent and selective targeted agents remains the major clinical challenge in AML. In fact, allogeneic stem cell transplantation remains the best curative treatment option for AML patients with high-risk features. This unmet clinical need may be addressed by novel approaches based on targeting networks of activated cancer signaling pathways (NACSPs) instead of only individual pathways. Signaling pathway activation in AML is not only related to class I mutations such as FLT3-ITD, but also to class II driver mutations. This is best exemplified by AML subtypes harboring non-random chromosomal aberrations, encoding driver mutations able to induce and maintain leukemia. Despite the contribution of class I mutations to a poor prognosis, targeting these lesions does not decisively contribute to the cure of AML in the great majority of the cases. AML with the translocation t(6;9)(p23;q34) encoding the related DEK/NUP214 fusion protein is a high risk group of AML patients characterized by young age and presence of FLT3-ITD in ~ 75% of the cases, which responds only transiently to FLT3-ITD inhibitors. Using this AML subtype as a model for high risk disease, we investigated the NACSPs activated by a class II driver mutation. In these patients FLT3-ITD represents the only established recurrent genetic aberration at diagnosis in addition to the t(6;9). We have shown that the driver mutation DEK/NUP214 transforms very immature hematopoietic stem cells with the contribution of activated STAT5, present also in FLT3-ITD-negative patients. Furthermore it has been reported that AKT/mTOR is activated in DEK/NUP214-positive cells.Here we investigated whether these signaling pathways are components of a leukemogenic NACSP and are therapeutically/clinically significant. We used different inhibitors to target either selectively or to determine a inhibition pattern of a.) PI3K/AKT/mTOR signaling (BKM120, BEZ2315, RAD001, Torin1 and AZD1208) at different levels; b.) receptor tyrosine kinases (RTK- ruxolitinib, sorafenib); and c.) members of the SRC kinase family (dasatinib, ponatinib, PF114, PP2). We employed four different models of t(6;9)-positive AML: U937 cells stably transfected with DEK/NUP214, t(6;9)-positive FKH1 cells, syngeneic DEK/NUP214-driven murine AML cells and primary t(6,9)-positive AML cells. Here we show that i.) STAT5 and AKT/mTOR activation was genetically determined by the t(6;9)-DEK/NUP214; ii.) STAT5 and AKT/mTOR activation were independent of JAK2 and PI3K activation, respectively; iii) selective inhibition of the AKT/mTOR cascades strongly increased STAT5 activation; iv.) both signaling pathways form a NACP, with activated members of the SRC kinase family (SKF - LYN and SRC) as a central node; v.) the NACSP was effectively targeted by inhibitors of the SRC-kinase activity (SKI) such as dasatinib, ponatinib, PF114 and the selective SKF inhibitor PP2, resulting in cell growth arrest and induction of apoptosis in t(6;9)-positive leukemic cells; vi.) SKI not only inhibited SKF/STAT5 but also the AKT/mTOR cascade; vii.) this NACP was independent of the activation of RTKs such as PDGFR, KDR, c-KIT, FLT3 a.o, as part of the target profile of many SKIs used above, as shown by the lack of activity of inhibitors such as ruxolitinib, sorafenib or ibrutinib; viii.) addition of AKT/mTOR inhibitors strongly increased effects of low dose dasatinib or ponatinib in primary t(6;9) leukaemic cells.Our findings implicate the t(6;9)-DEK/NUP214 oncogene as a central inductor of an NACSP including SFK, AKT/mTOR and STAT5 which is independent of the recurrent FLT3-ITD signaling in these patients. This NACSP is able to maintain the leukemia in the presence of effective inhibition of FLT3-ITD signaling by Sorafenib, AC220 and other inhibitors in clinical use. Furthermore, the increase of STAT5 activation upon AKT/mTOR inhibition, suggests that the use of such a treatment would not lead to the eradication of the disease, because of the role of activated STAT5 in the maintenance of leukemic stem cells. On the other hand, SKIs target the entire NACSP. In fact, hitting the central node of the NACSP abolished the activation of both STAT5 and AKT/mTOR.Taken together these data establish SKIs as a valid therapeutic concept not only in t(6;9)-positive AMLs but also in all other AML subtypes characterised by the same NACSP. DisclosuresOttmann:Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Fusion Pharma: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Ariad: Consultancy, Honoraria.

Editorial: "How to Improve Cord Blood Transplantation: By Enhancing Cell Counts or Engraftment?".

Editorial: "How to Improve Cord Blood Transplantation: By Enhancing Cell Counts or Engraftment?".

689. The Content of the More Immature Hematopoietic Stem Cells (HSCs) Is Dependent from the Underlying Genetic Diseases: Consequence on Transduction Efficiencies

Ex vivo gene transfer into hematopoietic CD34+ cells provides therapeutic benefits in primary immunodeficiencies. However, the CD34+ cell population is heterogeneous and is mainly composed of progenitors (CD34+lin-CD38+) and multilineage progenitors (CD34+lin-CD38-). This latter population can be divided in 3 subtypes regarding to the CD45RA and CD90 expression. CD45RA+ define cells with lymphoid potential, CD45RA-CD90- defined cells with myeloid potential and CD45RA-CD90+ define cells with long term repopulating capability (HSCs) which constitutes the relevant target for gene correction. We found that in bone marrow (BM) CD34+linneg, addition of CD133 helps to delimitate CD38neg population and we have analysed HSC, MLP and MPP population in BM from SCID-X1 (n=4), Wiskott Aldrich Syndrome (WAS) (n=2), Artemis (n=2), Chronic Granulomatous Disease (CGD) patients (n=3), sickle cell disease (SCD) (n=3) and age matched healthy controls (n=6).As expected, mean percentage of HSC represent 1% of CD34+ in healthy donors and similar results are obtained in CD34+ from SCID-X1 (1.76% ± 0.72), Artemis (1.98% ± 0.15), and SCD (2.16% ± 0.6) patients. On the contrary HSC percentage are lower in CD34+ from WAS (0.35% ± 0.15) and nearly undetectable in CGD patients. Moreover in multilineage progenitors, proportion of HSC, MLP and MPP varies from one pathology to another as shown in table1table1.Table 1Mean percentage of HSC, MLP and MPP in the linnegCD133+CD38neg CD34+ cellsHSCsMLPMPPHealthy Donor42%33%24%SCIDX137%45%28%WAS35%35%30%Artemis30%48%20%SCD36%19%44%Our aim was then to determine the ability of vectors used for gene therapy to transduce these different progenitors. We first analysed transduction of bulk CD34+ and HSCs by SIN retroviral vector for IL2RG in 2 SCIDX-1 patients enrolled in the clinical trial. After transduction, the 3 cell types were purified by flow cytometry and transduction determined in CFU after 14 days of culture in methyl cellulose in order to avoid detection of non-integrated vector. We show that in both patients, transduction efficiency was lower in HSCs than in bulk CD34+ cells. We were not able to recover enough CFU to determine VCN from MPP fraction and as expected, no CFU were obtained from MLP fraction. These results will be extended to transduction of CD34+ from WAS and SCD patients and efforts will be concentrated on transduction protocols in order to improve transduction of HSCs. Nevertheless low HSCs contents observed in CD34+ from CGD patients also represents a major difficulty to obtain long term reconstitution after gene therapy.

Notch signaling directs splenic resident hematopoietic stem cell fate decisions

Abstract Adult hematopoiesis occurs primarily in the bone marrow (BM), generating mature blood cells as well as maintaining a heterogeneous pool of self-renewing hematopoietic stem cells (HSCs). However, studies suggest that the BM may not be the only site of adult hematopoiesis: during times of BM hematopoietic distress stemming from radiation or disease, the spleen can independently reconstitute the blood. Given that the spleen does indeed contain a small, resident population of lineage-sca1+ckit+CD48-CD150+ long-term (LT)-HSCs with potent reconstitution ability, we investigated the peripheral blood cell contribution of these extra-medullary HSCs by transplanting UBC-GFP Tg reporter splenic fragments into a splenectomized syngenic wildtype mouse and tracking the blood cell production over time. While the spleen gives rise to both mature myeloid and lymphoid cells, we find that the proportion is skewed in favor of T cells. We see spleen-derived donor HSCs generating CD4+CD8+ double positive thymocytes for over five months post-transplant, suggesting that this peripheral T cell population derives from a long-lived stem or progenitor cell resident in the spleen. RNA sequencing demonstrates that splenic LT-HSCs are more responsive to Notch signaling than BM LT-HSCS, as indicated by increased levels of Hes1 transcription and decreased levels of the granulocyte/monocyte (GM) lineage priming genes Mpo, Gfi1, and Fcgr2b. Taken together with our finding that splenic HSCs have higher levels of cytoplasmic Notch2, we suggest that the Notch2-Hes1 axis is simultaneously repressing GM priming and enforcing T cell programming even among the most immature HSCs resident in the spleen.

How to Improve Cord Blood Engraftment?

Various factors make cord blood (CB) a significant source of hematopoietic stem cells (HSCs), including ease of procurement and lack of donor attrition, with the ability to process and store the donor cells long term. Importantly, high proliferative potential of the immature HSCs allows one log less use of cells compared to bone marrow or peripheral blood stem cells. As total nucleated cell (TNC) and CD34+ cell content of CB grafts are correlated to engraftment rate and speed, strategies to expand HSC and homing have been developed. This chapter will focus only on modalities such as intrabone administration, fucosylation, CD26 inhibition, prostaglandin E2 derivative or complement 3 exposure, and SDF-1/CXCR4/CXCL-12 pathway interventions that have been experimented successfully. Furthermore, increasing evidence in line with better recognition of CB progenitors that are involved in engraftment and homing will also be addressed.