scholarly journals Reconstruction of Human AML Reveals Stem Cell Origin and Therapeutic Targets for Treatment Resistant CD34-/Lo MLL-Rearranged Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3203-3203
Author(s):  
Tsz Kan Fung ◽  
Bernd Zeisig ◽  
Magdalena Zarowiecki ◽  
Huacheng Luo ◽  
Chiou Tsun Tsai ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Board Of Directors ◽  
Leukemic Cells ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Mouse Cells ◽  
Human Hscs ◽  
Human And Mouse

Identification of the origins of acute myeloid leukemia (AML) stem cells has been a holy grail for a better understanding of the developmental biology of the disease and the design of effective treatments. Studies using primary AML samples on patient-derived xenograft models identified AML stem cells in multiple different CD34/CD38 cellular fractions, which shared similar immunophenotypes of hematopoietic stem/progenitor cells including hematopoietic stem cells (HSCs), lymphoid-primed multipotent progenitors (LMPPs) and granulocyte-macrophage progenitors (GMPs). However inference of cells-of-origin based on the retrospective approach has major limitations as the reported HSC/LMPP/GMP-like AML stem cells are phenotypically different from their normal counterparts; and AML stem cells identified in late developmental stages do not necessarily maintain the same immunophenotypes of the disease initiating (pre-leukemic) cells, which can retain a relatively normal differentiation potential, and only their descendants acquire additional events becoming AML stem cells. While prospective disease modelling using mouse cells has provided unique insights into the potential origins of AML stem cells, human and mouse cells have different transformation requirements, distinct telomere biology, and a significant degree divergence of transcriptional regulation, chromatin state and gene regulatory networks that can profoundly affect their transformation potential and associated cancer biology. Therefore we reason that deconstruction of AML stem cell hierarchy from primary human samples followed by reconstruction of the corresponding human disease using candidate cell populations will give novel insights into this issue. Given that AML is a highly heterogeneous disease, our study initially focused on genetically well-defined MLL-rearranged AML frequently found in both infant and adult leukemia. By analyzing primary human AML patient samples, the current study unexpectedly revealed that AML stem cells driven by MLL fusions almost exclusively resided in immunophenotypically mature CD34-/loCD38+ compartments, as demonstrated by in vitro long-term culture initiating cells and in vivo limiting dilution xeno-transplantation assays into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Using highly purified populations of hematopoietic stem/progenitor cells from normal human umbilical cord blood (UCB) to reconstruct of the human disease, we found that only human HSCs and common myeloid progenitors (CMPs), but not developmentally branched LMPPs and GMPs, could be disease initiating cells, transformed by MLL-fusions and induced leukemia in vivo, suggesting a key difference between human and mouse leukemia. As revealed by RNA-sequencing, LMPPs and GMPs failed to activate stem cell transcriptional programs and genes essential for MLL-leukemia including MEIS1 and HMGA2 in the early phase of transformation. MLL-fusions transformed HSCs and CMPs were immunophenotypically indistinguishable to leukemic cells of human MLL-AML patients, and also had an enrichment of AML stem cells in CD34-/loCD38+ compartment. Using machine learning, a specific gene signature could stratify patients into HSCs- and CMPs-derived AML, in which HSCs-derived AML showed a significantly poorer prognosis. To further investigate the biology of HSCs/CMPs-derived MLL-AML related to treatment responses, we subjected HSC/CMP MLL-AML leukemia with chemotherapeutic drugs and inhibitor of Bromodomain and Extra-Terminal motif (BET) currently used in AML trial. Interestingly, CMP-derived MLL-AML was treatment sensitive and PDX models transplanted with CMP-like AML samples could be largely cured by chemotherapy or BET inhibitor targeted therapy. In contrast, human HSCs-derived AML was highly resistant to the treatments. Strikingly, shRNA-mediated knockdown or pharmacological inhibition by fidaxomicin targeting ATP-binding cassette (ABC) transporters, ABCC3 that is highly expressed in HSCs-derived MLL-AML could re-sensitize the cells to the current chemotherapy. Together, the current study not only for the first time functionally identifies the origin of human MLL-AML stem cells, but also provides a new actionable venue for overcoming stem cells-associated treatment resistance by repositioning an anti-diarrhea drug, fidaxomicin currently available in the clinics. Disclosures Mufti: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cellectis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

CML-CP Mouse Model of Genomic Instability.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1210-1210
Author(s):  
Elisabeth Bolton ◽  
Linda Kamp ◽  
Hardik Modi ◽  
Ravi Bhatia ◽  
Steffen Koschmieder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Stem Cell ◽  
Mouse Model ◽  
Board Of Directors ◽  
Genomic Instability ◽  
Oxidative Dna Damage ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Dependent Effect

Abstract Abstract 1210 Background: BCR-ABL1 transforms hematopoietic stem cells to induce chronic myeloid leukemia in chronic phase (CML-CP). Although CML is stem cell-derived, it is a progenitor cell-driven disease. In CML-CP, leukemia stem cells (LSCs) are characterized by elevated BCR-ABL1 expression in comparison to leukemia progenitor cells (LPCs). Increased expression of BCR-ABL1 kinase is also associated with progression from CML-CP to CML-blast phase. Previously we showed that BCR-ABL1 kinase stimulates reactive oxygen species (ROS)-dependent DNA damage resulting in genomic instability in vitro, which was responsible for acquired imatinib-resistance and accumulation of chromosomal aberrations (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). Result: To examine the effects of BCR-ABL1 expression on genomic instability during in vivo leukemogenesis we employed an inducible transgenic mouse model of CML-CP with targeted expression of p210BCR-ABL1 in hematopoietic stem and progenitor cells (Koschmieder et al., Blood, 2005). Mice exhibiting CML-CP-like disease resulting from BCR-ABL1 induction demonstrated splenomegaly, leukocytosis, and Gr1+/CD11b+ myeloid expansion in bone marrow, spleen and peripheral blood, as detected by FACS analysis. BCR-ABL1 mRNA expression was higher in Lin-c-Kit+Sca1+ stem-enriched cells than in Lin-c-Kit+Sca1- progenitor-enriched cells, thus reminiscent of CML-CP (LSCs>LPCs). BCR-ABL1 increased levels of ROS (hydrogen peroxide, hydroxyl radical) and oxidative DNA lesions (8-oxoG) in LSC-enriched Lin-c-Kit+Sca1+ cells. Preliminary data also suggested that quiescent (CFSEmax) Lin-c-Kit+Sca1+ cells from BCR-ABL1-induced mice exhibited greater ROS (superoxide) production than non-induced counter parts. Moreover, higher levels of ROS were detected in BCR-ABL1-positive Lin-c-Kit+Sca1+ stem-enriched population in comparison to BCR-ABL1-positive Lin-c-Kit+Sca1- progenitor population, suggesting a dosage-dependent effect of BCR-ABL1. To confirm that BCR-ABL1 exerts a dosage-dependent effect on ROS-induced oxidative DNA damage, we showed that the levels of ROS, 8-oxoG and DNA double-strand breaks were proportional to BCR-ABL1 kinase expression in murine 32Dc13 and human CD34+ cells. Conclusion: In summary, this mouse model recapitulates the BCR-ABL1 expression profile attributed to stem and progenitor populations in human CML-CP. It also shows that the BCR-ABL1-positive, stem cell-enriched Lin-c-Kit+Sca1+ population displays elevated levels of ROS and oxidative DNA damage in comparison to normal counterparts, which makes it suitable to study the mechanisms of genomic instability in LSCs. Single nucleotide polymorphism (SNP) arrays will shed more light on the genomic instability of this BCR-ABL1-induced transgenic model of CML-CP. Disclosures: Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.

Genome-Wide RNA Tomography of the Hematopoietic Stem Cell Niche in Zebrafish Reveals Unexpected Functional Macrophage-Stem Cell Interactions

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3882-3882
Author(s):  
Elliott J Hagedorn ◽  
Julie R Perlin ◽  
Clara Mao ◽  
Brian Li ◽  
Christopher D'Amato ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Cell Interactions ◽  
Cell Populations ◽  
Rna Seq ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract The challenges of visualizing the mammalian bone marrow have precluded a rigorous analysis of the dynamic cell-cell interactions that control hematopoietic stem and progenitor cell (HSPC) engraftment. The transparent zebrafish embryo provides an unparalleled opportunity to directly visualize HSPC-niche cell interactions in live animals. To identify genes expressed in the zebrafish caudal hematopoietic tissue (CHT) - an embryonic niche akin to the mammalian fetal liver - we employed a new technique called tomo-seq (RNA tomography). By pairing cryosectioning with RNA-seq, this technology permits spatial analysis of transcriptome-wide gene expression. Using tomo-seq we identified ~300 genes showing enriched expression in the CHT. In situ hybridization for 75 of 107 tested genes confirmed CHT expression. In parallel we performed RNA-seq on isolated cell populations, including endothelial cells, macrophages, neutrophils and erythrocytes, sorted from whole embryos. By cross-referencing these datasets we determined the cell types in which many of the 300 CHT-enriched genes were expressed. This analysis revealed several cell surface adhesion receptors enriched on macrophages in the CHT, including the integrin heterodimers itgam/itgb2, itgae/itgb7, itga4/itgb1b and itga4/itgb7. We examined whether known ligands for any of these integrins were present on HSPCs. In situ hybridization to vcam1 (ligand for itga4/itgb1b)showed punctate HSPC-like staining in the CHT. We then generated a vcam1:GFP promoter fusion, which we found was expressed in HSPCs. Using spinning disk confocal microscopy we imaged HSPCs and macrophages in the CHT and observed direct and specific physical interactions that preceded the engraftment of HSPCs. In a grooming-like behavior that lasts for 30-45 minutes, the HSPC is engaged by the macrophage, which moves all over the surface of the cell, before disengaging the HSPC, which then remains in the CHT. Between 48-72 hours post fertilization (hpf), 20% of HSPCs were engaged in this behavior with a macrophage. To evaluate the specificity of these interactions we established in vitro co-cultures using purified cell populations. In co-cultures between macrophages (mpeg1:mCherry) and HSPCs (cd41:GFP) we observed cell-cell interactions that were strikingly similar to those observed in vivo. In macrophage-HSPC co-cultures, 25% of cells were found to interact, whereas only 5% of cells were found to interact in macrophage-erythrocyte co-cultures. To functionally evaluate the macrophage-HSPC interactions in vivo, we depleted macrophages from zebrafish embryos at 55 hpf using clodronate liposomes and observed circulating HSPCs with a significant reduction in HSPC engraftment in the CHT (11/15 embryos, compared to the control where 14/14 embryos showed normal CHT engraftment). Together these studies establish a role for macrophages in promoting the niche engraftment of HSPCs. The results of this work could have important implications for the design of new therapies to improve engraftment during stem cell transplantation. Disclosures Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder.

scholarly journals High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 770-770
Author(s):  
Owen J. Tamplin ◽  
Ellen M. Durand ◽  
Logan A. Carr ◽  
Sarah J. Childs ◽  
Elliott H. Hagedorn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
High Resolution ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership

Abstract Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche. Disclosures Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Inhibition Of Microenvironmental Interleukin-1 Signaling Enhances TKI-Mediated Targeting Of Chronic Myelogenous Leukemia Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 512-512 ◽  
Author(s):  
Bin Zhang ◽  
Yin Wei Ho ◽  
Tessa L. Holyoake ◽  
Ravi Bhatia
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Chronic Myelogenous Leukemia ◽  
Interleukin 1 ◽  
Myelogenous Leukemia ◽  
Proliferation Index ◽  
Leukemia Stem Cells ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in chronic myelogenous leukemia (CML) patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. Most CML patients need continued TKI treatment to prevent disease relapse, and new strategies to eliminate residual leukemia stem cells are required to enhance possibility of achieving treatment-free remission. In previous studies we have shown that increased several cytokines expressed by leukemia cells may provide a selective growth advantage to CML compared with normal long term hematopoietic stem cells (LTHSC) within the CML BM microenvironment. Studies evaluating the effects of individual factors indicated that exposure to Interleukin-1α/β (IL-1α/β) at concentrations similar to those observed in CML BM resulted in significantly increased growth of CML compared with normal LTHSC (Cancer Cell 2012, 21:577). Consistent with previous reports (PNAS 2010, 107:16280), we observed that expression of the IL-1 receptor-associated protein (IL-1RAP), an important IL-1 signaling component, was increased in primitive CML cells, potentially explaining enhanced IL-1 sensitivity. To further evaluate the role of microenvironmental IL-1 in maintenance of CML LTHSC, we used recombinant IL-1 receptor antagonist (IL-1RA) to block IL-1 receptor signaling. IL-1RA is clinically approved for the treatment of rheumatoid arthritis. Purified LTHSC (Lin-Sca-1+Kit+Flt3-CD150+CD48- cells) from the SCL-tTA/BCR-ABL inducible mouse model of CML (CD45.1) and from congenic FVBN mice (CD45.2) were mixed in a 1:1 ratio and cultured with CML BM plasma, with and without IL-1RA. Culture with CML BM plasma for 7 days results in significantly increased growth of CML compared to normal LTHSC. The ratio of CML to normal cells was significantly reduced in the presence of IL-1RA (2.5μg/ml) (3.6:1 without IL-1RA, 1.7:1 with IL-1RA, p=0.0002), indicating that inhibition of IL-1 signaling reduced the growth advantage of CML LTHSC cultured in CML BM plasma. We next investigated the effect of IL-1RA on CML hematopoiesis in vivo. BM cells from CML mice (CD45.1) were transplanted into congenic FVBN mice (CD45.2) to generate CML-like disease in recipient mice. Four weeks after transplantation mice were treated with Nilotinib (NIL, 50mg/kg/d, gavage), IL-1RA (150mg/kg/d s.c.), the combination of NIL and IL-1RA, or vehicle (control) for 3 weeks. Treatment with NIL plus IL-1RA resulted in significantly greater reduction in CD45.1+ CML cells in blood, and in CML LTHSC, MPP, CMP and GMP in BM, compared with NIL alone (CML LTHSC/2 femurs: control 738±122, NIL 486±94, IL-1RA 525±49, combination 360±33, P=0.01 combination vs. Nilotinib). Mice treated with NIL plus IL-1RA also showed significantly prolonged survival after completion of treatment compared to mice treated with NIL alone (median survival 6 days for NIL alone versus 45 days for combination, p=0.02). Following transplantation of BM cells from treated mice into 2nd recipients (CD45.2), significantly lower CML cell engraftment in BM and reduced development of leukemia was seen after transplantation of cells from mice treated with the combination compared with NIL or untreated controls (8 out of 8 mice developed leukemia for control, 6 out of 8 for NIL, 5 out of 8 for IL-1RA, 3 out of 8 for the combination). We also studied the effect of treatment with NIL (5μm), IL-1RA (5μg/ml), NIL+IL-1RA, or vehicle for 72 hours on human CML and normal CD34+CD38+ and CD34+CD38- cells cultured with CML BM conditioned medium (CM). The combination of NIL and IL-1RA significantly reduced CML CD34+CD38+ and CD34+CD38- cell growth compared to Nilotinib alone (CD38- cells: NIL 23.7±10.1%, combination 13.1±8.9% of control, p<0.05), cell division (measured by CFSE labeling) (CD38- proliferation index: NIL 3.3±1.0, combination 2.4±0.6, p=0.06) and CFC frequency in methylcellulose progenitor assays (CD38- cells: NIL 67±22 per 1000 cells, combination 39±26, p<0.05); and moderately increased apoptosis of CML CD34+CD38- cells. We conclude that inhibition of microenvironmental IL-1 signaling using IL-1RA significantly increases inhibition of self-renewing murine and human CML stem cells in combination with NIL. Our results support further evaluation of IL-1 inhibition as a strategy to enhance elimination of CML LSC in TKI-treated patients. Disclosures: Holyoake: Novartis: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Ariad: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Myeloproliferative Neoplasm (MPN) Blastic Transformation Occurs at the Level of Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 101-101
Author(s):  
Xiaoli Wang ◽  
Cing Siang Hu ◽  
Joseph Tripodi ◽  
Vesna Najfeld ◽  
Bruce Petersen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Chromosomal Abnormalities ◽  
Myeloproliferative Neoplasm ◽  
Leukemic Cells ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Nsg Mice

Abstract Myeloproliferative neoplasm-blast phase (MPN-BP) and de novo acute myeloid leukemia (AML) each have distinct mutational patterns and clinical courses. MPN-BP patients have a particularly dismal prognosis with a median survival of less than 6 months with currently available therapies. So far, the cellular hierarchy that characterizes MPN-BP and the evolution of various leukemia-initiating clones (LIC) in MPN-BP have not been well delineated. We therefore established an in vivo MPN-BP xenograft model to address these questions. Among the 22 patients with MPN-BP studied 11 were cytogenetically normal while the remainder had multiple chromosomal abnormalities including del(5), del(20q), del(14), +1q, del(17p). 86% of the patients had at least 2 myeloid malignancy gene mutations including JAK2, ASXL1,TET2, MPL, SF3B1, RUNX1, U2AF1, PTPN11, IDH1/2, SRSF2 and TP53. These findings indicate that MPN-BP is characterized by multiple mutational events and cytogenetic abnormalities. T cell-depleted mononuclear cells from 8 of 14 patients engrafted in NSG mice {>0.5% hCD45+ cells in bone marrow (BM)}. Among them, samples from 6 patients resulted in a high degree of hCD45+ cell chimerism (34.6±6.4% in BM) and recapitulated numerous aspects of MPN-BP within 4 months, including the presence of at least 20% hCD45dimCD33+ cells or hCD34+ cells, or at least 20% blasts as detected by morphological examination of the marrow and leukemia cell dissemination to the spleen and PB. These mice had a 2.8±0.6- fold increase in splenic weight as compared to mice receiving PBS alone. The leukemic mice were characterized by reduced blood counts, suggesting that MPN-BP cells suppressed normal murine hematopoiesis, or led to cytopenias due to hyper-splenism. Moreover, the greater degrees of blast cell chimerism and the higher frequency of leukemia initiating cells as determined by limiting dilution analyses correlated with a shorter time to leukemia initiation and an inferior clinical outcome of the transplanted NSG mice. Grafts from each of these 6 MPN-BP patients produced a large number of donor-derived myeloid cells and a smaller number of lymphoid cells (mostly CD3+ and few CD19+). Cells belonging to each of these lineages and leukemic cells in primary recipients produced from Pts 4, 5, 6 and 11 had an identical proportion of chromosomally abnormal and mutated cells as primary cells [Pt 4: JAK2V617F, TET2 and PHF6; Pt 5 and 11: Del (20q), +8; Pt 6: +1q, del(17p)], except that a small proportion of T cells from Pts 5 and 11 lacked chromosomal abnormalities. Furthermore, the degree of MPN-BP engraftment and leukemic cell burden increased with the subsequent 3 serial transplantations even when the recipients received progressively smaller numbers of MPN-BP cells from the prior recipient. Primary Pt 6 originally had a JAK2V617F+ PV but lost JAK2V617F at the time the MPN-BP occurred at which time there were two clonal cell populations, one with +1q (12%) and the other del(17p) (80%), the site of the TP53 gene, as well as normal cells (8%). In the primary recipient NSG the donor derived cells were JAK2V617F- but contained +1q (1%) and del(17p) (98.5%) and cytogenetically normal (0.5%). +1q and JAK2V617F were not observed, while cells containing the TP53 deletion alone were detected in donor derived leukemic cells, mature myeloid and T cells in the secondary and subsequent serial recipients. Furthermore, del(17p) was found in phenotypically isolated HSCs, MPPs, MLPs, CMPs, GMPs, MEPs, and mature T cells within the CD33- cell fraction as well as CD45dimCD33+ AML blasts selected from primary MPN-BP cells from Pt6. However, +1q was found exclusively in purified MLPs and MEP. These observations establish that cytogenetic and mutational events that lead to MPN-BP occur at different stages along the developmental HSC hierarchy and that a small population of normal HSCs persist. Furthermore, in JAK2V617F+ MPNs that develop MPN-BP and lose JAK2V617F, additional cytogenetic events occur at different stages along the JAK2V617F- MPN-BP-stem cell hierarchy. Our ability to serially transplant the LIC from these patients has allowed us to create the first MPN-BP PDX model that will not only extend our understanding of MPN-BP stem cell biology but might also prove useful for screening drugs to treat MPN-BP. Disclosures Rampal: Jazz: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; Stemline: Research Funding; Constellation: Research Funding; Celgene: Honoraria. Mascarenhas:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche: Research Funding; Novartis: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Promedior: Research Funding; Janssen: Research Funding. Hoffman:Formation Biologics: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Incyte: Research Funding; Janssen: Research Funding.

Hematopoietic Stem Cell Niche Interactions In The Embryo Can Modulate The Size Of The Stem Cell Pool Into Adulthood

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 585-585
Author(s):  
Owen J. Tamplin ◽  
Ellen M. Durand ◽  
Logan A. Carr ◽  
Pulin Li ◽  
Leonard I. Zon
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
Hsc Niche ◽  
Equity Ownership ◽  
Stem Cell Pool

Abstract Hematopoietic stem cells (HSC) reside in the bone marrow niche and sustain the production of blood throughout life. The entire pool of these rare and important cells is generated during a brief window of embryonic development. HSC are produced by the hemogenic endothelium of the dorsal aorta, migrate to and expand in the fetal liver, and then migrate again to seed the bone marrow. The zebrafish is a highly conserved and well-established model for HSC development. Similar to mammals, HSC emerge from the dorsal aorta, but then colonize a vascular plexus in the tail of the embryo—the caudal hematopoietic tissue (CHT). It is difficult to directly observe the interactions between an endogenous HSC and its niche, so we have developed the CHT as a model for HSC-niche interactions. To track HSC in vivo we have generated a transgenic reporter using the previously described mouse Runx1 +23 kb intronic enhancer. The purity of the stem cell pool marked by this reporter was determined. Using adult-to-adult limiting dilution transplantation with as few as one Runx1+23 positive cell, we have estimated the HSC purity to be approximately 1/35 (without immune matching), or similar to Kit+Sca1+Lin- (KSL) in mouse. This is the most pure stem cell population defined in the zebrafish system. Using embryo-to-embryo transplantation, a technique that is unique to zebrafish, we sorted Runx1+23 positive cells from one group of embryos and transplanted them to another by injection directly into circulation. Embryos are then grown to adulthood and marrow is tested for long-term engraftment between 3 and 5 months. This transplantation technique precedes formation of the thymus, thereby removing any chance of immune rejection. Highly stringent dilution of HSC in our embryo-to-embryo transplants has estimated a stem cell purity of one in two cells. Next, we applied our highly specific reporter to visualize HSC migration to the CHT niche. After arrival of the HSC, we have described 5 distinct steps during colonization: 1) adherence; 2) extravasation; 3) abluminal migration; 4) endothelial niche formation (“cuddling”); and 5) cell fate decisions. Live imaging analysis of HSC together with endothelial and stromal transgenic reporters has allowed us to quantify the relationship between different cell types within the CHT. For example, we observe preferential localization of HSC in close proximity to cxcl12a positive stromal cells. Lastly, we have sought to identify the molecular mechanisms involved in interactions between HSC and their niche. A chemical genetic screen identified the natural product lycorine as a small molecule that increases hematopoiesis in the CHT and promotes HSC-endothelial cell interactions. Combined chemical treatment and live imaging revealed that lycorine significantly increased the residence time of HSC in the niche. To test if treatment during the window of CHT colonization (2-3 days post fertilization) had long-term effects on HSC and the stem cell pool, the compound was washed off at 3 days and the Runx1+23 positive population was quantified by FACS. At 7 days post fertilization, after colonization of the marrow, there was a sustained and significant increase in Runx1+23 positive HSC. Strikingly, after 3 months, when treated embryos were raised to adulthood, we discovered that the increased HSC-endothelial cell interactions we observed in the CHT niche had in fact had an impact on the number of HSC in the adult. Our studies establish that the Runx1+23 transgenic is a highly specific reporter of HSC both in the embryo and adult, and that we can use this reporter for in vivo observation of an endogenous HSC niche. Furthermore, we show that the size of the adult stem cell pool can be altered by a transient signal during development. Disclosures: Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

IKZF1 Mutation Mediate Resistance to IMiDs in Human Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3003-3003
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jing Wu ◽  
Markus Y Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Board Of Directors ◽  
Cell Expansion ◽  
Lineage Commitment ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Introduction: Previously we have shown that the immune modulatory drugs (IMiDs) downregulate GATA1 and PU.1 resulting in maturational arrest of granulocytes with accumulation of immature myeloid precursors and subsequent neutropenia. Our studies further revealed that similar to MM cells cereblon (CRBN) is critical for the mediation of the effects of IMiDS in hematopoietic stem cells (HSCs) and associated with decrease of IKZF1-dependent transcription factors such as GATA1 and PU.1, which are critical for development and maturation of neutrophils and erythrocytes as well as thrombocytes. Here we investigated the mechanism how IMIDs induce degradation of IKZF1 and confirmed our studies in vivo by using the humanized NOD/SCID/Gamma-c KO (NSG) mouse model. Methods and Results After we had shown that knockdown of CRBN in HCS mediates resistance to IMIDs (2014 ASH abstract 418) we assessed the impact of IKZF1 inhibition using two different approaches. First, we knocked down IKZF1 expression in CD34+ cells by shRNA lentivirus transduction. As expected, IKZF1 knockdown in CD34+ cells mimicked the effects of IMiDs resulting in increased CD34+ cell proliferation, CD33+ cell expansion (flow cytometry) and shift of lineage commitment from BFU-E to CFU-G (colony assay). Knockdown of IKZF1 was associated with decreased GATA1 and PU.1 expression at both mRNA and protein levels. Next, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. CD34+ cells were transduced with lentiviral constructs to overexpress IKZF1-WT or IKZF1-Q146H. POM failed to induce IKZF1 degradation in IKZF1-Q146H-OE CD34+ cells, indicating CRBN binding to IKZF1 and subsequent ubiquitination is critical in this process. Functional assays further confirmed that IKZF1-Q146H CD34+ cells were resistant to POM induced CD33+ cell expansion and shift in lineage commitment from BFU-E to CFU-G. Since conventional mouse models are not applicable to test IMIDs in vivo due to the fact that IMIDs do not bind to mouse CRBN (Kronke, Fink et al. 2015), we established a humanized mouse model resembling human hematopoiesis. In this model, NOD/SCID/Gamma-c KO (NSG) mice received human fetal thymus grafts and 105 CD34+ fetal liver cells to generate human hematopoiesis including functional T-cells. After establishing human hematopoiesis mice were injected with POM (0.3 mg/kg) i.v every 2 days for 3 weeks. Analysis of bone marrow revealed that POM treatment significantly induced granulocyte/macrophage progenitor cells (CD34+ CD38+ CD45RA+ cells) at the expense of common lymphoid progenitors (CD34+ CD10+ cells). The shift into myelopoiesis is consistent with our in vitro finding that IMiDs affect lineage commitment. Conclusion: In summary, our results demonstrate that IMiDs affect CD34+ cell fate via CRBN and IKZF1 mediated mechanism. These results will be helpful to elucidate the mechanism of IMiDs on lineage commitment and maturation in HSCs. Also establishment of the humanized xenograft mice model may provide an advanced platform for the analysis of human hematopoiesis and human immune responses to IMiDs as well development of secondary hematologic malignancies in vivo. Disclosures Lentzsch: Axiom: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

scholarly journals GPR84 sustains aberrant β-catenin signaling in leukemic stem cells for maintenance of MLL leukemogenesis

Blood ◽  
2014 ◽  
Vol 124 (22) ◽  
pp. 3284-3294 ◽  
Author(s):  
Philipp A. Dietrich ◽  
Chen Yang ◽  
Halina H. L. Leung ◽  
Jennifer R. Lynch ◽  
Estrella Gonzales ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Strong Dependence ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Transcription Program

Key Points GPR84 simultaneously augments β-catenin signaling and an oncogenic transcription program essential for establishment of MLL. Our study demonstrates a strong dependence of hematopoietic stem cell–derived MLL leukemic cells on GPR84 for disease maintenance in vivo.

scholarly journals A Network Of Epigenetic Regulators Guide Developmental Hematopoiesis In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1174-1174
Author(s):  
Katie L Kathrein ◽  
Hsuan-Ting Huang ◽  
Abby Barton ◽  
Zachary Gitlin ◽  
Yue-Hua Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Large Scale ◽  
Chromatin Domain ◽  
Advisory Committees ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Marker Expression

Abstract Long-term hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation into all mature hematopoietic lineages. This process is regulated by transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, we have undertaken the first large-scale in vivo reverse genetic screen targeting chromatin factors. We have designed and injected antisense morpholinos to knockdown expression of 488 zebrafish orthologs of conserved human chromatin factors. The resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC marker genes, c-myb and runx1, which are expressed in the developing blood stem cells. Morphants were categorized into five groups based on HSC marker expression, ranging from no change to mild, intermediate, or strong reduction in expression or an increase in expression. 29 morpholinos caused a complete or near complete knockdown of HSC marker expression, while 4 were found to increase HSC marker expression. As ubiquitous knockdown of chromatin factors could interfere with vascular development and the establishment of proper arterial identity, a crucial upstream event for HSC formation, we subsequently analyzed morphants with the most robust HSC phenotypes using two vascular markers: kdr for overall vasculogenesis and ephrinb2a for arterial formation. We found that of the 29 morpholinos that caused reduced marker expression, only 9 showed reduced overall vascular or arterial marker staining, suggesting that the majority of morphants with HSC phenotypes are specific to HSC formation. For the 4 morphants with increased HSC marker expression, vasculature appeared normal. These factors likely function as potent negative regulators of HSC development. Several genes known to be essential for HSC self-renewal and maintenance were identified in the screen. For example, knockdown of Mll or Dot1, which are also present in leukemia fusion proteins, fail to specify HSCs, as indicated by a nearly complete reduction in expression of the HSC markers in embryos tested. Of the remaining hits, many represent factors with no previous function ascribed in hematopoiesis. By incorporating protein interaction data, we have defined a handful of complexes necessary for HSC specification, including the SWI/SNF, ISWI, SET1/MLL, CBP/P300/HBO1/NuA4, HDAC/NuRD, and Polycomb complexes. As chromatin factors associated with the same complex likely share target binding sites, we analyzed 34 published ChIP-seq datasets in K562 erythroleukemia cells of chromatin factors tested in the screen, including hits from our screen: SIN3A, CHD4, HDAC1, TAF1, and JARID1C associated with the HDAC/NuRD complex and RNF2, SUZ12, CBX2, and CBX8 from the Polycomb complexes. We ranked triplet combinations of these factors together with all other groups of three factors based on the percent overlap of target genes. The HDAC/NuRD and PRC1/2 complex combinations predicted from our screen fell within the top 20% of all possible combinations of 3 factors, suggesting that our screen has identified chromatin factors that function in distinct complexes to regulate hematopoietic development. Our work has been compiled into a web-based database that will be made publicly available upon publication. Within this database, users can search by gene names and aliases, chromatin domain names and human or zebrafish genes. All experimental data, including experimental design, materials, protocols, images, and all further analyses of the 33 most robust morphants is included. Our large-scale genetic analysis of chromatin factors involved in HSC development provides a comprehensive view of the programs involved in epigenetic regulation of the blood program, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders and leukemia. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Valine as a Key Metabolic Regulator of Hematopoietic Stem Cell Maintenance

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-20-SCI-20
Author(s):  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Amino Acid ◽  
Board Of Directors ◽  
Bone Marrow Niche ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Conditioning Approach

Abstract Hematopoietic stem cells (HSCs) are maintained by a specialized bone marrow microenvironment (niche) and are largely quiescent during the steady-state conditions1. However, upon stimulation or transplantation, HSCs can expand and differentiate into any mature hematopoietic cell type. This ability of donor HSCs to reform a recipient's hematopoietic system is key to the success of HSC transplantation (HSCT). For donor HSCs to engraft, the recipient bone marrow niche must first be emptied via myeloablative irradiation or chemotherapy. However, myeloablative conditioning can cause severe complications and even mortality. As an alternative, we have recently developed a metabolic conditioning approach for HSCT. By screening the amino acid requirements of HSCs, we identified the essential amino acid valine as indispensable for the expansion and maintenance of HSCs2. Both mouse and human HSCs failed to expand when cultured in valine-restricted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within one week. Dietary valine restriction thereby emptied the mouse bone marrow niche and just a two-week diet afforded long-term donor-HSC engraftment without chemoirradiative myeloablation. We conclude that valine plays a critical role in HSC maintenance and suggest dietary valine restriction as a conditioning regimen that may reduce iatrogenic complications in HSCT. These findings, and recent efforts to optimize this metabolic conditioning approach through mechanistic understanding of the HSC valine dependency, will be presented. Sudo K, Ema H, Morita Y, Nakauchi H. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med. 2000;192:1273-1280.Taya Y, Ota Y, Wilkinson AC, et al. Depleting dietary valine permits nonmyeloablative mouse hematopoietic stem cell transplantation. Science. 354:1152-1155. Disclosures Nakauchi: ReproCELL Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Megakaryon Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; iBUKI Corp: Equity Ownership; iCELL Inc: Equity Ownership; Advanced Immunothearpy Inc: Equity Ownership.

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