Valine as a Key Metabolic Regulator of Hematopoietic Stem Cell Maintenance

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.

Download Full-text

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

Blood ◽

10.1182/blood.v124.21.770.770 ◽

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.

Download Full-text

Clinical Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Transfusion-Dependent β-Thalassemia Treated at the Bambino Gesù Children's Hospital, Rome, Italy

Blood ◽

10.1182/blood-2019-123440 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 969-969

Author(s):

Pietro Merli ◽

Annalisa Ruggeri ◽

Mattia Algeri ◽

Giuseppina Li Pira ◽

Giulia Ceglie ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Board Of Directors ◽

Graft Failure ◽

Unrelated Donor ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Sibling Donor ◽

Equity Ownership

β-thalassemia is one of the most common monogenic blood disorders worldwide, and is highly prevalent in Mediterranean countries. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been the only curative treatment for transfusion-dependent β-thalassemia (TDT; the most severe disease form) for many years, but it is limited by donor availability and has a significant risk of morbidity and mortality. We conducted a chart review of patients with β-thalassemia who underwent allo-HSCT (N=80) at the Bambino Gesù Children's Hospital, Rome, Italy, between March 2011 and August 2018. Median (range) age at allo-HSCT was 5.5 (0.3-20.0) years [<12 years: n=70 (87.5%), ≥12-18 years: n=7 (8.8%), ≥18 years: n=3 (3.8%)] and all patients but one had TDT [n=79 (98.8%)]. Prior to allo-HSCT, patients received a median (range) of 17.4 (4-52) transfusions per year (n=64) and had a median (range) serum ferritin concentration of 1217 (135-9123) ng/mL and a median (interquartile range) hemoglobin level of 10.3 (9.3-11.4) g/dL. All patients had received regular iron chelation therapy prior to transplantation. In total, 18 (22.5%), 28 (35.0%), and 34 (42.5%) patients received allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors, HLA-haploidentical donors, and unrelated donors (fully matched donor: n=28, donor with a single HLA disparity: n=6), respectively. Of these donors, 42 (52.5%) were carriers for thalassemia-associated mutations. In total, 53 (66.3%) donors and 35 (43.8%) recipients were cytomegalovirus-positive. Bone marrow was the stem cell source in 51 cases (63.8%), while 28 patients received an alphabeta T-cell depleted peripheral blood haploidentical HSCT (35.0%); the remaining child (1.3%) received both bone marrow and cord blood from the same related donor. All patients continued to receive transfusions immediately after allo-HSCT; however, only 7 (8.8%) received a transfusion in the 3 to 12-month post-transplantation period (2 due to underlying disease; 5 due to other reasons including GI bleeding). Median (range) time to reach transfusion-free status was 3.8 (1.1-47.8) weeks. Median (interquartile range) hemoglobin levels at 6 and 12 months after allo-HSCT were 10.9 (10.2-11.9) and 11.9 (10.6-13.0) g/dL, respectively. The cumulative incidences of primary and secondary graft failure were 10.0% and 12.5% at 24 months (HLA-identical donor: 0% and 11.1%, haploidentical donor: 17.9% and 3.6%, unrelated donor: 8.8% and 20.6%). Eleven out of 14 patients experiencing graft failure were successfully rescued with a second allograft, while 2 patients were not retransplanted due to parental decision and 1 patient died after the engraftment of the second allograft. Eight patients developed grade II-IV acute graft-versus-host disease (GVHD) and one patient developed moderate chronic GVHD. Cumulative incidence rates of grades II-IV and III-IV acute GVHD were 12.7% and 8.0% at 24 months (HLA-identical donor: 0% and 0%, haploidentical donor: 7.3% and 0%, unrelated donor: 23.8% and 18.8%). Three patients (3.8%) died of transplant-related causes (1 case each of hemophagocytic lymphohistiocytosis, sepsis, and multi-organ failure [the patient receiving the second allograft]) with a median (range) time from transplantation to death of 8.7 (3.7-11.0) months. Of these patients, all had been transplanted from an unrelated donor and 2 had reached sustained full-donor chimerism. The probability of overall and event-free (event defined as either death or primary/secondary graft failure) survival was 96.2% and 81.2% at 24 months (HLA-identical sibling donor: 100% and 88.9%, haploidentical donor: 100% and 78.6%, unrelated donor: 91.2% and 79.4%). The probability of thalassemia-free survival (event defined as either death or primary/secondary graft failure not rescued by a second allograft) was 93.7% at 24 months (HLA-identical sibling donor: 100%, haploidentical donor: 92.9%, unrelated donor: 91.2%). In this large single-center cohort of children with predominantly TDT, allo-HSCT led to beneficial outcomes for most patients, resulting in the discontinuation of transfusions with 93.7% of patients being thalassemia-free. Nevertheless, HSCT is still associated with GVHD, graft failure, and mortality, and only 22.5% of patients had an HLA-identical sibling donor, illustrating a key limitation of allo-HSCT. Emerging research is addressing such barriers to treatment. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Algeri:Miltenyi: Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Bluebird bio: Consultancy, Honoraria. Gruppioni:Bluebird bio: Employment, Equity Ownership. Kommera:Bluebird bio: Employment, Equity Ownership. Maa:Bluebird bio: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Download Full-text

Msi2 Maintains Normal Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood.v118.21.222.222 ◽

2011 ◽

Vol 118 (21) ◽

pp. 222-222 ◽

Cited By ~ 1

Author(s):

Michael G Kharas ◽

Christopher Lengner ◽

Fatima Al-Shahrour ◽

Benjamin L. Ebert ◽

George Q. Daley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Board Of Directors ◽

Bone Marrow Cells ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Marrow Cells

Abstract Abstract 222 Genes that regulate normal hematopoietic stem cells are commonly dysregulated in hematopoietic malignancies. Recently we published that the Msi2 RNA binding protein is an important modulator in both normal hematopoietic stem cells and leukemia (Kharas et al, Nat. Medicine 2010). The closely related Msi1 protein has been shown to regulate mRNA translation through binding to the 3'UTR. Based on the high homology in the RNA recognition motifs, Msi2 has been considered to have similar functions. Moreover, increased MSI2 expression in chronic myelogenous leukemia blast crisis and acute myeloid leukemia predicts a worse clinical prognosis. Previous studies have mainly utilized shRNAs to functionally assess the role of Msi2 in the hematopoietic compartment. However, it remains unclear how Msi2 affects hematopoietic stem cells (HSC) and what are its critical mRNA targets. To develop a model focusing on the HSC compartment and to avoid potential compensatory mechanisms during development, we created Msi2 conditional knockout mice and crossed them with Mx1-Cre mice. We induced excision with poly(I):poly(C), (pIpC), and tested the peripheral blood, bone marrow cells and splenocytes by Southern blotting and QPCR analysis to verify Msi2 deletion. Loss of Msi2 mRNA was confirmed in the Lineagelo, Sca1+ and c-Kit+ (LSK) population. Msi2 deleted bone marrow contained reduced myeloid colony forming capacity and replating efficiency. Mice conditionally deleted for Msi2 had normal white blood cell counts but smaller spleens. In addition, we observed normal percentages of the mature hematopoietic populations, including the myeloid and lymphoid compartments. Nevertheless, absolute numbers of long-term HSCs in the bone marrow were reduced by 3-fold. Bone marrow cells non-competitively transplanted into primary and secondary recipient mice showed a dramatic reduction in HSC chimerism. This defect was also observed when bone marrow was transplanted first to allow engraftment followed by Msi2 deletion. Furthermore, we were able to recapitulate this defect in vitro using the cobblestone-forming activity assay. These results indicate that Msi2 is both an important regulator of normal HSC maintenance and required for efficient engraftment. Most interestingly, Msi2 HSCs failed to maintain a normal quiescent HSC population. We performed microarrays to identify the pathways altered in the LSK population. The Msi2 deficient LSKs showed a reduced self-renewal and increased differentiation gene signature. Gene expression analysis indicates a defective self-renewal program in Msi2-deficient HSCs that is identical to the program gained in leukemic stem cells. These data suggest that MSI2 is a critical modulator of HSCs and may help explain its requirement in the most aggressive myeloid leukemias. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation

Blood ◽

10.1182/blood-2019-121763 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3199-3199

Author(s):

Ji Zha ◽

Lori Kunselman ◽

Hongbo Michael Xie ◽

Brian Ennis ◽

Jian-Meng Fan ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mouse Model ◽

Board Of Directors ◽

Hematopoietic Stem Cell ◽

Graft Failure ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Bm Niche ◽

Deficient Mice

Hematopoietic stem cell (HSC) transplantation (HSCT) is required for curative therapy for patients with high-risk hematologic malignancies, and a number of non-malignant disorders including inherited bone marrow failure syndromes (iBMFS). Strategies to enhance bone marrow (BM) niche capacity to engraft donor HSC have the potential to improve HSCT outcome by decreasing graft failure rates and enabling reduction in conditioning intensity and regimen-associated complications. Several studies in animal models of iBMFS have demonstrated that BM niche dysfunction contributes to both the pathogenesis of iBMFS, as well as impaired graft function after HSCT. We hypothesize that such iBMFS mouse models are useful tools for discovering targetable niche elements critical for donor engraftment after HSCT. Here, we report the development of a novel mouse model of Shwachman-Diamond Syndrome (SDS) driven by conditional Sbds deletion, which demonstrates profound impairment of healthy donor hematopoietic engraftment after HSCT due to pathway-specific dysfunctional signaling within SBDS-deficient recipient niches. We first attempted to delete Sbds specifically in mature osteoblasts by crossing Sbdsfl/flmice with Col1a1Cre+mice. However, the Col1a1CreSbdsExc progenies are embryonic lethal at E12-E15 stage due to developmental musculoskeletal abnormalities. Alternatively, we generated an inducible SDS mouse model by crossing Sbdsfl/flmice with Mx1Cre+ mice, and inducing Sbds deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by polyinosinic-polycytidilic acid (pIpC) administration. Compared with Sbdsfl/flcontrols, Mx1CreSbdsExc mice develop significantly decreased platelet counts, an inverted peripheral blood myeloid/lymphoid cell ratio, and reduced long-term HSC within BM, consistent with stress hematopoiesis seen in BMF and myelodysplastic syndromes. To assess whether inducible SBDS deficiency impacts niche function to engraft donor HSC, we transplanted GFP+ wildtype donor BM into pIpC-treated Mx1CreSbdsExc mice and Sbdsfl/flcontrols after 1100 cGy of total body irradiation (TBI). Following transplantation, Mx1CreSbdsExc recipient mice exhibit significantly higher mortality than controls (Figure 1). The decreased survival was related to primary graft failure, as Mx1CreSbdsExc mice exhibit persistent BM aplasia after HSCT and decreased GFP+ reconstitution in competitive secondary transplantation assays. We next sought to identify the molecular and cellular defects within BM niche cells that contribute to the engraftment deficits in SBDS-deficient mice. We performed RNA-seq analysis on the BM stromal cells from irradiated Mx1CreSbdsExc mice versus controls, and the results revealed that SBDS deficiency in BM niche cells caused disrupted gene expression within osteoclast differentiation, FcγR-mediated phagocytosis, and VEGF signaling pathways. Multiplex ELISA assays showed that the BM niche of irradiated Mx1CreSbdsExc mice expresses lower levels of CXCL12, P-selectin and IGF-1, along with higher levels of G-CSF, CCL3, osteopontin and CCL9 than controls. Together, these results suggest that poor donor HSC engraftment in SBDS-deficient mice is likely caused by alterations in niche-mediated donor HSC homing/retention, bone metabolism, host monocyte survival, signaling within IGF-1 and VEGF pathways, and an increased inflammatory state within BM niches. Moreover, flow cytometry analysis showed that compared to controls, the BM niche of irradiated Mx1CreSbdsExc mice contained far fewer megakaryocytes, a hematopoietic cell component of BM niches that we previously demonstrated to be critical in promoting osteoblastic niche expansion and donor HSC engraftment. Taken together, our data demonstrated that SBDS deficiency in BM niches results in reduced capacity to engraft donor HSC. We have identified multiple molecular and cellular defects in the SBDS-deficient niche contributing to this phenotype. Such niche signaling pathway-specific deficits implicate these pathways as critical for donor engraftment during HSCT, and suggest their potential role as targets of therapeutic approaches to enhance donor engraftment and improve HSCT outcome in any condition for which HSCT is required for cure. Disclosures Olson: Merck: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Download Full-text

Increased Early Mortality after Fludarabine and Melphalan Conditioning with Peripheral Blood Grafts in Haploidentical SCT with Post-Transplant Cyclophosphamide

Blood ◽

10.1182/blood-2019-129872 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4496-4496 ◽

Cited By ~ 1

Author(s):

Luke Eastburg ◽

David A. Russler-Germain ◽

Ramzi Abboud ◽

Peter Westervelt ◽

John F. DiPersio ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Early Mortality ◽

Cell Transplant ◽

Advisory Committees ◽

Post Transplant ◽

Equity Ownership

The use of post-transplant cyclophosphamide (PTCy) in the context of haploidentical stem cell transplant (haplo-SCT) has led to drastically reduced rates of Graft-vs-Host (GvH) disease through selective depletion of highly allo-reactive donor T-cells. Early trials utilized a reduced-intensity Flu/Cy/TBI preparative regimen and bone marrow grafts; however, relapse rates remained relatively high (Luznik et al. BBMT. 2008). This led to the increased use of myeloablative (MA) regimens for haplo-SCT, which have been associated with decreased relapse rates (Bashey et al. J Clin Oncol. 2013). Most studies have used a MA total body irradiation (TBI) based regimen for haplo-SCT. Preparative regimens using fludarabine and melphalan (FluMel), with or without thiotepa, ATG, and/or low dose TBI have also been reported using bone marrow grafts. Reports on the safety and toxicity of FluMel in the haplo-SCT setting with PTCy and peripheral blood stem cell (PBSC) grafts are lacking. In this two-center retrospective analysis, the safety/toxicity of FluMel as conditioning for haplo-SCT was evaluated. We report increased early mortality and toxicity using standard FluMel conditioning and PBSC grafts for patients undergoing haplo-SCT with PTCy. 38 patients at the University of Rochester Medical Center and the Washington University School of Medicine underwent haplo-SCT with FluMel conditioning and PBSC grafts between 2015-2019. Outcomes were measured by retrospective chart review through July 2019. 34 patients (89.5%) received FluMel(140 mg/m2). Two patients received FluMel(100 mg/m2) and two patients received FluMel(140 mg/m2) + ATG. The median age at time of haplo-SCT was 60 years (range 21-73). 20 patients were transplanted for AML, eight for MDS, two for PMF, two for NHL, and five for other malignancies. The median Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) score was 4 (≥3 indicates high risk). 11 patients had a history of prior stem cell transplant, and 16 patients had active disease prior to their haplo-SCT. Seven patients had sex mismatch with their stem cell donor. Median donor age was 42 (range 21-71). 20 patient deaths occurred by July 2019 with a median follow up of 244 days for surviving patients. Nine patients died before day +100 (D100, "early mortality"), with a D100 non-relapse mortality (NRM) rate of 24%. Median overall and relapse free survival (OS and RFS, respectively) were 197 days (95% CI 142-not reached) and 180 days (95% CI 141-not reached), respectively, for the entire cohort. The 1 year OS and NRM were 29% and 50%. The incidence of grades 2-4cytokine release syndrome (CRS) was 66%, and 52% of these patients were treated with tocilizumab. CRS was strongly associated with early mortality, with D100 NRM of 36% in patients with grade 2-4 CRS compared to 0% in those with grade 0-1. The incidence of acute kidney injury (AKI) was 64% in patients with grade 2-4 CRS, and 8% in those without (p < 0.001). 28% of patients with AKI required dialysis. Grade 2-4 CRS was seen in 54% of patients in remission prior to haplo-SCT and in 92% of those with active disease (p = 0.02). Of the 9 patients with early mortality, 89% had AKI, 44% needed dialysis, and 100% had grade 2-4 CRS, compared to 31%, 10%, and 55% in those without early mortality (p = 0.002, p = 0.02, p = 0.01). Early mortality was not significantly associated with age, HCT-CI score, second transplant, disease status at transplant, total dose of melphalan, volume overload/diuretic use, or post-transplant infection. In conclusion, we observed a very high rate of NRM with FluMel conditioning and PBSC grafts for haplo-SCT with PTCy. The pattern of toxicity was strongly associated with grade 2-4 CRS, AKI, and need for dialysis. These complications may be mediated by excessive inflammation in the context of allo-reactive donor T-cell over-activation. Consistent with this, multiple groups have shown that FluMel conditioning in haplo-SCT is safe when using bone marrow or T-cell depleted grafts. Based on our institutional experiences, we would discourage the use of FluMel as conditioning for haplo-SCT with PTCy with T-cell replete PBSC grafts. Alternative regimens or variations on melphalan-based regimens, such as fractionated melphalan dosing or inclusion of TBI may improve outcomes but further study and randomized controlled trials are needed. This study is limited in its retrospective design and sample size. Figure Disclosures DiPersio: WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Karyopharm Therapeutics: Consultancy; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Amphivena Therapeutics: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liesveld:Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.3229.3229 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3229-3229 ◽

Cited By ~ 1

Author(s):

Ivana N Micallef ◽

Eric Jacobsen ◽

Paul Shaughnessy ◽

Sachin Marulkar ◽

Purvi Mody ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cd34 Cells ◽

Low Platelet Count ◽

Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

CML-CP Mouse Model of Genomic Instability.

Blood ◽

10.1182/blood.v116.21.1210.1210 ◽

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.

Download Full-text

Epoxyeicosatrienoic Acids Regulate Hematopoietic Stem/Progenitor Cell Fate Decision During Stress Response and Embryonic Hematopoiesis

Blood ◽

10.1182/blood.v118.21.860.860 ◽

2011 ◽

Vol 118 (21) ◽

pp. 860-860

Author(s):

Pulin Li ◽

Emily K Pugach ◽

Elizabeth B Riley ◽

Dipak Panigrahy ◽

Garrett C Heffner ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Marrow Transplantation ◽

Mouse Bone Marrow ◽

Zebrafish Embryos ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Runx1 Expression

Abstract Abstract 860 During bone marrow transplantation, hematopoietic stem/progenitor cells (HSPCs) are exposed to various stress signals, and undergo homing, rapid proliferation and differentiation in order to achieve engraftment. To explore how fate decisions are made under such stress conditions, we developed a novel imaging-based competitive marrow transplantation in zebrafish. The feasibility of handling hundreds of zebrafish for transplantation per day allowed us to screen a library of 480 small molecules with known bioactivity, aimed at identifying new drugs and pathways regulating HSPC engraftment. Two structurally related eicosanoids, 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, were able to enhance GFP+ marrow engraftment compared to DsRed2+ engraftment in zebrafish. This remarkable effect of EETs on adult marrow prompted us to study the effect of EETs in embryonic hematopoiesis. Treating zebrafish embryos with 11,12-EET during definitive hematopoiesis increased the HSPC marker Runx1 expression in the AGM (Aorta-Gonad-Mesonephros), resulting in a significant increase of HSPC in the next hematopoietic site, caudal hematopoietic tissue, the equivalent of fetal liver/placenta in mammals. The same treatment condition also induced ectopic Runx1 expression in the tail mesenchyme, a non-hematopoietic tissue. Microarray analysis on EET-treated zebrafish embryos revealed an upregulation of genes involved in stress response, especially Activator Protein 1 (AP-1) family members. Genetic knockdown experiments confirmed AP-1 members, especially JunB and its binding partners, cFos and Fosl2, are required for Runx1 induction. Motif analysis also predicted several conserved AP-1 binding sites in the Runx1 enhancer regions. To understand how EETs induced AP-1 expression, a suppressor screen was performed in zebrafish embryos. The screen revealed that activation of both PI3K/Akt and Stat3 are required for induced AP-1 expression, and therefore Runx1 upregulation. Similarly, ex vivo treatment of mouse whole bone marrow with 11,12-EET resulted in a 2-fold increase of long-term repopulating units. Microarray data had previously shown that Cyp2j6, one of the cytochrome P450 enzymes involved in EET biosynthesis from arachidonic acid, is enriched in quiescent mouse long-term HSCs. To further increase the EET levels in HSPCs, human CYP2C8 enzyme was over-expressed in transgenic mice using the Tie2 promoter. These transgenic mice have a 4-fold increase of long-term multi-lineage repopulating unit compared to their wild-type siblings. In purified mouse HSPCs, EETs directly and cell-autonomously activate PI3K/AKT pathway. Co-treatment of mouse bone marrow with EET and a PI3K inhibitor, LY294,002, completely blocked EET-induced enhancement of mouse bone marrow engraftment. In conclusion, we performed the first competitive marrow transplantation-based chemical screen, leading to the discovery of arachidonic acid-cytochrome P450-EETs as a novel modulator of HSC cell fate decision. PI3K/Akt and Stat3 pathways activated by EETs are required for adult HSPC engraftment and/or embryonic HSC specification, partially through transcriptional regulation of AP-1. We also demonstrated the requirement of AP-1 family members for Runx1 expression during embryonic development. This discovery may have clinical application in marrow or cord blood transplantation. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Zon:Fate Therapeutics: Founder; Stemgent: Consultancy.

Download Full-text

The Zinc Finger Transcription Factor Growth Factor Independence 1b (Gfi1b) Regulates The Wnt/Beta-Catenin Signaling Pathway In Hematopoietic Stem Cells Through Interaction With Inhibitory Proteins

Blood ◽

10.1182/blood.v122.21.2417.2417 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2417-2417

Author(s):

Peiman Shooshtarizadeh ◽

Ryan Chen ◽

Tarik Moroy

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Amino Acid ◽

Hematopoietic Stem Cells ◽

Signaling Pathway ◽

Beta Catenin ◽

Hematopoietic Stem ◽

Endosteal Niche ◽

Canonical Wnt

Abstract Hematopoietic stem cells (HSCs) reside in the bone marrow in specific niches at the border between bone cells and the bone marrow (endosteal niche) or around blood vessels (perivascular niche). In the endosteal niche, HSCs are maintained at low oxygen levels in a quiescent (dormant) state by adhesion to niche cells. We have previously shown that Gfi1b restricts the expansion and proliferation of HSCs as well as their mobilization or re-localization into peripheral blood. We have proposed that Gfi1b exerts this function by regulating the expression of surface molecules such as integrins on HSCs that are required to maintain them in their bone marrow niche at a quiescent state. The objective of this study was to gain more insight into the precise molecular mechanisms by which Gfi1b regulates HSCs dormancy and mobilization and to obtain insights that may be exploited in the future to improve stem cell therapies or the expansion of human hematopoietic stem cells for clinical use. Immune precipitation and mass spectrometry identified a series of Gfi1b interacting proteins, most notably a group of regulators of the canonical Wnt/beta-catenin pathway. Independent protein IP validation of these findings suggested that Gfi1b can interact with several inhibitors of the canonical Wnt/beta catenin pathway namely with APC (Adenomatous polyposis coli) a tumor suppressor protein and important factor in the beta-catenin destruction complex, with the DNA helicase and chromatin remodeling factor CHD8, which silences beta catenin mediated transcription, with CtBP which antagonizes beta-catenin activity and is part of the LSD1/CoRest histone demethylase complex and with the direct beta-catenin inhibitors TLE1 and TLE3 (also called Groucho). Of particular interest was that the interactions between the Groucho proteins and Gfi1b were dependent on a previously unidentified Groucho binding domain (GBD) in Gfi1b. This is a well-conserved six-amino acid stretch that is found in the middle part of the Gfi1b protein. In addition, the binding of CtBP was dependent on the presence of the 20 amino acid N-terminal SNAG domain in Gfi1b that also mediates LSD1 binding. Using luciferase reporter gene assays (TOP/FOP reporter assay), we found that Gfi1b was able to significantly up-regulates TCF/beta-catenin-dependent transcription upon activation by LiCl or Wnt3A in HEK293 cells. This activity of Gfi1b was dependent on both the presence of the SNAG domain and the newly identified Groucho binding domain. Also, Gfi1b was able to reverse partially the inhibitory effect of CtBP and TLE3 on beta-catenin activity in the TOP/FOP reporter assays. To obtain further evidence that Gfi1b is indeed implicated in regulating the Wnt/beta catenin signaling pathway in hematopoietic stem cells, we FACS sorted Lin-Kit1+Sca+ hematopoietic progenitors (LSK cells) from wt and Gfi1b deficient mice and tested them for expression of Wnt effector genes using a Wnt signaling specific PCR array. We observed that the majority of Wnt target genes were significantly down regulated in Gfi1 deficient LSKs compared to wt LSKs. Among the genes affected the most were typical Wnt targets such as Axin2, Frz7, Tcf4, Klf5, Vegfa and Ccnd1. To show that Gfi1b is able to regulate Wnt pathway effectors in vivo in HSCs, we crossed Gfi1b flox/flox, Mx-Cre mice with animals that carry a NLS-lacZ reporter gene under the control of the endogenous Axin2 promoter/enhancer region. Treatment with pIpC, which deletes Gfi1b correlated with a significant decrease of Axin2 expression in HSCs and MPP1, which are high Gfi1b expressing cells. The Axin2 reporter was not affected by Gfi1b deletion in MPP2 or GMPs, which express low levels or no Gfi1b. The canonical Wnt/b-catenin signaling pathway is recognized as one of the elements that are critically important in the regulation of HSC function. Here we have identified Gfi1b as a potential new player in the Wnt-beta catenin signaling pathway. Our data suggest that Gfi1b acts on at least two inhibitory complexes of this pathway, on the TLE family of Groucho proteins and the CtBP/LSD1 complex and regulates effectors of the Wnt/beta-catenin signaling cascade. We propose therefore that Gfi1b may titer the level of activation of the Wnt/beta-catenin signaling pathway in HSCs, which offers an explanation of the hematopoietic stem cell phenotype seen in mice lacking Gfi1b. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

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

Blood ◽

10.1182/blood.v128.22.3882.3882 ◽

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.

Download Full-text