Investigational NEDD8-Activating Enzyme (NAE) Inhibitor, MLN4924, Demonstrates Activity Against Primary AML Blast, Progenitor and Stem Cell Populations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1414-1414
Author(s):  
Siddhartha Sen ◽  
Jeanne P. De Leon ◽  
Peter G Smith ◽  
Gail J. Roboz ◽  
Monica L. Guzman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stress Responses ◽  
Colony Formation ◽  
Specific Inhibitor ◽  
Myelogenous Leukemia ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

Abstract Abstract 1414 Acute myelogenous leukemia (AML) has a poor prognosis. Most patients die of either primary refractory or relapsed disease. It has been proposed that relapse may result from ineffective ablation of leukemia stem cells (LSCs) by chemotherapy. Therefore, in order to improve therapy, it is imperative to identify agents that eliminate leukemic progenitor and stem cell populations in addition to blasts. We have previously demonstrated that proteasome inhibition can result in selective ablation of LSCs, without harming normal hematopoietic stem cells (HSCs). Thus, we sought to investigate the activity of a more specific inhibitor of the ubiquitin-proteasome pathway, an investigational NEDD8-Activating Enzyme (NAE) inhibitor MLN4924. Treatment of primary human AML and blast crisis CML samples with MLN4924 resulted in a dose-dependent decrease in the total viability of the blast-cell populations, with an average LD50 of 0.367 μM (0.203–0.530 95% CI; N=17). The average LD50 for phenotypically described LSCs was 0.645μM (0.304–0.986; 95%CI; N=12). Interestingly, when performing progenitor/stem functional assays, we observed a 64% and 86% decrease in colony formation after treatment with 0.25μM and 1 μM of MLN4924 (p< 0.001; N=5; p< 0.001 N=8 respectively). Furthermore, MLN4924 had little to no effect on colony formation of normal hematopoietic cells. Thus, MLN4924 may impair the activity of AML stem/progenitor cells, but not normal cells, in vitro. Importantly, primary AML samples that were resistant to standard chemotherapeutics such as cytarabine in vitro were sensitive to MLN4924. Treatment of primary AML samples with MLN4924 showed increased γH2AX foci, indicating activation of stress responses. Together, these data suggest that MLN4924 can eliminate AML blasts, progenitor and stem cell populations in vitro. Disclosures: Smith: Millennium Pharmaceuticals: Employment.

scholarly journals Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2422-2430 ◽  
Author(s):  
FC Zeigler ◽  
BD Bennett ◽  
CT Jordan ◽  
SD Spencer ◽  
S Baumhueter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cell ◽  
Receptor Tyrosine Kinase ◽  
Lymphoid Cells ◽  
Stem Cell Population ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3- positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

scholarly journals In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (12) ◽  
pp. 4045-4052 ◽  
Author(s):  
FC Zeigler ◽  
F de Sauvage ◽  
HR Widmer ◽  
GA Keller ◽  
C Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Cell Suspension Cultures ◽  
Hematopoietic Stem ◽  
Kit Ligand ◽  
Stem Cell Populations

Recently, the ligand for c-mpl has been identified and cloned. Initial studies of this molecule indicate that it is the platelet regulatory factor, thrombopoietin (TPO). Previous work has indicated that c-mpl is expressed in very immature hematopoietic precursors and thus raised the possibility that TPO may act directly on the hematopoietic stem cell. Therefore, in these studies, we investigate the effects of TPO on hematopoietic stem cell populations isolated from the murine fetal liver and bone marrow. Cocultivation of stem cells with fetal liver stroma give rise to multilineage expansion of the stem cells but with little or no megakaryocytopoiesis. Addition of TPO to these cocultures gives significant megakaryocyte production. This production is enhanced in combination with Kit ligand or interleukin-3. The addition of TPO to stem cell suspension cultures produces a dynamic thrombopoietic system in which stem cells undergo differentiation to produce megakaryocytes and proplatelets. These experiments show that the megakaryocytopoietic and thrombopoietic activities of TPO are initiated at the level of an early progenitor cell or upon the hematopoietic stem cell.

scholarly journals The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-Specific Stem Cell Markers

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 154
Author(s):  
Hanluo Li ◽  
Federica Francesca Masieri ◽  
Marie Schneider ◽  
Alexander Bartella ◽  
Sebastian Gaus ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hair Follicle ◽  
Cell Populations ◽  
Stem Cell Markers ◽  
Outer Root Sheath ◽  
Root Sheath ◽  
Gene And Protein Expression ◽  
Stem Cell Populations

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells.

Leukemic and Normal Stem Cell Transcriptional Signatures Determined by Functional Assays Are Predictive of the Overall Survival of AML Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 389-389
Author(s):  
Kolja Eppert ◽  
Katsuto Takenaka ◽  
Björn Nilsson ◽  
Eric R Lechman ◽  
Vicki Ling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Overall Survival ◽  
Stem Cell ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Functional Assays ◽  
Stem Cell Populations

Abstract Abstract 389 Normal hematopoiesis and acute myeloid leukemia (AML) are organized as hierarchies with stem cells, which possess extensive self-renewal and proliferative capacity, at the apex. Although there is definitive evidence from experimental models for the existence of leukemic stem cells (LSC) in some human leukemias, the relevance of LSC to human disease progression is still lacking. While chemotherapeutic treatment of AML patients typically results in disease remission, the majority of patients will eventually relapse and succumb to the disease, indicating that residual LSC are not eliminated by current treatment. We hypothesize that stem cell derived gene expression profiles may be more clinically relevant than those derived from examination of bulk leukemia samples. Here we show the clinical significance of novel stem cell related expression profiles derived from 25 functionally validated human leukemia stem cell populations and 6 normal hematopoietic stem cell populations. Little is currently known about the molecular regulatory networks that govern human LSC or hematopoietic stem cells (HSC). Therefore, we have carried out global mRNA gene expression profiling of FACS sorted subpopulations of cells enriched for human stem cells, progenitor cells and mature cells from 16 AML primary patient samples and 3 cord blood samples to investigate these pathways. Similar to normal hematopoietic stem cells, leukemia stem, progenitor and mature cells can be sorted using CD34 and CD38 markers. Due to the heterogeneous nature of AML, it is vital that quantitative functional assays are used to characterize the LSC and progenitor activity in each sorted fraction. In vitro cell suspension cultures and methylcellulose colony formation assays were performed to characterize progenitor and blast populations. Importantly, we applied a novel and improved in vivo SCID leukemia initiating cell assay to substantiate the presence of LSC activity in each sorted fraction of 16 AML patient samples. With this enhanced assay, LSC were detected in the expected CD34+/CD38- population. However, in the majority of AML samples, LSC were detected in at least one additional fraction, demonstrating the importance of functional validation when interpreting global gene expression profiles of sorted stem cell populations. LSC and HSC specific signatures were identified following a statistical analysis that compared fractions with stem cell activity against those without (25 LSC vs 29 non-LSC; 6 HSC vs 6 non-HSC). When applied to an independent gene expression data set from 160 cytogenetically normal AML samples, a 25 probe LSC signature was the strongest predictor of overall survival (p<0.0001, HR=2.6, 95%CI 1.8-4.0, median survival 236 vs 999 days; Figure 1a). Furthermore, the 225 probe HSC specific signature derived from normal cells also provided a strong predictor of survival (p<0.0001, HR=2.3, 95%CI 1.5-3.4, median survival 238 vs 741 days; Figure 1b). We queried the gene expression-based chemical genomic database Connectivity Map with the LSC-related gene list and found a negative correlation between the genes in the LSC profile and the expression of genes that are transcriptionally induced following treatment with common chemotherapeutic compounds such as doxorubicin, suggesting resistance to chemotherapy as one possible mechanism for the correlation of the stem cell signatures with survival. Together these data support the hypothesis that the biological determinants that underlie stemness in both normal and leukemic cells are predictors of poor outcome, and are potential targets for novel therapy. Disclosures: No relevant conflicts of interest to declare.

MixL1 Expression Identifies Putative Hematopoietic Stem Cells In the Murine Allantois.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1591-1591
Author(s):  
Adam D Wolfe ◽  
Karen Downs
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Preliminary Evidence ◽  
Yolk Sac ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Poorly Differentiated ◽  
Stem Cell Populations

Abstract Abstract 1591 MixL1, a paired-type homeodomain transcription factor, is implicated in pre-hematopoietic commitment of stem cell populations. In poorly-differentiated human lymphoma and leukemia lines, MixL1 is inappropriately over-expressed (Drakos et al., Human Pathol 2007:38:500). When constitutively expressed in mice, MixL1 is sufficient to induce Acute Myeloid Leukemia (Glaser et al., PNAS 2006:103:16460). On the basis of these observations, we hypothesize that MixL1 plays an important role in gating the cellular decision to remain in a poorly differentiated and proliferative phase rather than proceeding to definitive hematopoietic stem cell (HSC) identity. Several years ago, the placenta was shown to be a major site of hematopoiesis (Gekas et al., Dev Cell 2005:8:365; Ottersbach and Dzierzak, Dev Cell 2005:8:377). The placenta is composed of the chorionic disc and the allantois, the latter of which matures into the umbilical component of the placenta. The allantois exhibits definitive hematopoietic potential (Ziegler et al., Development 2006:133:4183; Corbel et al., Dev Biol 2007:301:478), and has recently been demonstrated to contain a core of stem cells referred to as the Allantoic Core Domain, or ACD, where potential placental hematopoietic activity may originate (Downs et al., Dev Dyn 2009:238:532). Our immediate goal is to evaluate whether MixL1 is expressed in the allantois, and to establish its precise spatiotemporal whereabouts with respect to early markers of hematopoietic cells, such as Runx1 (Chen et al., Nature 2009:457:887). Using immunohistochemistry in conjunction with the LacZ/Runx1 reporter mouse (North et al., Development 1999:126:2563), we have demonstrated that MixL1 is strongly expressed in the blood islands of the yolk sac, as well as in a broad, contiguous posterior domain of the embryo that extends to include the ACD stem cell core of the allantois. This domain does not include Runx1, and MixL1 expression temporally precedes that of Runx1 in the allantois. As development proceeds, the MixL1 signal becomes most prominent in putative nascent blood cells budding off from the poorly-described blood vessel common to the allantois, yolk sac and dorsal aortae, which we have called the “Vessel of Confluence” (VOC). Here, fetal blood is shuttled into the umbilical cord to the chorion for exchange with the mother. Shortly thereafter, Runx1 begins to appear within VOC, and is co-expressed with MixL1. These findings provide preliminary evidence that MixL1 is expressed within the allantois, and within nascent blood cells derived from a specific arterial site common to the allantois, yolk sac, and fetus. Moreover, MixL1 expression appears to precede that of Runx1. Thus, MixL1 may identify one of the earliest hematopoietic precursor cell populations thus far known in mammals. Further, these data provide additional evidence that the allantois is a promising model system for the study of definitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Embryonic and hematopoietic stem cells express a novel SH2–containing inositol 5′-phosphatase isoform that partners with the Grb2 adapter protein

Blood ◽  
2001 ◽  
Vol 98 (7) ◽  
pp. 2028-2038 ◽  
Author(s):  
Zheng Tu ◽  
John M. Ninos ◽  
Zhengyu Ma ◽  
Jia-Wang Wang ◽  
Maria P. Lemos ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Cell Populations ◽  
Adapter Protein ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

SH2–containing inositol 5′-phosphatase (SHIP) modulates the activation of immune cells after recruitment to the membrane by Shc and the cytoplasmic tails of receptors. A novel SHIP isoform of approximately 104 kd expressed in primitive stem cell populations (s-SHIP) is described. It was found that s-SHIP is expressed in totipotent embryonic stem cells to the exclusion of the 145-kd SHIP isoform expressed in differentiated hematopoietic cells. s-SHIP is also expressed in primitive hematopoietic stem cells, but not in lineage-committed hematopoietic cells. In embryonic stem cells, s-SHIP partners with the adapter protein Grb2 without tyrosine phosphorylation and is present constitutively at the cell membrane. It is postulated that s-SHIP modulates the activation threshold of primitive stem cell populations.

scholarly journals Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 4201-4208 ◽  
Author(s):  
Xiuli Wang ◽  
Shundi Ge ◽  
George McNamara ◽  
Qian-Lin Hao ◽  
Gay M. Crooks ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Liver Damage ◽  
Human Albumin ◽  
Cell Populations ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

AbstractRodent bone marrow cells can contribute to liver. If these findings are applicable to humans, marrow stem cells could theoretically be harvested from a patient and used to repair his/her damaged liver. To explore this potential, CD34+ or highly purified CD34+CD38−CD7− human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice. One month after transplantation, carbon tetrachloride (CCl4) was administered into the mice to induce liver damage and hepatocyte proliferation. Mice were analyzed in comparison with CCl4-injured mice that did not receive transplants and noninjured controls that received transplants with the same stem cell populations, one month after liver damage. Human-specific albumin mRNA and protein were expressed in the mouse liver and human albumin was detected in the serum of mice that had received CCl4 injury. Human alpha-fetoprotein was never expressed, but in some mice, human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin-secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations in injured mice that did not receive transplants nor in noninjured mice that had received transplants of human stem cells. Human albumin expression was detected only in CCl4-treated mice that received transplants of human stem cells, and recovery was increased by administration of human hepatocyte growth factor 48 hours after the CCl4-mediated liver injury. Our studies provide evidence that human “hematopoietic” stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression.

Stem Cells in Paediatric Leukaemia Are Resistant to Parthenolide.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1429-1429
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Functional Capacity ◽  
Cell Populations ◽  
Minimal Effect ◽  
Childhood All ◽  
Stem Cell Populations

Abstract Abstract 1429 Poster Board I-452 The concept of cancer stem cells as developmentally early cells that are capable of continued growth and expansion in haematopoietic malignancies and solid tumours has been substantiated in recent years. Consequently these cells may be responsible for disease maintenance and relapse. Acute lymphoblastic leukaemia (ALL) is the most common paediatric cancer with survival rates around 80-85%. However, a significant proportion of patients relapse, often with disease that is highly refractory to further therapeutic intervention. Leukaemia stem cells have been described in childhood ALL that can proliferate to initiate and sustain the disease in vivo. In addition these leukaemia stem cells have also been shown to be refractory to commonly used clinical agents. Therefore it is important to investigate ALL stem cells to understand their biological properties and to identify the most appropriate agents that are capable of eradicating these cells. The sesquiterpene lactone Parthenolide (PTL) has been shown to induce apoptosis in malignant cells by inducing oxidative stress and inhibiting NF-κB activity. Importantly PTL has been shown to be effective against stem cell populations in acute myeloid leukaemia and in chronic lymphocytic leukaemia with minimal effect on normal haemopoietic cells. In this study we have attempted to assess the effects of PTL on stem cell populations in paediatric ALL. Primary cells from 20 childhood ALL cases from mixed prognostic subgroups were used in this investigation. Cells from B-ALL cases were sorted on the basis of expression of CD34/CD19, while CD34/CD7 antigens were used to sort cells from T-ALL cases. Sorted and unsorted populations were co-cultured with and without PTL at 7.5μM and 10μM for 18-24 hours. Subsequently cell viability and apoptosis were determined by flow cytometry using Annexin V and PI staining. Antibodies against phosphorylated IKKα and IKKβ were used to assess NF-κB activity in treated and untreated cells. The functional ability of the treated cells was assessed in some cases using long-term in vitro and in vivo assays. Both concentrations of PTL resulted in a significant reduction in viability in unsorted ALL cells (28±4% and 23±5% respectively). Similar results were observed with CD34+/CD19+, CD34+/CD7+ and CD34- subfractions, with viability reduced to 14-39%. In contrast the phenotypically primitive CD34+/CD19- (85±11% viable) and CD34+/CD7- (83±5% viable) populations were significantly more resistant to 10μM PTL than unsorted cells and other sorted populations (P≤0.002). FISH analyses were performed at the end of the time-course and confirmed that leukaemia cells were surviving PTL treatment. It was not possible to detect phosphorylated IKKα/β in the CD34+/CD19- and CD34+/CD7- populations, in cases examined to date, suggesting NF-kB may not be active in these subpopulations. Of note PTL treatment seemed to have minimal effect on the long-term proliferative ability of ALL cells. There were no significant differences in the absolute cell numbers generated in cultures of PTL treated CD34+/CD19- or CD34+/CD7- cells compared to untreated cells at all time points assayed up to the end of culture at week 6 (P≥0.23). Interestingly, similar results were observed with the unsorted cells and all other sorted populations. From week 3 of culture there was no difference in the absolute cell counts when growth from treated and untreated cells was compared (P>0.47), albeit they proliferated to a much lesser extent than the phenotypically primitive populations. In addition PTL treated cells were capable of engrafting NOD/SCID mice. The levels of leukaemia engraftment obtained using PTL treated unsorted (0.2-5% CD45+), CD34+/CD19- (2-10% CD45+) and CD34+/CD7- (1.5-9% CD45+) populations were similar to their respective untreated controls. These data demonstrate that while PTL showed promising effects on the bulk leukaemia cells, the effects on CD34+/CD19- B-ALL cells and CD34+/CD7- T-ALL cells were insignificant. This may be due in part to lack of NF-kB activity in leukaemia stem cells. However, the functional capacity of every ALL population evaluated in vitro was not significantly impaired by the short course of PTL treatment. These findings further highlight the importance of evaluating new therapeutic agents on leukaemia stem cell populations in addition to the bulk leukaemia and the significance of investigating the functional capacity of drug treated cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cellular and molecular characterization of the role of the flk-2/flt-3 receptor tyrosine kinase in hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 84 (8) ◽  
pp. 2422-2430 ◽  
Author(s):  
FC Zeigler ◽  
BD Bennett ◽  
CT Jordan ◽  
SD Spencer ◽  
S Baumhueter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cell ◽  
Receptor Tyrosine Kinase ◽  
Lymphoid Cells ◽  
Stem Cell Population ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Stem Cell Populations

Abstract The flk-2/flt-3 receptor tyrosine kinase was cloned from a hematopoietic stem cell population and is considered to play a potential role in the developmental fate of the stem cell. Using antibodies derived against the extracellular domain of the receptor, we show that stem cells from both murine fetal liver and bone marrow can express flk-2/flt-3. However, in both these tissues, there are stem cell populations that do not express the receptor. Cell cycle analysis shows that stem cells that do not express the receptor have a greater percentage of the population in G0 when compared with the flk-2/flt-3- positive population. Development of agonist antibodies to the receptor shows a proliferative role for the receptor in stem cell populations. Stimulation with an agonist antibody gives rise to an expansion of both myeloid and lymphoid cells and this effect is enhanced by the addition of kit ligand. These studies serve to further illustrate the importance of the flk-2/flt-3 receptor in the regulation of the hematopoietic stem cell.

scholarly journals Non-Genotoxic Conditioning Efficiently Depletes Host Hematopoietic Stem Cells and Facilitates Robust Multi-Lineage Engraftment in a Mouse Model of Fanconi Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2041-2041
Author(s):  
Meera Srikanthan ◽  
Kevin G. Haworth ◽  
Christina M. Ironside ◽  
Jerry Chen ◽  
Adam J. Hartigan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Marrow Transplant ◽  
Cell Populations ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Equity Ownership ◽  
Stem Cell Populations

Abstract Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow prior to a bone marrow transplant to facilitate engraftment of donor cells. While effective, some major issues with chemotherapy remain which includes off-target genotoxic effects increasing the risk of secondary malignancies. These complications are compounded in disease settings that arise from a deficit in DNA repair pathways where cytotoxic treatments can result in oncogenic transformation, such as Fanconi anemia (FA). FA is an inherited bone marrow failure disorder resulting from an intrinsic defect in DNA repair affecting approximately 130,000 children each year. Currently mutations in 22 genes have been implicated in the pathogenesis of FA. While novel gene-therapy based protocols are showing early promise for this patient populations, the standard treatment for the hematologic complications for FA is a bone marrow transplant. However, secondarily to an underlying sensitivity to DNA damage, these patients are unable to receive standard myeloablative conditioning, which can reduce the efficiency of reconstitution. Avoiding alkylating agents could improve outcomes and success rates in these patients. Furthermore, this methodology could be translated to the allogeneic bone marrow transplantation setting, decreasing the toxicity of this treatment modality. Our approach for characterizing an alternative conditioning regimen for FA patients utilizes immunotoxin conjugates specifically targeting hematopoietic stem cell populations. These non-genotoxic antibody-based drug conjugates utilize saporin (SAP), a ribosomal toxin, to eliminate cells in a targeted manner while leaving the remainder of the marrow compartment intact. Antibodies targeting either CD45 or CD117 were used in a mouse model of FA where expression of their FANCA gene, one of the most common mutations in humans, has been knocked out. Mice conditioned with either of the drugs received various doses of whole marrow from healthy heterozygous littermates. On the day of transplant, mice conditioned with either immunotoxin demonstrated significantly reduced LSK stem cell populations in the marrow similar to cyclophosphamide (Cy) controls (Figure 1A). Peripheral engraftment of donor cells was monitored for 6 months, after which mice were sacrificed for complete analysis of engraftment in the bone marrow compartment. No significant difference was observed in engraftment between Cy and immunotoxin conditioned mice (Figure 1B), and all treatment groups exhibited robust multilineage reconstitution in a cell dose dependent manner. Additionally, Cy treated mice demonstrated greater and sustained weight loss and lower gastrointestinal losses compared to immunotoxin treated mice. Ongoing clonal analysis studies of engrafted cells has demonstrated polyclonal reconstitution, indicating a large number of donor stem cells are actively contributing to hematopoiesis. In this study, we demonstrate that non-genotoxic conditioning approaches both facilitate multilineage engraftment of donor marrow and significantly deplete host hematopoietic stem cell populations. These are crucial since persistence of host hematopoiesis could eventually result in clonal evolution and leukemogenesis in post-transplant FA patients. Achieving both of these conditions through targeted elimination with immunotoxin conjugates represents a major advancement in bone marrow transplantation for FA. We now are initiating studies using immunotoxin-based conditioning for the transplantation of gene-modified syngeneic stem cells and allogeneic cells. We think these studies will inform future clinical trials and provide the groundwork for the next-generation of therapy for FA patients. Disclosures Hartigan: Magenta Therapeutics: Employment. Palchaudhuri:Harvard University: Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy.

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