Combined G-CSF and Plerixafor enhance hematopoietic recovery of CD34+ cells from poor mobilizer patients in NSG mice

2020 ◽  
Vol 86 ◽  
pp. 15-20.e2
Author(s):  
Marie-Laure Arcangeli ◽  
Philippe Brault ◽  
Jean-Henri Bourhis ◽  
Frédérique Kuhnowskie ◽  
Elia Henry ◽  
...  
Keyword(s):  
Nsg Mice ◽  
Hematopoietic Recovery ◽  
Cd34 Cells ◽  
Poor Mobilizer

scholarly journals MGTA-456, a First-in-Class Cell Therapy Produced from a Single Cord Blood Unit, Enables a Reduced Intensity Conditioning Regimen and Enhances Speed and Level of Human Microglia Engraftment in the Brains of NSG Mice

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 115-115
Author(s):  
Kevin A. Goncalves ◽  
Shuping Li ◽  
Melissa L. Brooks ◽  
Sharon L. Hyzy ◽  
Anthony E. Boitano ◽  
...  
Keyword(s):  
Fold Increase ◽  
High Dose ◽  
Reduced Intensity Conditioning ◽  
Employment Equity ◽  
Nsg Mice ◽  
Hematopoietic Recovery ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
The Brain ◽  
Reduced Intensity

Abstract Background. Allogeneic bone marrow transplant (BMT) is a promising, curative approach for patients with inherited metabolic disorders (IMDs), a class of pediatric diseases characterized by a single enzyme deficiency. The goal of transplant is to provide cells that produce functional enzymes otherwise deficient in these patients, and thereby prevent or ameliorate neurological complications associated with selected IMDs. Donor-derived microglial cells are protective, limiting neurological disease progression. For IMD patients who do not have an HLA matched, non-carrier related donor, cord blood (CB) is the preferred HSPC source given its rapid availability and superior clinical outcomes compared to other graft sources. CB, however, is associated with delayed hematopoietic recovery and relatively poor engraftment due to the limited numbers of hematopoietic stem cells (HSCs) in a CB unit, delaying enzyme/protein reconstitution and cross-correction of non-hematopoietic cells. An aryl hydrocarbon receptor antagonist (AHRa)-based culture has been shown to expand CB CD34+ and CD34+CD90+ cells 330-fold and 100-fold, respectively, leading to rapid hematopoietic recovery after infusion of the clinical product, MGTA-456 (Wagner et al., Cell Stem Cell 2016 and Orchard et al., ASH 2018). As microglia are thought to be derived from HSCs, we hypothesized that MGTA-456 might lead to faster and greater microglial engraftment and potentially enable reduced intensity conditioning. Here, we assessed human hematopoietic and brain engraftment in NSG mice after transplant with MGTA-456 and showed that microglia engrafted faster with MGTA-456, less conditioning was needed, and that, mechanistically, these cells are derived from the CD34+CD90+ cell fraction. Methods. CB CD34+ cells were expanded in growth factor-supplemented media with or without an AHRa for 10 days. NSG mice were transplanted with unmanipulated CB CD34+ cells or the expanded product after 200 cGy total body irradiation or busulfan (BU) dosed at 20 or 40 mg/kg ip. Microglial engraftment was measured by flow cytometry of homogenized brains, quantitating the number of CD45+CD11b+Iba1+ cells, and by immunohistochemistry of brain sections. Results. Relative to naïve, unmanipulated CB CD34+ cells, transplant of MGTA-456 into sublethally irradiated mice led to an 8-fold increase in hematopoietic engraftment and a 10-fold increase in microglial engraftment in the brain (p<0.0001, n=15 mice), with histology consistent with engrafting microglia. As high dose BU enables enhanced microglia engraftment relative to irradiation by crossing the blood brain barrier and clearing host microglia (Capotondo et al., PNAS 2013), we evaluated the effectiveness of MGTA-456 after BU conditioning at 20 or 40 mg/kg. Transplant of MGTA-456 led to a 37-fold increase in engraftment relative to mice transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). Notably, transplant of MGTA-456 into mice conditioned with low-dose BU (20 mg/kg) led to a 15-fold increase in engraftment relative to high-dose BU (40 mg/kg)-conditioned animals transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). To evaluate speed of microglial engraftment, we evaluated brains weekly to 16 weeks after transplant. A 28-fold increase in microglial engraftment was demonstrated as early as 2 weeks post-transplant with MGTA-456 (p<0.0001, n=8). Number of engrafting hematopoietic cells in the periphery correlated with number of engrafting microglia in the brain (p<0.0001). Lastly, subpopulations of MGTA-456 were evaluated to determine the source of microglial engraftment. Only CD34+CD90+ cells, but not CD34+CD90- or CD34- cells, led to brain engraftment, consistent with the subpopulation of cells that result in hematopoietic engraftment following transplant of unexpanded cells (Radtke et al., Sci Trans Med 2017 and Goncalves et al., Blood 2017 130:659). Conclusions. These studies demonstrate that microglial engraftment is faster and greater in recipients of MGTA-456 even after lower dose BU conditioning, that microglial engraftment correlates with peripheral blood recovery, and that microglia cells are derived from CD34+CD90+ cells. These results suggest that lower dose BU may improve safety without jeopardizing efficacy in IMD recipients of MGTA-456. A Phase 2 clinical trial is ongoing to evaluate transplant of MGTA-456 in patients with select IMDs. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

MURINE PGK PROMOTER IN A LENTIVIRAL VECTOR IN CANINE LEUKOCYTE ADHESION DEFICIENCY AND IN HUMAN LAD-1 CD34+CELLS IN NSG MICE

2012 ◽  
Vol 07 (01) ◽  
pp. 1250001
Author(s):  
MICHAEL J. HUNTER ◽  
UIMOOK CHOI ◽  
LAURA M. TUSCHONG ◽  
HUIFEN ZHAO ◽  
SHERRY KOONTZ ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Leukocyte Adhesion ◽  
Leukocyte Adhesion Deficiency ◽  
Nsg Mice ◽  
Cd34 Cells

scholarly journals Antagonizing PPARγ Expands Human Hematopoietic Stem and Progenitor Cells By Switching on FBP1-Repressed Glycolysis and Preventing Differentiation

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 709-709
Author(s):  
Bin Guo ◽  
Xinxin Huang ◽  
Hal E. Broxmeyer
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Metabolic Reprogramming ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ppar Γ ◽  
Control Shrna ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Allogeneic hematopoietic cell transplantation (HCT) is widely used as a life-saving treatment for malignant and non-malignant blood disorders. Hematopoietic stem cells (HSCs) are a major contributing cell population for a successful HCT. While cord blood (CB) is an acceptable source of HSCs for clinical HCTbecause of its many advantages including prompt availability, lower incidence of GvHD and virus infection, CB HCT is usually associated with slower time to engraftment especially in adult patients when compared with other cell sources; this is partly due to limiting numbers of HSCs in single cord units. In order to overcome this limitation, ex vivo expansion of CB HSCs has been evaluated in preclinical and clinical studies for improvement of the clinical efficacy of CB HCT. While a number of different ways have been evaluated to ex-vivo expand human HSCs, little is known about the mechanisms involved, and whether efficient expansion of CB HSCs could be achieved by metabolic reprogramming. In a compound screen for potential candidates which could promote ex vivo expansion of CB HSCs, we found that PPARγ antagonist GW9662 treatment significantly enhanced ex vivo expansion of CB phenotypic HSCs (~5 fold) and progenitor cells (HPCs) (~6.8 fold) in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and cytokines (SCF, FL, TPO) when compared with vehicle control. GW9662 significantly increased numbers of CB colony-forming unit (CFU) granulocyte/macrophage (GM) (~1.8 fold) and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) (~3.2 fold) progenitors after 4 days ex vivo culture. To assess whether the ex vivo expanded CB HSCs enhanced by the PPARγ antagonist were functional in vivo, we performed both primary and secondary transplantation in immunocompromised NSG mice. Engraftment of CB CD34+ cells in primary recipients was significantly increased (~3 fold) both in bone marrow (BM) and peripheral blood (PB) by the cultured cells treated with GW9662. The percentages of both myeloid and lymphoid lineages were enhanced in BM of primary recipients transplanted with GW9662-treated CB CD34+ cells. We also transplanted CB CD34+ cells transfected with control shRNA or PPAR γ shRNA into NSG mice, and consistently found that both myeloid and lymphoid chimerism was enhanced in BM of recipients which were infused with PPAR γ shRNA transfected-CD34+ cells compared with control shRNA transfected-CD34+ cells. Long term reconstituting and self-renewing capability of GW9662-treated CB CD34+ cells with both enhanced myeloid and lymphoid chimerism, was confirmed in PB and BM in secondary recipients. Limiting dilution analysis was performed to calculate SCID-repopulating cells (SRC), a measure of the number of functional human HSCs. The SRC frequency of GW9662-cultured CB CD34+ cells was 4 fold greater than that of day 0 uncultured CD34+ cells, and 5 fold increased above that of vehicle-treated CD34+ cells with cytokines alone. To gain mechanistic insight into how PPARγ antagonism enhances expansion of human CB HSCs and HPCs, we performed RNA-seq analysis. Antagonizing PPARγ in CB CD34+ cells resulted in downregulation of a number of differentiation associated genes, including CD38, CD1d, HIC1, FAM20C, DUSP4, DHRS3 and ALDH1A2, which suggests that PPARγ antagonist may maintain stemness of CB CD34+ cells partly by preventing differentiation. Of interest, we found that FBP1, encoding fructose 1, 6-bisphosphatase, a negative regulator of glycolysis, was significantly down-regulated by GW9662, which was further confirmed by RT-PCR, western blot and flow cytometry analysis. GW9662 significantly enhanced glucose metabolism in CB HSCs and HPCs without compromising mitochondrial respiration. Enhanced expansion of CB HSCs by antagonizing PPARγ was totally suppressed by removal of glucose or by inhibition of glycolysis. Importantly, suppression of FBP1 greatly promoted glycolysis and ex vivo expansion of long-term repopulating CB HSCs (~3.2 fold). Overexpression of FBP1 significantly suppressed enhancedexpansion and engraftment of CB HSCs by PPARγ antagonist. Our study demonstrates that PPARγ antagonism drives ex vivo expansion of human CB HSCs and HPCs by switching on FBP1 repressed glucose metabolism and by preventing differentiation. This provides new insight into human HSC self-renewal, and suggests a novel and simple means by which metabolic reprogramming may improve the efficacy of CB HCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 659-659
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Jennifer L. Proctor ◽  
Hillary L. Adams ◽  
Sharon L. Hyzy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Small Molecule ◽  
Hdac Inhibitors ◽  
Employment Equity ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (&gt;3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Daily Measurements of Blood CD34+ Cells After Stem Cell Mobilization Predict Stem Cell Yield and Posttransplant Hematopoietic Recovery

1997 ◽  
Vol 6 (1) ◽  
pp. 13-19 ◽  
Author(s):  
KARI REMES ◽  
IRMA MATINLAURI ◽  
SEIJA GRENMAN ◽  
MAIJA ITÄLÄ ◽  
MARJUT KAUPPILA ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Mobilization ◽  
Cell Yield ◽  
Hematopoietic Recovery ◽  
Cd34 Cells ◽  
Cell Mobilization

Pegfilgrastim Versus Filgrastim To Accelerate Hematopoietic Recovery after High Dose Melphalan and Autologous Hematopoietic Stem Cell Transplant (ASCT) for Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5193-5193
Author(s):  
Rebecca L. Olin ◽  
Selina M. Luger ◽  
David L. Porter ◽  
Stephen J. Schuster ◽  
Donald Tsai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cohort Study ◽  
Stem Cell ◽  
Retrospective Cohort Study ◽  
Retrospective Cohort ◽  
Median Number ◽  
High Dose ◽  
Hematopoietic Recovery ◽  
Cd34 Cells ◽  
High Dose Melphalan

Abstract High-dose melphalan followed by ASCT is a common component of the early treatment for patients with multiple myeloma. Daily subcutaneous injections of filgrastim (Neupogen) at 5 ug/kg/day until ANC &gt; 500/ul are routinely administered at our center from day +4 following ASCT, in order to accelerate hematopoietic recovery and lessen neutropenic complications. Pegfilgrastim (Neulasta) as a single 6 mg fixed dose subcutaneous injection has been shown to have similar efficacy and ease of use when compared to filgrastim in the non-transplant setting, but little data is available in the transplant setting. We began using pegfilgrastim day +1 following ASCT for patients with multiple myeloma and performed a retrospective cohort study comparing those who received filgrastim (n=6) with those who received pegfilgrastim (n=11). Transplants occurred between July 2002 and January 2004 and included all patients transplanted for myeloma in that time period for whom sufficient data was available. All patients had at least 2 x 106 CD34+ cells/kg peripheral stem cells harvested after cytoxan and filgrastim mobilization. Main outcome measures were: days from stem cell infusion to WBC nadir, days to ANC&gt;500/ul, and days to ANC&gt;1000/ul. Subjects were excluded if CBCs were drawn less frequently than every four days. There were no significant differences between the filgrastim and pegfilgrastim groups with respect to the following demographic variables: age, gender, hemoglobin, creatinine, calcium, albumin and beta-2 microglobulin at diagnosis. The groups were also balanced with respect to SPEP, UPEP, presence of lytic lesions and number of prior lines of therapy. The median number of CD34+ cells infused was similar: 5.7 x 106 in the filgrastim group vs 4.8 x 106 in the pegfilgrastim group (p=0.28). After transplant, median number of days to WBC nadir in the filgrastim group (FG) was 7 (range 5–9) vs 6 (range 5–8) in the pegfilgrastim group (PG) (p=0.31). However, median number of days to ANC&gt;500/ul in the FG was 11.5 (range 11–17) vs 10 (range 9–12) for PG (p=0.02). Similarly, median number of days to ANC&gt;1000/ul was 12 (range 11–17) for FG vs 11 (range 10–13) for PG (p=0.03). Five of six patients in the FG had neutropenic fever after transplant, compared to five of eleven patients in the PG (p=0.30). Currently, no significant differences in infection or relapse rates between groups have been noted and there were no deaths in either group. In this retrospective cohort study, pegfilgrastim was safe and at least equivalent to filgrastim for accelerating hematopoiesis after ASCT for multiple myeloma. Furthermore, there was no significant difference in the incidence of neutropenic fever, infection and survival, suggesting a similar clinical utility.

Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3722-3722
Author(s):  
Li Ming Ong ◽  
Xiubo Fan ◽  
Pak Yan Chu ◽  
Florence Gay ◽  
Justina Ang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Insulin Growth Factor ◽  
Nsg Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 514-514 ◽  
Author(s):  
Bin Zhang ◽  
David Irvine ◽  
Yin Wei Ho ◽  
Silvia Buonamici ◽  
Paul Manley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Colony Formation ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Advisory Committees ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Hh Signaling

Abstract Abstract 514 Background: Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo. Methods and Results: 1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone. Conclusions: We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients. Disclosures: Buonamici: Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.

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