ALBERT

Description: Background: Maintaining T cell function and survival after ex vivo manipulation remains a major challenge in adoptive immunotherapy. We previously demonstrated that murine T cells activated and expanded ex-vivo with anti-CD3 and anti-CD28 antibody-coated magnetic beads (CD3/CD28 beads) exhibit reduced GVHD-inducing potential compared to naïve (unmanipulated) T cells in a murine allogeneic BMT model. Blood diagnostics are commonly used to help identify patterns of biomarkers in multiple disease states. Objective: Determine if plasma profiles of 59 different analytes are altered in murine allogeneic BMT recipients who receive ex-vivo activated, suicide gene transduced and selected (Td) T cells compared with animals that received naïve suicide gene expressing T cells. Methods: Murine T cells were Td with a chimeric CD34-thymidine kinase (CD34-TK) fusion suicide gene. High efficiency (70%) gene transfer of CD34-TK to C57BL/6 (B6) murine T cells was accomplished 24 h after CD3/CD28 bead activation and gene-modified cells were purified to 〉 96% by CD34 immunomagnetic selection 2 days post-infection. Naïve B6 CD34-TK-expressing T cells were purified from the spleens of CD34-TK transgenic mice the day of BMT. To induce GVHD, lethally irradiated BALB/c allogeneic recipients were given T cell depleted (TCD) B6 BM supplemented with or without B6 CD34-TK purified naïve or Td T cells. Animals were bled 1 week after BMT and EDTA plasma samples were prepared and frozen. Quantitative measurements of 59 analytes were obtained using a rodent multi-analyte profile test (MAP test; Charles River Lab) on the plasma of 2 mice that received naïve T cells and died from GVHD on days 16 and 21 after BMT, 2 mice that received Td T cells and died from GVHD on day 34 after BMT, and 2 mice that received TCD BM only and survived 〉 100 days. Results: As before, we found that ex vivo activation of the donor T cells before BMT significantly prolonged the survival of mice transplanted with the Td T cells compared with mice receiving naïve T cells (p = 0.0028). Eighteen analytes, including IFN-γ, IL-2, IL-3, IL-4, IL-7, IL-11, and TNF-α were below the detection limits of the rodent MAP test for all samples analyzed. Twenty-six analytes, including VEGF, SCF, MIP-2, MIP-1α, MIP-3β, MCP-5, IL-1β, IL-18 and GM-CSF were detectable but not significantly different than the BM only controls for either the naïve or Td T cell groups. Nine analytes, including eotaxin, MIP-1γ, MCP-3, MCP-1, and IL-10 were similarly increased 2- to 3-fold in both the Td and naïve T cell groups compared to the BM only control. Interestingly, recipients of naive T cells exhibited increased plasma levels of growth hormone (≥14-fold), MIP-1β (4.6-fold), IP-10 (2.3-fold), and lymphotactin (2-fold), as well as decreased levels of leptin (≥14-fold), compared to both the BM only and Td T cell groups. This extreme modulation of growth hormone and leptin levels is particularly interesting given the fact that leptin influences local growth hormone secretion from lymphocytes and that both of these analytes have multiple biologic effects on T cells. Finally, only a single analyte, monocyte derived chemokine (MDC), was increased (4-fold) in mice that received Td T cells compared to the naïve T cell recipients. Conclusion: These results indicate that several plasma analytes with important immunological functions are altered when donor T cells are manipulated ex-vivo. These alterations may account for the decreased GVHD-inducing potential of ex vivo manipulated T cells after allogeneic BMT.

Description: Background: Current clinical protocols use granulocyte colony-stimulating factor (G-CSF) to mobilize normal hematopoietic stem cells (HSCs) from the bone marrow (BM) to the peripheral blood. Unfortunately, this process requires from 4 to 6 days of G-CSF injection and is associated with significant morbidity, most notably bone pain. We are evaluating a novel method for the mobilization of HSCs using a direct antagonist of the CXCR4/SDF-1 interaction called Plerixafor (AMD3100). Methods: Human CD34+ cells were collected from three different studies at our institution. In the first study, fifteen healthy allogeneic related donors were initially mobilized with increasing doses of intravenous (IV) AMD3100 (80, 160, 240, or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single subcutaneous (s.c.) dose of 240 μg/kg AMD3100 and collected cells were used as a source of HSCs for transplantation. In the second study, ten healthy donors were mobilized with 5 days of s.c. injection of G-CSF (10 μg/kg/day), and leukapheresed on day 5. In the final study, eight individual normal donors were mobilized sequentially with AMD3100 and G-CSF. Donors received 1 s.c. injection of 240 μg/kg AMD3100, followed by leukaphersis beginning 4 h after drug treatment. After 10 days of drug clearance, the same donors were mobilized with 5 days s.c. injection of 10 μg/kg/day G-CSF, and leukapheresed on day 5. Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection (MACS) CD34 isolation kit and total RNA was isolated using RNeasy Mini Kit columns (Qiagen). The purity (〉85% for all experiments) and phenotype of isolated CD34+ cells was quantified by flow cytometry. RNA profiling analyses were performed using Affymatrix U133+2 arrays. Results: Peak mobilization of CD34+ cells occurred 4 to 6 hours after both IV and s.c. dosing, however, patients given IV doses had higher peak levels of CD34/μl at every time point. There was a clear doseresponse relationship of IV AMD3100 on mobilization of CD34+ HSCs in normal donors, with the 320 μg/kg dose yielding a maximum increase in circulating CD34+ cells from 3.3 ± 1.8 CD34+/μl at baseline to 28.8 ± 4.7 CD34+/μl at 6 h after injection. Although the magnitude of neutrophil, monocyte, and T lymphocyte mobilization by IV AMD3100 was less than that observed for CD34+ cells (2 to 3 fold increase over baseline), the kinetics of their mobilization were similar to the CD34+ HSCs (peak mobilization 4 to 6 h after AMD3100). In contrast, B-lymphocytes were mobilized more rapidly (4.5 ± 1.7-fold at 15 min post-AMD3100) and efficiently (6.6 ± 2.6-fold at 2 h post-AMD3100) by IV AMD3100. This rapid mobilization of B-lymphocytes correlates with our pharmacokinetic studies, which showed that peak levels of AMD3100 occur between 15 and 30 minutes after IV infusion. The gene signature of AMD3100-mobilized human CD34+ HSCs is distinct from that of G-CSF-mobilized CD34+ cells. Of note, EMR1, GIMAP8, PIM1, S100A8, SOCS3, and TMEM49 were expressed more abundantly in all GCSF-mobilized CD34+ cells while BCL-2, CLC, CXCR4, C200rf118, DNTT, IRF8, PRG2, RASD1, RNASE6, and UHRF1 were more abundantly expressed in all AMD3100-mobilized CD34+ cells. Interestingly, the RNA profile of CD34+ HSCs obtained from the BM of three healthy donors clustered with AMD3100-mobilized CD34+ HSCs rather than GCSF mobilized HSCs. Using flow cytometry, we identified a CD34dimCD45RA+ hematopoietic precursor cell that is uniquely enriched in nearly 60% of the AMD3100 products evaluated to date (6/10 patients). In contrast to G-CSF mobilized products, where

Description: Abstract 32 Background: Plerixafor (Mozobil®) is a CXCR4 antagonist that was recently approved by the Food and Drug Administration for use in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSCs) in patients with non-Hodgkin's lymphoma and multiple myeloma undergoing autologous transplantation. As part of our ongoing clinical evaluation of plerixafor, we observed that the drug efficiently mobilized a CD34dim population of HSCs. Here, we characterize the CD34dim population and assess its relative frequency following mobilization with G-CSF and/or plerixafor in apheresis samples and in non-mobilized cord blood samples. Methods: Aliquots of apheresis products were obtained from 16 healthy donors following treatment with a single injection of plerixafor or 5 days of G-CSF (10 μg/kg/day). In a separate study, five patients with lymphoma were mobilized sequentially with plerixafor (240 μg/kg), G-CSF (10 μg/kg/day), and plerixafor + G-CSF. These patients received an injection of plerixafor on day −5. Twenty-four hours later, patients were treated with G-CSF for 4 days. On the fifth day (day 0), patients received a dose of G-CSF and a dose of plerixafor followed four hours later by apheresis. Samples were collected after treatment with plerixafor on day −5 as well as immediately before and 4 hrs after plerixafor administration on day 0. Cord blood units were obtained from the St. Louis Cord Blood Bank. Human CD34+ cells were purified by positive selection with a Magnetic Affinity Cell Selection CD34 isolation kit. We used Affymetrix Human Genome U133+2 arrays to generate gene expression profiles for 24 samples of CD34+ cells collected following mobilization of healthy donors with either Plerixafor (n=12) or G-CSF (n=12). Results: Human CD34+ cells can be divided into three distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Rα): (i.) CD34+CD45RA−CD123+/− primitive HSCs, (ii.) CD34+CD45RA+CD123+/− committed progenitors, and (iii.) CD34dimCD45RA+CD123hi cells. Table 1 shows the relative frequencies of each of these CD34+ cell subsets in apheresis products obtained from healthy donors mobilized with G-CSF or plerixafor as well as in cord blood units. Strikingly, we observed that each graft type was significantly enriched for one of the CD34+ cell subsets compared to the other two grafts. G-CSF mobilized grafts contained more CD45RA−CD123+/− primitive HSCs, cord blood units were enriched with CD45RA+CD123+/− committed progenitors and plerixafor mobilized products had significantly more CD34dimCD45RA+CD123hi cells. Table 1 also shows the CD34 cell subset distribution following sequential mobilization of lymphoma donors with plerixafor, G-CSF, and plerixafor + G-CSF (PL+G). This data agrees with the results we obtained from healthy donors and confirms that G-CSF grafts are enriched with CD34+CD45RA−CD123+/− cells while plerixafor preferentially mobilizes the CD34dimCD45RA+CD123hi subset. Extensive FACS and functional analyses determined that the CD34dim population represents a plasmacytoid pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their CD45RA+CD123hiBDCA−2+BDCA−4+CD36+CXCR4hiCD4dimCD25−CD13− phenotype and inability to form CFU-GM colonies in vitro. Finally, we found that the gene signature of plerixafor-mobilized CD34+ cells is distinct from that of G-CSF-mobilized CD34+ cells. Plerixafor-mobilized CD34+ cells expressed significantly more of the specific transcriptional regulator of plasmacytoid DC (pDC) development, E2-2, as well as additional transcriptional factors (SpiB, IRF7, IRF8) and cell surface markers (BDCA-2, ILT7) that are specific to the pDC lineage. Conclusions: This data suggest that the CD34dimCD45RA+CD123hi cells preferentially mobilized by plerixafor are precursor pDCs. Further study is required to determine the impact these cells have on the engraftment and function of plerixafor mobilized grafts after hematopoietic stem cell transplantation. Disclosures: DiPersio: Genzyme Corp.: Honoraria.

Description: Abstract 787 The interaction between leukemic blasts and the bone marrow microenvironment is postulated to be an important mediator of chemoresistance in AML. Although many candidate receptor/ligand pairs have been implicated, the CXCR4 / SDF-1 axis functions as the principal regulator of homing and retention of both normal and malignant hematopoietic cells in the marrow. We hypothesized that disruption of the CXCR4 / SDF-1 axis with plerixafor (Mozobil®), a small molecule inhibitor of CXCR4, may sensitize AML to the effects of chemotherapy. We conducted an open label, phase I/II study in patients with relapsed or refractory AML in which plerixafor was administered prior to salvage chemotherapy. A test dose of plerixafor was administered SQ followed by a 24 hour observation period to analyze its effects on AML blasts in the absence of chemotherapy. Plerixafor was then given 4 hours prior to MEC chemotherapy (mitoxantrone 8 mg/m2/d, etoposide 100 mg/m2/d and cytarabine 1,000 mg/m2/d) daily for 5 days. Forty pts have been enrolled in the study with median age of 49 yrs (range 19-71). Baseline characteristics include 6 pts (15%) with secondary AML, 4 (10%) with prior transplant, 24 (60%) with intermediate and 10 (25%) with poor risk cytogenetics. Thirty-six pts (90%) received plerixafor + MEC as their 1st salvage regimen for relapsed disease with 21 (53%) having a CR1 duration of 〈 12 months and 9 pts (6%) for primary refractory disease. The remaining four pts (10%) received the regimen as their 2nd salvage regimen. Three dose levels of plerixafor: 80 mcg/kg, 160 mcg/kg and 240 mcg/kg were tested in the phase I dose escalation. In the phase II, a total of 34 patients have been treated at the 240 mcg/kg dose level. Common grade ≥ 3 adverse events consisted primarily of cytopenias and infections. No evidence of hyperleukocytosis or significant delays in neutrophil recovery (ANC 〉500/mm3, median 27d, range 21-37) or platelet recovery (plt 〉50k/mm3, median 26d, range 20-40d) were observed. Of the 32 pts currently evaluable for response at the 240 mcg/kg dose level, a complete remission (CR+CRi) has been achieved in 50% of pts (CR=13, CRi=3) which compares favorably to historical CR rates of 25-35%. Treatment failure was due to persistent disease in 14 pts (44%) and early death due to complications from infection in 2 pts (6%). One year KM estimate of overall survival is currently 56%. Correlative studies demonstrate that plerixafor mobilizes AML blasts (mean 2.5-fold increase, range 0.9-7.3 fold) into the peripheral circulation peaking at 6-8 hours after administration. FISH performed in pts with informative cytogenetic abnormalities indicates that mobilization occurs equally in both non-leukemic and leukemic populations. Higher baseline surface CXCR4 expression correlated with increased mobilization of AML blasts (Pearson's r=0.53, p=0.023) into the PB at 6 hrs post-plerixafor. In addition, a strong correlation was also observed between baseline CXCR4 expression and % SDF-1 migration in transwell assays (r=0.84, p=0.0013). Furthermore analysis of AML PB blasts at 6 hrs post-plerixafor demonstrate increased CXCR4 expression as well as increased chemotaxis in response to an SDF-1 gradient in transwell assays compared to baseline (64% vs 38%, p=0.0006). We conclude that plerixafor can be safely administered in combination with cytotoxic chemotherapy in patients with AML. Based on encouraging preliminary evidence of efficacy and in vivo evidence of CXCR4/SDF-1 blockade, confirmatory randomized studies of plerixafor for chemosensitization in AML are being planned. Disclosures: Uy: Genzyme: Consultancy, Speakers Bureau. Off Label Use: Plerixafor for AML. Abboud:Genzyme: Consultancy. Vij:Genzyme: Consultancy. DiPersio:Genzyme: Consultancy, Honoraria.

Description: Background: Mobilization of hematopoietic stem cells (HSCs) in mice and man can be induced using G-CSF (G) or by the use of chemokines and chemokine receptor antagonists. Recent preclinical and clinical data using the bicyclam AMD3100 (A) suggests that the combination of G+A results in significantly improved yields of HSCs compared to G alone in both mice and man (Blood.2005;106:1867). In humans, optimal mobilization of HSCs occurs 6–9 h after subcutaneous (s.c.) A. These mobilization kinetics necessitate the somewhat undesirable administration of A the evening before the first day of apheresis. Methods: We assessed the kinetics of HSC mobilization in mice and man after IV or s.c. administration of A. Human donors were initially mobilized with increasing doses of IV A (80, 160, 240 or 320 μg/kg). After 4 days of drug clearance, the same donors were then mobilized with a single s.c. dose of A (240 μg/kg) and collected cells were used as a source of HSCs for HLA-matched sibling recipients undergoing single dose (550 cGy) TBI and cytoxan allogeneic HSC transplantation (Devine et. al. Blood. 2006;108:53). Results: Peak mobilization of mouse CFU-GM occurred 3 h after s.c A (5 mg/kg; 15–18-fold; n=5) and 0.5–1 h after IV A (1–3 μg/kg; 10–12-fold; n=3). Identical CFU-GM kinetics were observed after mice were first given G (250 μg/kg) for 4 days then administered either s.c. A (250–500 fold in 3 h; n=3) or IV A (170–350 fold n=3). To date, 6 normal HLA-matched sib donors have been treated with 80 μg/kg (n=3) or 160 μg/kg (n=3) IV A over 30 minutes followed 4 days later by 240 μg/kg s.c. A. Peak mobilization of CD34+ cells occurred 1–4 h after 80 μg/kg IV A, 4–6 h after 160 μg/kg IV A, and 9 h after s.c. A. The more rapid kinetics of CD34 mobilization after IV A is in contrast to the kinetics of s.c. A in autologous transplant patients receiving both G and A (J Clin. Oncol.2004;22:1095) and in normal volunteers receiving A alone (∼9 hours; Blood. 2003;102:2728). This data suggests that IV A not only mobilizes HSCs from mouse and man more quickly than s.c. A, but also, that the peak of mobilization may be enhanced (6.4-fold at 160 uμg/kg IV vs. 5.2-fold for 240 μg/kg s.c.) and prolonged (5.7 fold at 160 μg/kg IV vs. 3.8 fold for 240 μg/kg s.c. at 6 h post-A) after IV A. IV A also induced: (i.) a more rapid and greater peak of CD19+ B cell mobilization (6-fold at 1 h and 6.7-fold at 4 h) than s.c. A (2.2-fold at 6 h), (ii.) a modest increase in CD4+ and CD8+ CD3+ T cell mobilization (3.2-fold vs. 1.1-fold for sc A at 6h) and (iii.) no significant changes in Treg, NK, CMV-specific CD8, and γδ T cells. To identify genes that are differentially expressed following G or A mobilization, we performed RNA profiling analyses using Affymatrix U133+2 arrays and RNA isolated from purified (〉95%) CD34+ HSCs obtained from 5 individual normal donors mobilized sequentially with A and G. Of note, CXCR4, CCR9, ALCAM, ICAM, and CEECAM1 were expressed more abundantly in all A mobilized CD34+ cells while mucin, Integrin α 6, Integrin α 2b, and CEACAM1 were more abundantly expressed in all G-mobilized CD34+ cells. Conclusions: IV A results in more rapid and prolonged mobilization of mouse HSC and human CD34+ cells compared to s.c. A. IV A was not associated with any adverse events in the 6 donors mobilized at the first two IV dose levels. The enhanced and more rapid mobilization of CD34+ cells by IV A may have significant applications for the optimal collection of peripheral blood HSC products.

Description: Background: The mechanisms by which AML cells and other hematologic malignancies interact with the BM microenvironment to provide a chemoprotective effect remain largely unknown. Our hypothesis is that resistance and relapse of AML may be related to altered AML-BM niche interactions, and that interruption of these interactions might enhance chemosensitivity thus overcoming chemotherapy resistance. In the current studies we have generated an in vitro model of chemotherapy protection mediated by a bone marrow (BM) stromal cell line and attempted to determine soluble and cell associated factors that mediate this protection against genotoxic stresses such as chemotherapy.sought to answer if murine stromal cells provide chemoprotection to mCGPR/+ APL cells in vitro. Methods: APL cells generated from mice in which a single copy of the human PML-RARa was knocked into the murine cathepsin G locus (mCGPR/+; Westervelt et al, Blood. 2003 Sep 1;102(5):1857-65) were cultured in 12-well plates (normal or 0.4 μm pore transwell) with or without murine M2-10B4 stromal cells. After 24 h, APL cells were left untreated or treated with AraC (40 mg/ml) or daunorubicin (DNR; 40 ng/ml) for 2 days. FACS was used to test APL cell viability, cell cycle status, and proliferation kinetics using annexin V/PI, acridine orange, and CFSE assays, respectively. Results: We observed a significant survival benefit for mCGPR/+ APL cells co-cultured with M2-10B4 stromal cells (Table 1). This survival advantage was observed in both the absence (untreated) and presence of chemotherapeutic agents. Optimal protection against both chemotherapy agents (95% daunorubicin and 92% Ara-C) was seen when APL cells were in direct contact with stromal cells. Interestingly, the survival benefit afforded by the stromal cells was largely maintained following separation of the APL cells from the M20-10B4 cells by a 0.4 um transwell (Table 1). This observation suggests that the anti-apoptotic stimulus provided by the M2-10B4 cells was mediated, at least in part, by a soluble factor released from the stromal cells. To begin to identify candidate molecules that might mediate this anti-apoptotic effect, we harvested media from wells incubated 72 h in the presence or absence of M2-10B4 cells and measured the levels of 64 different molecules using a luminex bead assay. The five abundant molecules detected in this initial screen were VCAM-1 (22-fold), SCF (29-fold), VEGF (747-fold), MCP-1 (979-fold), and MCP-3 (907-fold). It should be noted that the APL binding to stroma was not associated with alterations of cell cycle (except increase in G1a) nor decreased proliferation kinetics (no change in CFSE striping profiles relative to untreated cells). Conclusion: These results demonstrate that murine stromal cells confer a chemotherapy survival advantage for mCGPR/+ APL cells in vitro. Furthermore, antibodies and small molecules (such as mobilizing agents) that interrupt direct binding of leukemic cells to the BM microenvironment or block signaling pathways mediated by physical attachment or anti-apoptotic soluble stromal factors may be rationally used to sensitize leukemia cells to chemotherapy in vivo. Table 1. M2-10B4 stromal cells provide a survival advantage to mCG PR/+ APL cells % Apoptotic cells(AnnexinV+/PI-) Untreated Ara-C (40ng/mL) DNR (40ng/mL) No stroma 27% (SD 1.3) 80.6% (SD 1.9) 80% (SD 2.2) Transwell 4.3% (SD 4.1) 12.2% (SD 0.2) 10.1% (SD 2.8) Stroma 3.3% (SD 0.9) 8.2% (SD 0.9) 5.3% (SD 0.3)

Description: Background: Hematopoietic stem cells (HSC) interact with stromal cells, osteoblasts and matrix proteins in the hematopoietic niche. This interaction plays an important role in HSC trafficking, proliferation and differentiation. Significant data support the roles of both the SDF-1/CXCR4 and the VCAM1/VLA-4 axes in HSC homing and mobilization. Little is known regarding the kinetics and effect of combining small molecule inhibitors of CXCR4 and VLA-4 on normal HSC and APL mobilization. Here we examine this question and assess the role that the spleen plays in HSC and APL mobilization and in the progression of APL cells in vivo. Methods: To evaluate mobilization of HSC progenitors, normal and splenectomized 8 week old 129/B6 F1 mice were treated with AMD3100 5 mg/kg sc and/or a small molecule inhibitor of VLA-4 (AMD15057 1mg/kg iv). Mobilization of APL cells was performed using the same doses and schedules of AMD3100 and/or AMD15057 by first injecting normal and splenectomized 8 week old 129/B6 mice iv with 106 banked APL cells from leukemic mice in which a single copy of PML-RARa was inserted into the murine cathepsin G locus (Westervelt, Blood. 2003;102:1857–65). These cells were transfected with murine oncoretroviruses carrying a CBR-luciferase-eGFP fusion gene to allow tracking by both bioluminescence (BLI) and FACS. Mobilization of APL cells was assessed 10 days after injection of normal mice and 12 days after injection of splenectomized mice when 1–2% of the peripheral blood of syngeneic recipients was leukemic as measured by FACS for the characteristic phenotypic markers of murine APL (CD34+/GR-1+). Engraftment and expansion of APL in normal and splenectomized mice was assessed by WBC, BLI and overall survival. Results: A single sc injection of AMD3100 or iv injection of AMD15057 resulted in maximum mobilization of HSC and APL in 3 hours in normal mice, and in 1–3 hours in splenectomized mice. Dramatic synergism of both normal HSC (130 fold over baseline as measured by CFU-GM/mL of peripheral blood) and APL (22 fold over baseline) mobilization was seen only when AMD3100 and AMD15057 were co-administered to normal and not splenectomized mice. Furthermore, the magnitude of AMD3100 or combination-induced mobilization of HSC and APL was dramatically greater from normal vs splenectomized mice (3.0 to 11 fold, CFU-GM) suggesting that the mouse spleen (vascular niche) represents the major site of both normal HSC and APL mobilization by AMD3100. In contrast, similar mobilization of both HSC and APL was seen in normal and splenectomized mice after treatment with AMD15057 suggesting that the marrow (osteoblastic niche) is the major site for mobilization after VLA4 blockade. After injection of 106 APL cells into syngeneic recipients, unsplenectomized controls compared to splenectomized mice exhibited: decreased overall leukemia burden (as measured by whole body BLI), increased expansion of APLLuc cells in the BM (femurs), and increased overall survival (median survival time increased from 14d to 22d; P=0.0003). Conclusions: Rapid mobilization of both normal progenitors and leukemia cells can be induced in vivo by AMD3100 and AMD15057. Co-administration of CXCR4 and VLA4 small molecule antagonists have a synergistic effect on the rapid mobilization of both HSC and APL from the vascular and osteoblastic niches, respectively. These results may have important clinical implications in patients undergoing chemotherapy for acute leukemia and stem cell transplantation.

Description: CXCR4/SDF-1 axis regulates the trafficking of normal stem cells to and from the bone marrow (BM) microenvironment. SDF-1 is a chemokine widely expressed by many tissues especially BM stromal cells and osteoblasts. AMD3100 (AMD) is a novel bicyclam molecule that is a competitive inhibitor of SDF-1/CXCR4 binding and has been used to enhance stem cell mobilization when combined with G-CSF in mouse, dog and man. We are interested in evaluating whether leukemic cells “mobilize” similar to normal stem cells after treatment with AMD, and if so, whether this mobilization increases the efficacy of chemotherapy. Therefore, we utilized a mouse model of human acute promyelocytic leukemia (APL) in which the PML-RARα transgene was knocked into a single allele of the murine cathepsin G locus. To more efficiently track the leukemic cells, we transduced banked APL tumors with a dual function reporter gene that encodes a fusion protein comprised of click beetle red (CBR) luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (CBR/EGFP). We generated large numbers of CBR/EGFP+ APL cells by isolating EGFP+ cells using a MoFlo cell sorter, and passaging them in secondary syngeneic recipients. Importantly, the secondary recipients developed a rapidly fatal acute leukemia after intravenously (iv) or intraperitoneal injection, which displayed an APL phenotype (CD34/GR1 co-expression) and exhibited luciferase activity. Upon iv injection into syngeneic recipients, the CBR/EGFP+ APL cells rapidly migrated to the BM microenvironment, as evidenced by the significantly increased BLI signal in the femurs, spine, ribs, and skull of recipients at 4 days after injection. Over the next 2–3 days the CBR/EGFP+ cells migrated to the spleen followed rapidly by widespread dissemination and death due to leukostasis by 14–16 days. To our knowledge, this represents the only mouse leukemia model in which leukemia cells home preferentially to the BM microenvironment in a manner that is similar to what is seen in human AML. Therefore, we used this model to study the effect of AMD on the “mobilization” of APL cells into the peripheral blood (PB) and on their sensitivity to chemotherapeutic agents that are known to affect the proliferation of these cells. Surprisingly, injection of AMD (5 mg/kg) immediately at the time of APL infusion had no impact on the engraftment (short term or long term) of either normal BM stem cells or the leukemic cells. However, we observed rapid mobilization of the leukemic cells when AMD was administered 11 days after APL injection. In fact, 40% of mice that received a single dose of AMD on day +11 after APL injection died 2 to 4 hours after AMD injection as a result of the rapid and massive mobilization of blasts. Overall, we found that AMD treatment on day +11 induced a 3-fold increase in total WBC counts with a 10-fold increase in the leukemic blasts into PB. Interestingly, the administration of AMD concomitant with cytarabine (AraC) (200 mg/kg) on day +11 significantly prolonged the overall survival of mice, compared with mice treated only with AraC. In summary, we developed a mouse model to study the APL cell trafficking, and we have shown leukemia cell mobilize from the BM into PB after AMD administration. We propose that CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. In these preliminary results, we observed that AMD sensitizes APL cells to AraC.

Description: The CXCR4-SDF-1 axis possesses a central role in the trafficking and retention of both normal and malignant stem cells in the bone marrow. Previous work from our laboratory established that in a murine model, a single dose of the CXCR4 antagonist, AMD3100, sensitizes AML blasts to chemotherapy supporting the premise that the interaction between AML blasts and the marrow microenvironment confers resistance to genotoxic stress (Nervi et al., ASH 2006). Here we examine the effects of repetitive dosing of AMD3100 on the kinetics of normal and leukemic mobilization. Following SQ injection of AMD3100 5mg/kg into B6/129 F1 mice daily for 5 days (n=8), we observed a 2.4 fold increase in total leukocyte counts with a 12.4 increase in CFU-GM when compared to 3 hours post injection (Fig 1A). No differences were seen in the degree of mobilization between d1 and d5 with WBC and CFU-GM counts returning to baseline after 24 hours. We next tested repetitive doses of AMD3100 in our mouse model of AML in which 106 blasts derived from leukemic mice carrying the PML-RARα fusion gene in the murine cathepsin G locus are adoptively transferred into genetically compatible secondary recipients. AMD3100 at 5mg/kg was then administered to these AML mice for 4 consecutive days. At 3 hrs post AMD3100 injection, we observed a 1.8 fold increase in peripheral leukocyte counts with a 4.5 fold increase in circulating blasts compared to baseline (n=3). Again, no significant differences are seen in the degree of mobilization from d1 to d4 (Fig 1B). Based on these preclinical data, we have initiated a phase I/II trial of AMD3100 plus mitoxantrone, etoposide and cytarabine (MEC) in relapsed or refractory AML in which AMD3100 is administered 4 hours prior to MEC daily for 5 consecutive days. To study the kinetics of human AML mobilization, we administered AMD3100 by SQ injection followed by 24hr observation period prior to chemotherapy. Two patients have been treated at the first dose level of AMD3100, 80 μg/kg. In pt #1 following AMD3100 mobilization, total WBC increased from 3 × 103/mm3 to a peak of 17 × 103/mm3 at 6 hours post-AMD3100 representing a 5.7 fold increase in total white count (Fig 2). In addition, the blasts (CD45dim, SSlow) increased by 7.3 fold. Similarly in pt #2, we observed a 2 fold increase in the total WBC from 2.5 to 5.1 × 103/mm3 with a 2.3 fold increase in blasts (CD45dim, SSlow). Mobilization of AML was confirmed in both patients through informative FISH for 11q23 (MLL). No adverse events have been observed during mobilization. These data provide the preclinical rationale for repetitive dosing of AMD3100 and direct clinical evidence that AMD3100 mobilizes human AML blasts into the peripheral circulation. Our trial of AMD3100 plus MEC in relapsed or refractory AML is ongoing. Figure 1. AMD3100 induced mobilization of (A) normal progenitors and (B) AML blasts Figure 1. AMD3100 induced mobilization of (A) normal progenitors and (B) AML blasts Figure 2. AMD3100 mobilization of human AML Figure 2. AMD3100 mobilization of human AML

Description: Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and interact with stroma cells and extracellular matrix. CXCR4/SDF-1 axis regulates the trafficking of HSC to and from the BM. We utilized a PML-RARα knock-in mouse model of human acute promyelocytic leukemia (APL) to study APL interaction with the normal BM. We have previously shown there is a rapid mobilization of APL cells from the BM into peripheral blood (PB) after administration of AMD3100, a competitive inhibitor of CXCR4. We hypothesize that we can sensitize these tumor cells to chemotherapy by interrupting the interaction between APL and the BM stroma. We transduced banked APL cells with a dual function reporter gene that encodes a fusion protein comprised of Click Beetle Red luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (Luc/EGFP). Upon iv injection into genetically compatible recipients (F1 129/B6 mice), APL rapidly migrated to the BM with increased BLI signal in the femurs, spine, ribs, and skull, at 4 days after injection, followed by spleen infiltration and death due to leukostasis by day 15. 129/B6 F1 mice (n=28) were injected iv with 106 APL cells. By day 12 all mice had ±5% APL cells in PB. 8 mice received AraC (500mg/kg/sq) on days 12 and 13, and another 8 mice received AraC+AMD (5mg/kg/sq) 1 hour before and 3 hours after each AraC injection. 6 mice received only AMD and 6 control mice were observed. Total body BLI signal, WBC, and blasts per μl of blood on days 19 and 23 were higher in AraC versus AraC+AMD (p