Mobilization and Chemosensitization of AML with the CXCR4 Antagonist Plerixafor (AMD3100): A Phase I/II Study of AMD3100+MEC in Patients with Relapsed or Refractory Disease.

Abstract The interaction of leukemic blasts with the bone marrow microenvironment is postulated to be an important mediator of chemoresistance in AML. Although a number of 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. Plerixafor (AMD3100) is a bicyclam molecule which reversibly blocks CXCR4 binding to SDF-1 and is being developed clinically as a mobilization agent for hematopoietic stem cell transplantation. Preclinical data from our group has demonstrated that in murine models, plerixafor can disrupt the interaction of leukemic cells with the marrow microenvironment and sensitize blasts to the effect of chemotherapy. Based on these data, we have initiated a phase I/II study in patients with relapsed or refractory AML in which plerixafor is administered prior to salvage chemotherapy. Subjects were required to have AML which is primary refractory to at least 2 induction regimens, in 1st relapse with an initial remission duration of < 12 months, in 1st relapse having failed ≥ 1 salvage regimens, or in 2nd relapse or higher. Plerixafor is administered by SQ injection followed by a 24 hour observation period to analyze its effects on leukemic cell mobilization. Then plerixafor is given daily 4 hours prior to chemotherapy consisting of mitoxantrone 8mg/m2/d, etoposide 100 mg/m2/d and cytarabine 1000 mg/m2/d x 5 days. To date, 19 patients have been treated at 3 dose levels of plerixafor: 80, 160 and 240 mcg/kg/day. We find that plerixafor can modestly mobilize leukemic cells (~ 2-fold increase) into the peripheral circulation at a peak of 6–8 hours after administration. FISH studies performed from informative samples demonstrates that this mobilization occurs equally in both non-leukemic and leukemic populations. While CXCR4 expression is increased on the surface of mobilized blasts, no clear relationship has been observed between CXCR4 expression or plerixafor dose and mobilization. At the 80 and 160 mcg/kg dose levels, a complete response (CR+CRi) was observed in 2 of 6 patients (33%). At the plerixafor 240 mcg/kg dose level, a complete response (CR+CRi) was achieved in 6 of 8 evaluable patients (75%) in this historically chemorefractory population. Plerixafor was well tolerated with no evidence of hyperleukocytosis or significant delays in neutrophil recovery (median 30 days, range 24–40). Based on this encouraging evidence of safety and efficacy, expansion of a phase II cohort is ongoing.

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Phase I study of temozolomide in children and adolescents with recurrent solid tumors: a report from the Children's Cancer Group.

Journal of Clinical Oncology ◽

10.1200/jco.1998.16.9.3037 ◽

1998 ◽

Vol 16 (9) ◽

pp. 3037-3043 ◽

Cited By ~ 70

Author(s):

H S Nicholson ◽

M Krailo ◽

M M Ames ◽

N L Seibel ◽

J M Reid ◽

...

Keyword(s):

Phase I ◽

Children And Adolescents ◽

Complete Response ◽

Maximum Tolerated Dose ◽

Hematologic Toxicity ◽

Cancer Group ◽

Grade 3 ◽

Dose Levels ◽

Dose Limiting Toxicity

PURPOSE The Children's Cancer Group conducted a phase I trial of temozolomide stratified by prior craniospinal irradiation (CSI). PATIENTS AND METHODS Children and adolescents with recurrent or progressive cancer were enrolled. Temozolomide was administered orally daily for 5 days, with subsequent courses administered every 21 to 28 days after full hematologic recovery. Dose levels tested included 100, 150, 180, 215, 245, and 260 mg/m2 daily. RESULTS Twenty-seven patients on the non-CSI stratum were assessable for hematologic toxicity. During the first three dose levels (100, 150, and 180 mg/m2 daily), only grades 1 and 2 hematologic toxicity occurred. One patient at 215 mg/m2 daily had grade 3 hematologic toxicity. Three of eight patients (38%) treated at 245 to 260 mg/m2 daily had dose-limiting toxicity (DLT), which included both neutropenia and thrombocytopenia. Twenty-two patients on the CSI stratum were assessable for hematologic toxicity. Hematologic DLT occurred in one of six patients (17%) at 100 mg/m2 daily and in two of four patients (50%) at 215 mg/m2 daily. No nonhematologic DLT occurred; nausea and vomiting occurred in more than half of the patients. After two courses of temozolomide, 10 patients had stable disease (SD), and three patients had a partial response (PR), one of whom subsequently had a complete response (CR) that persists through 24 months of follow-up. CONCLUSION The maximum-tolerated dose (MTD) of temozolomide for children and adolescents without prior CSI is 215 mg/m2 daily and for those with prior CSI is 180 mg/m2 daily for 5 days, with subsequent courses that begin on day 28. Temozolomide is well tolerated and should undergo phase II testing in children and adolescents.

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Phase I Study for Poor Prognosis Lymphoma: Modification of the “BEAM” (BCNU +Etoposide+ARA-C+Melphalan) Regimen with Escalating-Dose Melphalan (MEL) Using Amifostine (AF) Cytoprotection and Autologous Hematopoietic Stem Cell Transplantation (AHSCT).

Blood ◽

10.1182/blood.v108.11.3076.3076 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3076-3076

Author(s):

Gordon L. Phillips ◽

Camille N. Abboud ◽

Steven H. Bernstein ◽

Jonathan W. Friedberg ◽

Michael W. Becker ◽

...

Keyword(s):

Phase I ◽

Maximum Tolerated Dose ◽

High Dose ◽

Hematopoietic Stem ◽

Hematopoietic Reconstitution ◽

Conditioning Regimens ◽

Artery Disease ◽

D 12 ◽

Dose Levels ◽

Pet Scans

Abstract Background: AHSCT is standard therapy for recurrent or progressive, yet chemosensitive lymphoma, both non-Hodgkin’s (NHL) and Hodgkin’s (HL). However, relapse is frequent, and augmentation of existing conditioning regimens (e.g. BEAM) is one approach to reducing these relapses. Since BEAM has considerable regimen related toxicity (RRT), methods to allow safe dose augmentation are needed. Prior experience (BBMT2004;10(7): 473–83) indicates that AF may be useful in protecting vs RRT produced by high-dose MEL used alone; thus, we postulated a cytoprotective effect in this situation as well. Goals: Determine the maximum tolerated dose (MTD) of escalated dose MEL in BEAM, using AF and AHSCT, in a classical Phase I trial. Methods: We utilized AF 740mg/m2 daily before and during BEAM (i.e., −7 to −1) with MEL, starting at 140 mg/m2 (level A) and escalating by 20 mg/m2 per 4 - pt. cohorts. AHSCT and other supportive care measures were routine. (Thiotepa was used in patients with a history or at high risk of central nervous system (CNS) disease.) The Berman (JCO1989; 7(9): 1288–94) scale of grading RRT was used to determine the MTD in 4- pt. cohorts; those who died of non-RRT causes were deemed non-evaluable and replaced. Results: Between 07/30/2003 and 06/28/2006, we entered 18 patients (NHL13 /HL 5), med age 60 (range 23 to 72) years. All had progressed or relapsed; 12 were chemosensitive. To date, 6 patients at level A (140mg/m2), 4 patients at B (160mg/m2), 5 patients at C (180mg/m2) and 3 patients at D (200mg/m2) have been evaluated >D +30. Three patients (2 at level A and 1 at level C) were non-evaluable due to: Removal (pt’s request) during BEAM, Death due to CNS bleeding; and MI (with known pre-existing coronary artery disease), and were replaced. None of the remaining 15 had Bearman RRT >II; 2 had none and 10 had only grade I RRT. All (save one case of hepatic II RRT) were stomatitis and/or gastrointestinal. All had CD34+ > 2.0 x 10e6/kg and prompt hematopoietic reconstitution: ANC >0.5 and platelets >20K at median D+ 11 (range +9 to +12) and D+ 12 (range +9 to +24), respectively. No late (i.e., >D+ 30) hematologic or non-hematologic toxicities were noted. At present, 11 patients are alive, 7 in CR, one too early to evaluate at median D+ 469, range + 27 to 999. Conversely, 7 are dead, due to relapse (5), MI (one), and CNS bleeding (1). Responses occurred at all dose levels. In evaluable patients < CR before AHSCT, 9/14 achieved CR (mostly confirmed by imaging studies, notably PET scans) by D +100; 6 remain in CR at median D +593, range +36 to +614, including one patient who had a “consolidation” allogeneic HSCT. Another patient in CR at AHSCT remains in CR at D +514. Conclusion: Although not strictly proven, we believe the use of AF allows the safe use of escalated doses of MEL in the BEAM regimen >/= 180mg/m2. Dose escalation will continue until a MTD is found.

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Multi-Agent Phase I Trials in Childhood Acute Lymphoblastic Leukemia (ALL).

Blood ◽

10.1182/blood.v108.11.4528.4528 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4528-4528

Author(s):

Richard Sposto ◽

Elizabeth A. Raetz ◽

Charles P. Reynolds ◽

Paul S. Gaynon

Keyword(s):

Phase I ◽

Phase I Trial ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Complete Response ◽

Maximum Tolerated Dose ◽

Cell Transplant ◽

Phase I Trials ◽

Hematopoietic Stem ◽

The Impact

Abstract Background: Single agent phase I trials with conventional methodology may not be suitable for children with relapsed leukemia. Accrual of children with ALL in relapse to single-agent phase I trials is poor due to clinical urgency and a > 30% likelihood of complete response (CR) with a variety conventional agents combinations (Br J Haematol.2005; 131(5): 579) with the option of hematopoietic stem cell transplant in remission. As most drugs are ultimately used in combination, a Phase I trial testing a new agent in combination with conventional agents would seem most useful and might increase accrual. However, with conventional phase I methodologies determination of a maximum tolerated dose is complicated by the toxicities of the accompanying conventional agents and by the background morbidity of relapsed leukemia. Methods: The Children’s Oncology Group (COG) study, AALL01P2, employed vincristine, prednisone, doxorubicin, and pegylated asparaginase for children with ALL in first marrow relapse. We determined the incidence of conventional non-hematologic dose limiting toxicities (DLT’s) and modeled the impact on a hypothetical phase I trial of a candidate agent with no additional toxicity. Results: Among 111 patients on AALL01P2, 19% had conventional non-hematologic DLT’s. Induction therapy was judged clinically acceptable. With a traditional Phase I escalation scheme that accepts 0/3 and 1/6 DLT’s at a dose-level and rejects 2/3 and 2/6 DLT’s, an agent that adds no morbidity would be rejected as too toxic at any dose 30% of the time. Conclusion: Background morbidity confounds identification of an acceptable dose of a non-toxic new agent tested in combination with conventional drugs for recurrent ALL. We propose a modification to the traditional Phase I design that increases the DLT thresholds to 1/3 and 2/6, which effectively compensates for background toxicity and reduces the chance of falsely rejecting an acceptable agent.

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Kinetics of Human and Murine Mobilization of Acute Myeloid Leukemia in Response to AMD3100.

Blood ◽

10.1182/blood.v110.11.867.867 ◽

2007 ◽

Vol 110 (11) ◽

pp. 867-867 ◽

Cited By ~ 3

Author(s):

Geoffrey L. Uy ◽

Michael P. Rettig ◽

Pablo Ramirez ◽

Bruno Nervi ◽

Camille N. Abboud ◽

...

Keyword(s):

Fusion Gene ◽

Fold Increase ◽

Genotoxic Stress ◽

Peripheral Circulation ◽

Cathepsin G ◽

Cxcr4 Antagonist ◽

Leukocyte Counts ◽

Kinetics Of ◽

Refractory Aml ◽

Observation Period

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

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HERG K+ Channel Is Essential for Leukemic Cell Migration Induced by SDF-1.

Blood ◽

10.1182/blood.v112.11.1637.1637 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1637-1637

Author(s):

Huiyu Li ◽

Yi-Mei Du ◽

Linlin Guo ◽

Tiannan Guo ◽

Shenghua Jie ◽

...

Keyword(s):

Stem Cells ◽

Cell Migration ◽

Leukemic Cell ◽

Cxcr4 Expression ◽

Leukemic Cells ◽

K Channel ◽

Leukemic Stem Cells ◽

Lower Chamber ◽

Herg Current ◽

K Current

Abstract Background: Recent studies suggest that HERG K+ channel is an important regulator of non excitable cell proliferation and migration, and has been found in tumor cells including acute myeloid leukemia(AML), where HERG K+ channel is generally considered to be absent from their healthy counterparts. Bone marrow stromal cells constitutively secrete the stromal cell-derived factor-1 (SDF-1) which is a homeostatic chemokine that signals through CXCR4, SDF-1/CXCR4 axis and plays an important role in hematopoiesis development and leukemic cells migration. In this study, we investigated whether SDF-1-induced leukemic cell migration associated with HERG K+ channel. Methods: primary CD34+/CD38− leukemic stem cells (LSCs) were isolated by cell sorting using a FACS Vantage. Transwell was used to assess the effect of E-4031, a specific HERG K+ channel inhibitor, on leukemic cell migration, the lower chamber was filled with serum-free RPMI-1640 with 100ng/ml SDF-1. Flow cytometry was used to analyze the CXCR4 expression as well as phenotypical analysis of leukemia samples. HERG K+ channels were expressed in Xenopus oocyte by microinjection and the resulting currents were measured using the standard two microelectrode voltage clamp techniques. Results: numbers of HL-60 cells with and without E-4031 treatment migrated towards SDF-1 in the lower chamber were 1.58±0.98 ×104 and 3.47±0.81 ×104 respectively, indicating E-4031 significantly blocked the cell migration induced by SDF-1. The similar results were also observed in primary leukemic cells (n=7) and leukemic stem cells(n=3). From a holding potential of −80 mV varying potentials from −70 mV to +50 mV in 10 mV increments (2s) were applied to elicit activating currents. Each pulse was followed by a constant return pulse to −50 mV (2s) to evoke outward tail currents. 100 ng/ml SDF-1 increased HERG K+ current expressed in oocytes, for example, at +50 mV, HERG current increased about 30% (n=5). The HERG K+ current increase by SDF-1 might contribute to the mechanism of SDF-1 induced leukemic cell migration. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless of untreated and treated with E-4031 for 24 hours (p>0.05), suggesting that the leukemic cell migration induced by SDF-1 were specifically associated with HERG K+ channel, not by regulating CXCR4 expression. Conclusion: the data showed that HERG K+ channel was essential for leukemic cell migration induced by SDF-1. SDF-1 enhanced herg current suggested that SDF-1 promotes leukemic cell migration. Blocking HERG K+ channel with specific inhibitor could decrease leukemic cell and leukemic stem cell migration caused by SDF-1. Prospectively, HERG K+ channel may be a potential therapeutic target with specific inhibitors in leukemia treatment.

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Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽

10.1182/blood.v112.11.2423.2423 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2423-2423

Author(s):

Sergej Konoplev ◽

Hongbo Lu ◽

Michael A Fiegl ◽

Zhihong Zeng ◽

Wenjing Chen ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Leukemic Cells ◽

Human Leukemia ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Leukemic Cell Lines ◽

And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

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Disruption of CXCR4 Utilizing AMD3100 Bypasses Mobilization Deficiency Exhibited by CD26−/− Mice and Results in Efficient Mobilization of Myeloid Progenitors

Blood ◽

10.1182/blood.v112.11.3492.3492 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3492-3492

Author(s):

Laura A. Paganessi ◽

Andrew L. Walker ◽

Stephanie A. Gregory ◽

Henry C. Fung ◽

Kent W. Christopherson

Keyword(s):

Bone Marrow ◽

Blood Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Fold Increase ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Genetically Engineered Mice ◽

Colony Forming Units ◽

Myeloid Progenitors

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

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Essential Role for the MAML1 Co-Activator In T-ALL

Blood ◽

10.1182/blood.v116.21.2501.2501 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2501-2501

Author(s):

Chunxia Cao ◽

Liang Tian ◽

Jian-Liang Li ◽

James D. Griffin ◽

Suming Huang ◽

...

Keyword(s):

Cell Growth ◽

Notch Signaling ◽

Leukemic Cell ◽

Genetic Alterations ◽

Leukemic Cells ◽

Cell Renewal ◽

Hematopoietic Stem ◽

Growth And Survival ◽

Oncogenic Pathways ◽

Cell Growth And Survival

Abstract Abstract 2501 T cell acute lymphoblastic leukemia (T-ALL) is the most common malignancy in children and accounts for nearly one third of all pediatric cancers. In this type of leukemia, lymphoid progenitor cells that are responsible for the generation of mature lymphocytes become genetically altered, leading to deregulated proliferation, survival, and clonal expansion. Two common genetic alterations frequently associated with this disease are mutations in the NOTCH1 cell-surface receptor and aberrant expression of the TAL1 transcription factor, with each abnormality detected in more than half of human T-ALL patients. The mutations in the NOTCH1 gene result in the aberrant activation of Notch signaling, a highly conserved signal transduction pathway that is critical for lymphocyte growth, maturation and survival. The constitutive activation of Notch signaling induces leukemia in mouse models and is required for human T-ALL leukemic cell growth and survival. On the other hand, TAL1 is required for the functions of hematopoietic stem cells and is essential for the generation of the erythroid and myeloid lineages. The ectopic activation of the TAL1 gene deregulates normal hematopoietic stem cell renewal and differentiation, leading to leukemia in cooperation with other oncogenes. Therefore, Notch and TAL1 oncogenic activities are critical for the initiation and maintenance of T-ALL. In this study, we investigated the role of a transcriptional co-activator, MAML1, in regulating NOTCH1 and TAL1 transforming activities in leukemic cells. In addition to its known function in co-activating Notch signaling, we found that MAML1 is a novel interacting partner for TAL1. MAML1 also enhanced TAL1 transcriptional activities, suggesting a role for MAML1 in TAL1-regulated transcription and leukemogenesis. A subset of T-ALL leukemic cells exhibit aberrant activation in both the NOTCH1 and TAL1 activities; thus, it suggests that these two genetic alterations cooperate in promoting leukemic cell growth and survival. Indeed, we found that the combined inhibition of both the pathways (via the pharmacological blockade of Notch signaling and shRNA-mediated TAL1 knockdown) results in synergistic responses in leukemic cells that carry genetic alterations in both the NOTCH1 and TAL1 genes, indicating that the two pathways synergize in promoting T-ALL. Since MAML1 appears to be a common key regulator for both TAL1 and Notch1 pathways, we next determined whether MAML1 expression level affects leukemic cell growth and survival. Gene knockdown studies suggest that MAML1 is essential for leukemic cell growth and survival by possibly regulating NOTCH1 and TAL1-mediated transcription. Overall, our data reveals a novel common regulatory mechanism for both NOTCH1 and TAL1 oncogenic pathways, and suggest that the manipulation of MAML1 expression or functional activities will affect leukemia initiation and progression. Therefore, our current studies focus on assessing the MAML1 co-activator as a target for these two oncogenic pathways. Disclosures: Griffin: Novartis: Consultancy, Research Funding.

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Osteocytes Embedded Inside Bone Are Essential for G-CSF-Induced Hematopoietic Stem/Progenitor Mobilization

Blood ◽

10.1182/blood.v118.21.721.721 ◽

2011 ◽

Vol 118 (21) ◽

pp. 721-721 ◽

Cited By ~ 2

Author(s):

Noboru Asada ◽

Yoshio Katayama ◽

Mari Sato ◽

Kentaro Minagawa ◽

Kanako Wakahashi ◽

...

Keyword(s):

Bone Cells ◽

Conflicts Of Interest ◽

Morphological Changes ◽

Fibroblast Growth Factor 23 ◽

Bone Matrix ◽

Fold Increase ◽

Nerve Fibers ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Endosteal Niche

Abstract Abstract 721 Hematopoietic stem/progenitor cells (HSPCs) are released from the bone marrow (BM) to the circulation by granulocyte-colony stimulating factor (G-CSF) via sympathetic nervous system (SNS)-mediated osteoblast suppression (Katayama et al. Cell 2006). We further elucidated that vitamin D receptor is essential for this neuronal control of endosteal niche (Kawamori et al. Blood 2010). Osteoblasts are known to adopt three fates: die by apoptosis, become bone-lining cells, or become embedded in osteoid and then in mineralized bone matrix to terminally differentiate into osteocytes, which constitute more than 95% of bone cells. Osteocytes have been shown to control the functional balance between osteoblast and osteoclast via mechanotransduction. In order to address the role of bone-embedded osteocytes in HSPCs niche function, we first quantified mRNA expression of bone-related genes in the femur of wild-type (WT) mice to examine if osteocytic function changes during G-CSF treatment (125μg/kg/dose, 8 divided doses, every 12 hours). Whereas markers relating to osteoblast function, osteocalcin and osteopontin, started to decrease late at 6 doses of G-CSF administration when mild mobilization of HSPCs had occurred, osteocyte-specific genes, including neuropeptide y, SOST, MEPE, E11/gp38 and Phex, were rapidly suppressed at 1 dose when no mobilization was observed. These data suggest that osteocytes respond to G-CSF with altered gene expression much earlier than osteoblasts. Further, the number and thickness of osteocyte projections extending toward the endosteal surface were markedly reduced, as assessed by fluorescently labeled phalloidin, at 8 doses of G-CSF treatment when full mobilization was achieved; these morphological changes were observed specifically in newly-embedded osteoid osteocytes, but not in mature osteocytes embedded deep inside mineralized bone. These findings suggest that osteoid osteocytes may sense the signal triggered by G-CSF. We confirmed the presence of β2-adrenergic receptor in osteoid osteocytes and tyrosine hydroxylase-positive nerve fibers in the vicinity by immunofluorecence staining, suggesting that osteoid osteocytes may be regulated by SNS. To directly address osteocyte involvement in G-CSF-induced mobilization, we utilized a transgenic (TG) mice in which inducible and specific ablation of osteocytes is achieved through targeted expression of diphtheria toxin (DT) receptor under DMP-1 promoter. A single injection of DT in TG mice generates “osteocyte-less (OL)” mice. We found that mobilization by G-CSF was drastically impaired in OL mice for progenitors (CFU-Cs, mean±SEM, WT vs Tg: 1673±271 vs 242±94/ml blood, n=6-13, p<0.01; lineage-Sca-1+c-kit+ (LSK) cells, WT vs Tg: 6878±1209/ml vs 1763±502/ml, n=6-13, p<0.01) and stem cells (repopulating units at 4 months, WT vs Tg: 2.5±0.7 vs 0.5±0.2, n=6-7, p<0.05), while the OL BM showed normal HSPC number. The levels of CXCL12 mRNA and protein in BM and bone were markedly decreased during G-CSF treatment even in OL mice despite the mobilization defect, and a CXCR4 antagonist AMD3100 induced mobilization normally in the absence of osteocytes. Thus, osteocytes embedded within the bone are indispensable for G-CSF-induced mobilization through a CXCL12-independent mechanism. Although most of bone-related genes exhibited drastic decreases following G-CSF treatment, we found that fibroblast growth factor 23 (fgf23) mRNA displayed a 4-fold increase at 6 doses of G-CSF. FGF23 is mainly produced by osteocytes and Klotho is an obligate coreceptor for FGF23 to bind and activate FGF receptors. Since we confirmed that klotho hypomorphic (kl/kl) mice showed remarkably disrupted osteocyte network, we injected G-CSF into these mice. As we expected, G-CSF induced virtually no mobilization in kl/kl mice while the number of HSPCs in the BM remained comparable to control mice. Collectively, our results demonstrate a novel function of bone-embedded osteocytes as a critical regulator of HSPC trafficking perhaps by controlling the endosteal niche and establish the important physiologic function of skeletal tissue for hematopoietic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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A Phase I/II Study of the Combination of Oral Rigosertib and Azacitidine in Patients with Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v124.21.3252.3252 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3252-3252 ◽

Cited By ~ 3

Author(s):

Shyamala C. Navada ◽

Guillermo Garcia-Manero ◽

Francois Wilhelm ◽

Katherine Hearn ◽

Rosalie Odchimar-Reissig ◽

...

Keyword(s):

Phase I ◽

Phase Ii ◽

Stable Disease ◽

Blood Count ◽

Single Agent ◽

Complete Response ◽

Leukemic Cells ◽

Cell Transplant ◽

Transfusion Requirements ◽

Count Recovery

Abstract Background:Rigosertib is a small molecule anti-cancer agent targeting PI3/polo-like kinase pathways that promotes G2/M arrest and has effects on the B-Raf and Ras pathways. It is currently being tested as a single agent with the intravenous (IV) formulation in patients (pts) who have relapsed or are refractory to hypomethylating agents (HMAs) as well as with the oral formulation in lower-risk, red-cell transfusion-dependent MDS patients. Azacitidine (AZA) is first-line therapy for pts with higher-risk MDS. In vitro, the combination of rigosertib with AZA acts synergistically to inhibit growth and induce apoptosis of leukemic cells (Skidan et al 2006). This effect appears to be sequence dependent, requiring exposure to rigosertib first, followed by AZA. These nonclinical results provided the rationale to combine the 2 agents in a phase I/II study in pts with MDS and AML. Methods: Pts with MDS and non-proliferative AML, who were previously untreated or had failed or progressed on an HMA were included in the phase I component of the study. Oral rigosertib was administered twice daily from day 1 through day 21 of a 28-d cycle. AZA 75 mg/m2/d was administered for 7 days starting on day 8 of the 28-d cycle. Pts were entered in 3 escalating-dose cohorts of rigosertib in a classic 3+3 design: [1] 140 mg twice daily; [2] 280 mg twice daily; [3] 560 mg qAM and 280 mg qPM. A CBC was performed weekly and a bone marrow (BM) aspirate and/or biopsy was performed at baseline and every 4-8 weeks afterwards. Results: Eighteen pts have been treated with the combination of oral rigosertib and AZA. Pts had diagnoses of intermediate-1 MDS (3), intermediate-2 MDS (6), high-risk MDS (2), CMML (1), and AML (6); median age was 70.5 years; 61% of pts were male. Pts have received 1-10+ cycles of treatment with the total number of cycles administered thus far being 58. Cytogenetic profiles by IPSS were good (8 pts), poor (8 pts), and intermediate (2 pts). 11of 18 patients were transfusion dependent at baseline [RBC (11), platelet (6)]. One patient became RBC transfusion independent after 3 cycles of treatment. 5 additional patients have had a reduction in their RBC and platelet transfusion requirements. 56% of patients received prior treatment with HMAs: AZA (6 pts), decitabine (4 pts). The most frequent adverse events (AEs) in Cycle 1 included constipation, diarrhea, nausea, fatigue, hypotension, and pneumonia. The AEs did not differ significantly among the 3 cohorts. Elevation in creatinine in 1 pt in cohort 1 was a possibly related grade 3 dose-limiting toxicity that required subsequent expansion of the cohort. Drug-related dysuria/cystitis was not reported in this pt population. Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: Complete Response (CR) (1 pt), Cri (CR with incomplete blood count recovery) (4 pts), stable disease (2), hematologic improvement-erythroid (1). Six pts received fewer than 4 cycles of treatment and are too early to evaluate. Six pts came off study for the following reasons: progression of disease (1), pt request (1), death from pneumonia (2), received stem cell transplant (1), persistent fungal pneumonia (1). Two evaluable pts have responded to the combination after progression or failure on HMA alone. Conclusions: The combination oforalrigosertib at 560/280 mg BID (recommended phase II dose) and standard-dose AZA can be safely administered and appears to be well tolerated in repetitive cycles in pts with MDS and non-proliferative AML. The AE profile does not differ significantly from that of AZA alone. Data from the Phase I component of this study suggest activity in patients with MDS after HMA failure. Additional data are required to evaluate this observation. The Phase II segment of this study is underway to further assess the response of the combination. Table Patient ID Diagnosis Prior HMA % Blasts in BM at Baseline % Blasts in BM after Treatment IWG Response 1 MDS No 2 1 CRi 2 AML No 40 0 CRi 3 AML No 22 N/A NE 4 MDS Azacitidine 0 0 NE 5 AML No 59 N/A NE 6 MDS No 21 <5 CRi 7 MDS No 2 1 CR 8 MDS No 2.5 2 SD 9 AML Decitabine 25 N/A NE 10 MDS Decitabine 12 3 CRi 11 CMML Azacitidine 2 3 SD 12 MDS Azacitidine 4 1 HI-E 13 AML Azacitidine 47 40 TE 14 AML Decitabine 7 7 TE 15 MDS No 9 5 TE 16 AML No 25 6 TE 17 AML No 15 19 TE 18 AML Azacitidine 64 45 TE IWG = International Working Group CR = Complete Response CRi = Complete Response with incomplete blood count recovery NE = Not Evaluable SD = Stable Disease HI-E = Hematologic Improvement - Erythroid TE = Too Early Disclosures Wilhelm: Onconova Therapeutics, Inc: Employment, Equity Ownership. Demakos:Onconova: Consultancy. Azarnia:Onconova Therapeutics, Inc: Employment. Silverman:Onconova: with Icahn School of Medicine at Mount Sinai Patents & Royalties.

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