Leukemia-bearing Mice Research Articles

Effects of CCL2/CCR2 Blockade in Acute Myeloid Leukemia

Background: A role for the Chemokine (C-C motif) ligand 2 (CCL2) in attracting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC) and infiltrating monocytes has been described for many solid tumors in which they play an essential role in modifying the adaptive immune response, ultimately favoring tumor progression. Unfortunately, little is known about the importance of this mechanism for the progression of AML. We recently identified CCL2 as the most prominent chemokine produced by bone marrow (BM) mesenchymal stromal cells (BM-MSC) in response to the interaction with myeloid leukemia cells (PMID: 24599548). In addition, elevated CCL2 plasma levels have been reported in patients of AML (PMID: 17822317), ALL (PMID: 21298741) and CLL (PMID: 22397722) when compared to normal controls. In this study we assessed the effects of blocking the CCR2-CCL2 axis on the migration and signaling of hematopoietic cells as well as on the infiltration of immune-suppressive cells in leukemia-bearing mice.Results: We first studied the efficacy and potency of agents at inhibiting CCL2-mediated migration, using the human monocytic leukemia cell line THP-1. Migration towards human recombinant CCL2 (5 ng/ml) was significantly inhibited by as little as 1 nM of NOX-E36, a human-specific CCL2 Spiegelmer (NOXXON Pharma, Berlin). Spiegelmers are RNA-like molecules built from L-ribose units that are able to bind molecules such as peptides and proteins with an affinity in the pico-to nanomolar range. Similar results were obtained with a CCR2 antagonist (100 ng/ml; Santa Cruz). In anticipation of in vivo studies in mice, we next confirmed the ability of a mouse-specific CCL2 Spiegelmer (mNOX-E36) to inhibit migration and signaling pathway activation in murine hematopoietic cells. For this purpose, we cloned and overexpressed via lentiviral transduction the murine CCL2 receptor (CCR2) in Ba/F3 cells (a murine pro-B cell line). Stimulation of Ba/F3-CCR2 cells with 5 ng/ml of mouse recombinant CCL2 induced a ~2000 fold increase in migration of Ba/F3-CCR2 cells and was successfully blocked with mNOX-E36 in a concentration-dependent manner. Western blot analysis of protein lysates from mCCL2-stimulated cells (30 minutes treatment) indicated activation of AKT, ERK and p38-MAPK. The CCL2-induced phosphorylation of these molecules was completely abrogated by pre-treatment with mNOX-E36.Subsequently, we determined whether the expression of CCL2 by stromal cells in leukemia-resident organs triggers the infiltration of TAMs and possibly other immune-suppressive cells into those organs. We conducted preliminary in vivo studies in non-irradiated immunocompetent C57BL/6 mice (n=5 per group) injected with syngeneic AML1/ETO9a-expressing primary murine leukemia cells (PMID: 19339691). After confirmation of leukemia engraftment by IVIS imaging, mice were treated with mNOX-E36 (14.4 mg/kg, s.c., three times per week) or vehicle control for 3 weeks. At this point, all animals were sacrificed and their tissues (spleens and BM from femurs) were collected for analysis. Although we did not observe differences in leukemia burden by imaging between vehicle and mNOX-E36 treated groups, flow cytometry analysis revealed an increase in the frequency of CD11b+ Ly6Clow MHC IIlow macrophages (2 to 7 fold increase) in spleens of mice engrafted with leukemia (vehicle-treated group) when compared to spleens collected from healthy mice. These MHC IIlow macrophages were previously identified as immunosuppressive M2-like macrophages as opposed to MHC IIhi macrophages which show a pro-inflammatory M1-like phenotype (PMID: 20570887). Importantly, CCL2 inhibitor mNOX-E36 abrogated this macrophage infiltration within the leukemia microenvironment.Conclusions: Our results indicate that blockade of the CCR2-CCL2 axis not only affects migration and signaling of treated cells in vitro, but also interferes with the infiltration of M2-like macrophages into spleens of leukemia-bearing mice. Current in vivo experiments using a combination of standard chemotherapy with mNOX-E36 in AML immunocompetent models are undergoing. We expect that in vivo modulation of CCL2 will improve response to chemotherapy of AML by reducing the marrow infiltration of infiltrating monocytes and tumor-associated macrophages, which would facilitate translation of this novel concept into clinical trials in AML. DisclosuresZuber:Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

Analysis of redox and apoptotic effects of anthracyclines to delineate a cardioprotective strategy.

Cardiotoxic side effects of anthracyclines limit their use as effective chemotherapeutics. One mechanistic model of anthracycline-induced cardiotoxicity is attributed to the generation of intracellular reactive oxygen species (ROS). However, this theory has been questioned because several cardioprotective strategies have included the use of antioxidants without significant clinical benefit. We sought to determine whether measurement of intracellular reactive oxygen species after anthracycline exposure in vivo and in vitro could provide a means for designing more effective antioxidant-based cardioprotective schemes. Intracellular levels of ROS were assessed in peripheral blood mononuclear cells from leukemia bearing mice exposed to anthracyclines and in patients receiving anthracyclines. Comparison of cell death induction and ROS levels were also conducted in vitro in cardiomyocyte and leukemia lines. ROS blockade using antioxidants was conducted, and effects on cell death were assessed. Elevated ROS in blood of mice and representative patient samples correlated with cardiomyocyte necrosis and decreased ejection fraction. In vitro, comparison of the cytotoxic effects of anthracyclines in acute leukemia cells and in cardiomyocytes revealed distinct kinetics of cell death induction and dependence upon oxidative stress. Although apoptotic cell death was observed in both acute leukemia cells and cardiomyocytes, the antioxidant N-acetylcysteine protected cardiomyocytes but not acute leukemia cells from anthracycline cytotoxicity. Our findings point toward revisiting the use of NAC as a cardioprotective agent since it does not appear to interfere with the cytotoxic action of anthracyclines. NAC has been evaluated clinically for cardioprotective activity but future trials must ensure that adequate dose, scheduling and incorporation of markers of oxidative stress are included.

AML Genotype-Specific and Non-Specific Regulation of Mesenchymal Stromal Cell Transcriptome in the Bone Marrow Microenvironment

Acute myeloid leukemia (AML) cells can modify the bone marrow (BM) microenvironment and “educate” it to promote leukemia progression; e.g., leukemia cells induce transcriptional changes in BM mesenchymal stromal cells (BM-MSC) that support growth advantage and chemoresistance of leukemia cells (Jacamo, R et al., Blood 2014). Given the genetic heterogeneity of AML, and the variable responses of individual patients to therapy, a better understanding of how different AML genotypes can influence their BM microenvironment is needed. We inoculated C57BL/6 mice with syngeneic murine leukemia cells harboring one of the following human AML genotypes: AML1-ETO, MLL-ENL, or MLL-ENL/FLT3-ITD (further divided into p53 wt or p53-/-) (Zuber, J et al., Genes Dev 2009). Leukemia engraftment was confirmed by live imaging (IVIS) and allowed to progress for 2 to 4 weeks, according to leukemia burden. Multiple mice from each condition were sacrificed to allow isolation of sufficient BM-MSC by flow cytometry based on cell surface markers: CD105+, Sca1+, CD106+, PDGF-Rα+ (CD140), and CD45-. Gene expression profiling with Illumina mouse WGv2 BeadArrays was performed on duplicate pools of mice to identify differences between BM-MSC from various AML-bearing mice and BM-MSC from control mice.Heat mapping of highly-variant genes showed that samples clustered according to AML genotype, confirming data quality and suggesting that changes induced in BM-MSC differ with AML genotype: the two BM-MSC samples derived from the MLL-ENL/FLT3-ITD leukemia-bearing mice were nearest neighbors, and also clustered next to the sample from mice injected with MLL-ENL alone. 942 genes were upregulated in the AML1-ETO BM-MSC samples at least 2-fold as compared to control BM-MSC and those from other AML genotypes. The most highly upregulated genes among these included Tenm4 (an inhibitor of chondrogenesis also known as ODZ4) and Plxnd1, a regulator of angiogenesis. For both MLL-ENL/FLT3-ITD samples, 632 genes were upregulated at least 2-fold as compared to control MSC and other AML genotypes, notably including Cited4 (a repressor of HIF-1α transactivation), Bmp1, and Spon2(a TGF-beta-regulated extracellular matrix (ECM) protein upregulated in multiple types of cancer). Ingenuity Pathway Analysis (IPA) implicated upregulation of genes involved in aryl hydrocarbon receptor signaling, a potential inhibitor of HIF-1α by competition for HIF-1β. For genes differing between BM-MSC exposed to p53 WT and -/- forms of MLL-ENL/FLT3-ITD leukemia, IPA suggested that p53 status differentially affects the BM-stroma transcriptome through effects possibly mediated by CFS3, IL-3 and TGF-β1.We also observed sets of genes that were consistently up- or downregulated in BM-MSC by all four AML genotypes. At a minimal 2-fold change, 57 BM-MSC genes were upregulated in all 4 samples, and 60 genes downregulated, by exposure to AML in vivo. Notable among the upregulated genes were Ctgf (12 and 33 fold increase [FI] by 2 array probes), genes related to complement (C4a, C4b, Serping1), Igfbp5 (FI = ~12 by 3 probes), Mgp (matrix Gla protein, an ECM regulator of multiple types of mesenchymal differentiation, FI = ~8) and Cxcl12 (FI = ~6). The latter, the ligand for CXCR4, is expressed by a subset of BM stromal cells and is essential for the maintenance of normal hematopoietic precursors. Gene Set Enrichment Analysis (GSEA) implicated upregulation of genes involved in ECM receptor interaction and cancer stem cells. Common downregulated genes included Ltf, a tumor suppressor and inhibitor of AKT signaling, and PDCD4, a suppressor of tumor-permissive stromal changes downregulated by miR-21, consistent with our finding of increased mir-21 in AML-derived BM-MSC compared to normal donor-derived BM-MSC.In summary, these results support the hypothesis that AML regulates the transcriptomes of BM-MSC, in both shared and genotype-specific ways. Further analysis of the data is ongoing. DisclosuresNo relevant conflicts of interest to declare.

MLAA-34: A New Leukemia-Associated Antigen for Immunotherapy Against Acute Monocytic Leukemia

Introduction: Relapse after chemotherapy is inevitable in the majority of acute myeloid leukemia patients without allogeneic transplantation. Thus, it is necessary to develop novel therapies that have different antileukemic mechanisms. Recent advances in immunology and identification of promising leukemia-associated antigens open the possibilities for eradicating minimal residual diseases by antigen-specific immunotherapy after chemotherapy.Identification of CTL epitopes that can induce CD8+ T cells to kill tumor cells is the fundamental step for development of cancer peptide vaccines. Our previous research showed that monocytic leukemia associated antigen-34(MLAA-34) was an anti-apoptotic molecule and overexpressed in patients with acute monocytic leukemia (M5), indicating MLAA-34 was a promising antigen for immunotherapy of M5. HLA-A*0201 is the main HLA class-I antigen in Chinese population. Therefore, we focused on identifying HLA-A*0201-restricted CTL epitopes of MLAA-34.Methods: MLAA-34 HLA-A*0201 restricted CTL epitopes were screened by bioinformatics prediction. The actual affinity of predicted peptides to HLA-A*0201 molecules was evaluated by T2 cell surface HLA class I stabilization assay. Mature DCs were induced and cultured from HLA-A*0201+ PBMCs using recombinant GM-CSF, IL-4 and TNF-α. High-purity CD8+ T cells were selected from non-adherent cells of HLA-A*0201+ PBMCs by magnetic activated cell sorting. CTLs were induced by three stimulations with DCs pulsed with the MLAA-34-derived peptides. CTL activity was tested using IFN-γ ELISPOT assay and LDH release assay. Humanized immunity was reconstructed in SCID mice by HLA-A*0201 human peripheral blood lymphocytes (hu-PBL). Human acute monocytic leukemia cell THP-1-bearing hu-PBL-SCID mouse model was used for in vivo assay. MLAA-34 peptide vaccine was composed of MLAA-34 epitope CP701 (236ILDRHNFAI244), Th epitope (830QYIKANSKFIGITE843) and incomplete adjuvant. Different vaccines were applied to leukemia bearing hu-PBL-SCID mice to observe the tumor size, survival of mice and to determine the antileukemic mechanisms and effects of vaccines.Results: 10 HLA-A*0201 restricted epitopes of MLAA-34 were predicted and synthesized. CP693 (293ILLKNQPKL301), CP700 (324YLIKQIRDL332) and CP701 (236ILDRHNFAI244) showed the strongest HLA-A*0201 binding affinity. IFN-γ ELISPOT assay showed that peptides CP701, CP700 and positive peptide CML28 (173-181)-specific CTLs produced a higher amount of IFN-γ than CTLs stimulated with peptide CP693 and negative control. Comparing with peptide CP700 and CP 693-induced CTLs, peptide CP701-induced CTLs presented the stronger cytotoxic effect on THP-1 cells (HLA-A2+MLAA34+) and T2 cells pulsed with peptide CP701 at various effector to target ratios. The CP701-induced CTLs had significant higher specific lysis on THP-1 cells (HLA-A2+MLAA34+) than that of human monocyte leukemia cell line U937 (HLA-A2-MLAA-34+), human breast cancer cells MCF-7 (HLA-A2+MLAA-34-) and human lung adenocarcinoma cell A549 (HLA-A2- MLAA-34-). Leukemia bearing success rate of hu-PBL-SCID mice was 100%. MLAA-34 peptide vaccine could inhibit the tumor growth in leukemia-bearing mice. The mean survival time of tumor-bearing mice in MLAA-34 peptide vaccine group was significantly longer than that of WT1 peptide group, inactivated THP-1 cells group, Th peptide group and incomplete Freund’s adjuvant group. At different effector to target ratios, killing efficacy of CTLs and NK cells derived from MLAA-34 peptide vaccinated group were significantly higher than other therapeutic groups. CTLs and NK cell derived from MLAA-34 peptide showed specific cytotoxicity to THP-1 cells (HLA-A2+MLAA34+), but not to U937 (HLA-A2-MLAA-34+), MCF-7 (HLA-A2+ MLAA-34-) or A549 (HLA-A2-MLAA-34-). Comparing with other therapeutic groups, there were significant higher percentage of CD3+CD8+ T cells and lower percentage of Treg cells in MLAA-34 peptide vaccine group. Furthermore, MLAA-34 peptide-stimulated T cells showed increased IFN-γ and IL-2 and decreased IL-10 and IL-4 expression compared to controls.Conclusions: MLAA-34 peptide CP701 (236ILDRHNFAI244) is an effective HLA-A*0201-restricted CTL epitope and that it may serve as a promising strategy in designing antigen-specific immunotherapy against MLAA-34-positive leukemia. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1421: Overexpression of the pro-glycolytic transcription factor MondoA enhances malignant potential of ALL in vivo

Abstract Introduction. Most cancer cells use glycolysis for energy production regardless of whether they are under normoxic or hypoxic condition (Koppenol et al., 2011). Warburg described this metabolic hallmark of cancer cells, named aerobic glycolysis. The high dependence of tumor cells on glucose utilization may be exploited for therapeutic intervention. One of the genes, which have effects on glucose metabolism, is MondoA. It is a transcription factor (Stoltzman et al., 2008) highly over-expressed in ALL (Wernicke et al. 2012). MondoA:MLX heterodimers have been discussed as a parallel network to MYC:MAX, which have broad effects on cellular growth, proliferation and survival (Kaadige et al., 2010; Sloan and Ayer, 2010). Knockdown experiments revealed that MondoA impacts on metabolic activity of leukemic cells and their glucose utilization. Furthermore MondoA knockdown resulted in a more differentiated leukemic phenotype and shifted gene expression towards a signature favoring apoptosis and thus reducing the signs of aggressiveness of leukemia cells in vitro (Wernicke et al. 2012). Based on our previous work, we hypothesized that assessing survival of leukemia bearing mice may reveal the role of MondoA in ALL in vivo. Methods. The murine/human xeno-transplantation model of leukemia was used (Richter et al. 2009). NALM-6 ALL cell lines were transduced by retroviral gene transfer of constructs, harboring specific for MondoA short hairpin RNA (shRNA), and injected into immune deficient Rag2-/-γC-/- mice. The tumor burden were measured and compared to the one in control mice bearing leukemia cells transduced with nonfunctional siRNA. Tumor burden of mice were evaluated by cyto- and histochemistry and by measuring the CD10+ blast cells content in blood, spleen and bone marrow by flow cytometry. Results. These experiments showed that MondoA knockdown significantly reduced leukemia cells' proliferation in mice as compared to control group. Although signs of leukemia engraftment were observed in all mice, the differences in amount of CD10+ blasts cells in blood, bone marrow and spleens in the controls and in the MondoA knockdown group were significant (p= 0,0079). Experimental results also showed a decrease of the leukemic infiltration-induced swelling of the spleen in the group with MondoA knockdown. Median spleen weight was 0.22g and 0.08g in the control and in the experimental group. Conclusion. These findings demonstrate that the downregulation of MondoA by RNA interference significantly delays leukemic burden and malignancy of B cell ALL in vivo and further confirm that MondoA is an attractive target for targeted and immune therapy of ALL. Citation Format: Alexandra A. Sipol, Günther H.S. Richter, Caroline M. Wernicke, Thomas G.P. Grunewald, Stefan Burdach. Overexpression of the pro-glycolytic transcription factor MondoA enhances malignant potential of ALL in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1421. doi:10.1158/1538-7445.AM2014-1421

Augmented efficacy with the combination of blockade of the Notch-1 pathway, bortezomib and romidepsin in a murine MT-1 adult T-cell leukemia model.

Adult T-cell leukemia (ATL) is an aggressive malignancy caused by human T-cell lymphotropic virus I (HTLV-1). There is no accepted curative therapy for ATL. We have reported that certain ATL patients have increased Notch-1 signaling along with constitutive activation of the NF-κB pathway. Physical and functional interaction between these two pathways provides the rationale to combine the γ-secretase inhibitor Compound E with the proteasome inhibitor Bortezomib. Moreover, Romidepsin, a histone deacetylase inhibitor, has demonstrated major antitumor action in leukemia/lymphoma. In this study, we investigated the therapeutic efficacy of the single agents and combinations of these agents in a murine model of human ATL, the MT-1 model. Single and double agents inhibited tumor growth as monitored by tumor size (P < .05), and prolonged survival of leukemia-bearing mice (P < .05) compared with the control group. The combination of three agents significantly enhanced the antitumor efficacy as assessed by tumor size, tumor markers in the serum (human sIL-2Rα and β2M), and survival of the MT-1 tumor bearing mice, compared with all other treatment groups (P < .05). Improved therapeutic efficacy obtained by combining Compound E, Bortezomib and Romidepsin supports a clinical trial of this combination in the treatment of ATL.

Pharmacology, immunogenicity, and efficacy of a novel pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase.

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an indispensable component used in the treatment of acute lymphoblastic leukemia (ALL) and certain lymphoma entities. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been approved as a second-line drug for treating patients exhibiting allergy syndromes to native and pegylated Escherichia coli-derived ASNase (EC-ASNase). However, it still induces hypersensitivity in at least 17 % of treated patients. In the present study, we investigated the pharmacological activity, immunogenicity and anti-leukemic activity of a new pegylated recombinant crisantaspase (PEG-r-crisantaspase). The results demonstrate that when compared to n-crisantaspase in vivo, PEG-r-crisantaspase maintains a complete depletion of plasma Asn for up to 72 h with a 50-fold lower dose. In mice receiving PEG-r-crisantaspase, specific antibodies against the enzyme were undetectable, indicating a lower immunogenicity of the pegylated enzyme. In vitro, PEG-r-crisantaspase exhibits similar cytotoxic effects (EC50 < 5 × 10(-4) U/mL for the most sensitive cell lines) to n-crisantaspase on various leukemia and lymphoma cells and was shown to be more efficient than EC-ASNase. Three repeated PEG-r-crisantaspase injections (2-20 U/Kg) prevented leukemia development in leukemia-bearing mice for 17 days and significantly prolonged animal survival to 7-12 days. Therefore, PEG-r-crisantaspase appears to be a promising drug candidate for ALL treatment and should be further explored in experimental and clinical trials.

Immunomagnetic selection or irradiation eliminates alloreactive cells but also reduces anti-tumor potential of cytokine-induced killer cells: implications for unmanipulated cytokine-induced killer cell infusion

Background aimsCytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential. MethodsIn this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells. ResultsIn vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro. ConclusionsOur study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.

Potent Effects Of The Vascular Disrupting Agent, ASA404 (DMXAA) On The Marrow Microenvironment In Preclinical Human Leukemia and Lymphoma Models

BackgroundTumor vascular disrupting agents (VDAs) represent a novel class of anti-cancer therapeutics with the unique ability to rapidly destroy established tumor vasculature and induce ischemic necrosis. In contrast to most VDAs which are derivatives of anti-tubulin agents, ASA404 (5,6-dimethylxanthenone-4-acetic acid, DMXAA) is an unique non-steroidal anti-inflammatory VDA which induces potent host immune responses and disrupts tumor vasculature in solid tumor mouse models. MethodsThe overall aim of these studies was to examine the effects of ASA404 treatment on tumor growth in preclinical human acute myeloid leukemia (HEL, HL60) and lymphoma (Raji) xenograft models. We specifically investigated the effects of drug treatment on host immune and inflammatory responses, marrow vasculature, and initial tumor engraftment. Disease progression in vivo was measured using small animal magnetic resonance imaging and bioluminescent imaging of luciferase-transfected human tumor cells. Host cytokine and chemokine levels in the plasma and other tissues (marrow, spleen, liver) were determined by multiplex flow cytometric assays. ResultsIn local acute myeloid leukemia (HEL-luciferase) xenografts, a single dose of ASA404 (25 mg/kg) markedly enhanced tumor vascular permeability, as determined by magnetic resonance imaging at 4 hours. This was followed by vascular disruption and central necrosis at 24 hours, as determined by immunohistochemistry. In systemic leukemia xenografts, a single dose of ASA404 enhanced marrow vascular permeability and induced several-fold elevations in host chemokine levels (G-CSF, IL-6, MCP-1, KC) in circulating plasma as well as bone marrow and spleen cells (MIG, G-CSF, MIP-1alpha, IP-10). Administration of ASA404 4-24 hours prior to inoculation of human acute myeloid leukemia (HEL) or lymphoma (Raji) cells in immunodeficient mice delayed disease progression and prolonged overall survival. Continuous ASA404 treatment (20 mg/kg twice weekly) for up to three weeks in established systemic leukemia and lymphoma xenografts resulted in marrow vascular disruption and significant inhibition of tumor growth. Prolonged ASA404 treatment was associated with elevations in circulating host TNF-a levels and increased numbers of macrophages within the leukemic marrow microenvironment. Combination ASA404 and doxorubicin therapy further improved the overall survival of HEL and Raji-engrafted mice as compared with AS404 or doxorubicin alone. Quantification of doxorubicin levels by HPLC in local tumor xenografts and leukemic marrow samples revealed several-fold higher doxorubicin levels following ASA404 vs. vehicle (PBS) treatment, consistent with ASA404-mediated improvements in leukemia drug delivery. Preliminary data suggest a therapeutic benefit of combination ASA404 and cytarabine therapy in the same xenograft models. ConclusionsOur results demonstrate that ASA404 exerts anti-tumor activity in human acute myeloid leukemia and lymphoma xenografts via myriad unique effects on the marrow microenvironment. Single dose administration of ASA404 results in rapid and marked alterations in multiple host cytokine/chemokine levels in association with enhanced tumor vascular permeability and impaired in vivo leukemia cell engraftment. Prolonged drug administration leads to pronounced vascular disrupting effects and inhibition of tumor growth. Combination regimens of ASA404 with anthracycline or cytarabine lead to further improvements in anti-tumor activity and overall survival in leukemia-bearing mice. These data suggest that ASA404/DMXAA may be worthy of further clinical investigation for the treatment of aggressive hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

The Plasma Cell Transcription Factor XBP1 is Required To Mitigate The Unfolded Protein Response In Ph+ ALL

BackgroundThe transcription factors X-box binding protein 1 (XBP1) and PR domain zinc finger protein 1 (PRDM1) are critical for terminal differentiation of B cells into plasma cells but have no known role at earlier stages of B-cell development. Additionally, XBP1 is a critical effector molecule during the unfolded protein response (UPR), which is required to salvage ER stress and to promote cell survival. Previous work identified XBP1 and PRDM1 as central requirements for plasma cell development and as critical mediators of cell survival in plasma cell-derived multiple myeloma. ResultsWe report the unexpected finding that XBP1 and PRDM1 genes are hypermethylated at their promoters and not expressed in normal pre-B cells, whereas pre-B cell-derived Ph+ ALL cells express both molecules at surprisingly high levels. Interestingly, XBP1 expression levels are linked to BCR-ABL1 kinase activity and transcriptionally activated by STAT5 and repressed by the tumor suppressor BACH2.Furthermore, high expression levels of XBP1 at diagnosis predict poor overall survival (OS) and relapse-free survival (RFS) of leukemia patients in two clinical trials for patients with high risk acute lymphoblastic leukemia (n=207; COG P9906 trial; p=8.95e-06 and ECOG E2993; n=215; p=0.017). A multivariate analysis showed that high expression of XBP1 is an independent predictor of poor outcome regardless of other established risk factors such as white blood cell count (WBC) and minimal residual disease (MRD) (n=207; COG P9906 trial; WBC; p=8.95e-06 and MRD; p=5.85e-05). Patients with a high expression of XBP1 and low expression of BACH2 profile had the worst RFS. In addition, high levels of XBP1also correlated with positive MRD status at day 29 after onset of chemotherapy.To study the function of Xbp1 in Ph+ ALL, we developed a genetic system for inducible deletion of Xbp1 in a mouse model of Ph+ ALL based on bone marrow B cells from mice carrying loxP-flanked allele of Xbp1 (Xbp1fl/fl). On the basis of this model, bone marrow B cell precursors were transformed by BCR-ABL1 in the presence of IL7. Inducible Cre-mediated deletion of Xbp1 was achieved by transduction of leukemia cells with tamoxifen (4-OHT)-inducible Cre. Interestingly, 4-OHT-induced deletion of Xbp1 in Ph+ ALL-like leukemia cells caused rapid cell death within two days of induction. Xbp1-deletion resulted in extensive apoptosis, cellular senescence and cell cycle arrest associated with increased levels of p53, p21 and Arf. Similarly, in an in vivo setting, deletion of Xbp1 substantially prolonged survival of NOD-SCID transplant recipient mice (n=7; p=0.007). Mechanistically, deletion of Xbp1leads to increased expression of the pro-apoptotic molecule CHOP and phosphorylation of the stress MAP kinases p38 and JNK. Clinical relevanceTo investigate the potential clinical relevance of these findings, we used recently identified small-molecule inhibitors for XBP1 activation (Papandreou et al., 2011; Volkmann et al., 2011; Kriss et al., 2012). Small-molecule inhibition of XBP1 activation overall mimicked the findings in genetic experiments. Importantly, pharmacological targeting of XBP1 also induced cell death in multiple patient-derived cases of Ph+ ALL carrying the T315I mutations, which confers far-reaching TKI-resistance and caused reduction of leukemia-initiating cells (LICs) in patient-derived TKI-resistant Ph+ ALL cells in vivo and significantly prolonged survival of leukemia-bearing NOD-SCI mice (n=5 per group). ConclusionsCollectively, these studies identify XBP1 as an important survival factor and as a potential therapeutic target to overcome drug-resistance in Ph+ ALL. Like transformed plasma cells in multiple myeloma, Ph+ ALL cells are selectively sensitive to ER stress and critically dependent on XBP1 and likely other factors of the UPR pathway. Clinical validation of this concept could lead to improved treatment options for patients with TKI-resistant Ph+ ALL. Disclosures:Glimcher:Bristol-Myers Squibb: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Markedly additive antitumor activity with the combination of a selective survivin suppressant YM155 and alemtuzumab in adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. Currently, there is no accepted curative therapy for ATL. In gene expression profiling, the antiapoptotic protein survivin (BIRC5) demonstrated a striking increase in ATL, and its expression was increased in patient ATL cells resistant to the anti-CD52 monoclonal antibody alemtuzumab (Campath-1H). In this study, we investigated the antitumor activity of a small-molecule survivin suppressant YM155 alone and in combination with alemtuzumab in a murine model of human ATL (MET-1). Both YM155 alone and its combination with alemtuzumab demonstrated therapeutic efficacy by lowering serum soluble IL-2Rα (sIL-2Rα) levels (P < .001) and prolonged the survival of tumor-bearing mice (P < .0001). Moreover, the combination of YM155 with alemtuzumab demonstrated markedly additive antitumor activity by significantly lowering serum sIL-2Rα levels and improving the survival of leukemia-bearing mice compared with monotherapy with either YM155 (P < .001) or alemtuzumab (P < .05). More significantly, all mice that received the combination therapy survived and were tumor free >6 months after treatment. Our data support a clinical trial of the combination of YM155 with alemtuzumab in ATL. This trial was registered at www.clinicaltrials.gov as #NCT00061048.

Anti-Leukemic Activity of a Novel Pegylated Recombinant Erwinia Chrysanthemi-Derived L-Asparaginase On Lymphoid Cell Lines and Leukemia-Bearing Mouse Models.

AbstractAbstract 2571 Background:Bacterial-derived L-asparaginase (ASNase) is an indispensable component of the therapy of acute lymphoblastic leukemia (ALL). Despite the high rate of successful treatment, hypersensitivity reactions occur in 20–40 % of patients receiving this non-human protein, which limits the use of ASNase. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been used for treating patients exhibiting allergic symptoms to native and PEGylated Escherichia coli-derived ASNases (EC-ASNase). However, hypersensitivity reactions are still observed in at least 17 % of patients receiving n-crisantaspase. A PEGylated recombinant Erwinia chrysanthemi-derived ASNase (PEG-r-crisantaspase) with improved pharmacokinetic and pharmacodynamics properties and reduced immunogenicity has been developed recently (Allas et al., abstracts #2003, #2034, ASH 2009). We present here the in vitro and in vivo evaluation of PEG-r-crisantaspase on leukemia and lymphoma cell lines and on a leukemia-bearing mouse model, respectively. Material and methods:Different cell lines (ALL, B, T and NK/T lymphoma) and bone marrow aspiration samples obtained from patients with B-ALL or T-ALL were exposed in vitro to increasing doses (0.00005 to 5 U/ml) of PEG-r-crisantaspase, n-crisantaspase, or native EC-ASNase for 3 days. The cytotoxicity of each molecule was evaluated using 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. For the in vivo study, 5 millions of ASNase-sensitive cells, RS(4,11) were xenografted by intravenous injection (iv) into immunodeficient NOD/SCID mice. When 5% of leukemic blasts were observed in the blood of leukemia-bearing mice, 2, 5, 10 or 20 U/kg of PEG-r-crisantaspase or vehicle was injected intravenously in those mice. Two additional injections were performed with an interval of 7 days. Mice having significant weight loss (>20%) and/or extensive expansion of leukemic cells in blood (> 25%) and/or impaired general condition were sacrificed. Results:In vitro, PEG-r-crisantaspase and n-crisantaspase exhibited similar half maximal effective concentration (EC50) values for inhibiting the proliferation of leukemia and lymphoma cells. PEG-r-crisantaspase had a greater cytotoxicity effect on two high-sensitive cell lines than n-crisantaspase did, as shown by 4.5 and 8.1-fold-weaker EC50 values of PEG-r-crisantaspase relative to n-crisantaspase. PEG-r-crisantaspase, r-crisantaspase and n-crisantaspase were more efficient than EC-ASNase on less sensitive cell lines, which might be related to the 10-fold-greater glutaminase activity of crisantaspases than EC-ASNase. In vivo, after the first administration of PEG-r-crisantaspase in animals with ≥ 5% of leukemic cells, the leukemic cells were reduced to almost 0 % in mice within 4 days with all doses of PEG-r-crisantaspase tested, whereas the leukemic cells kept multiplying in the mice receiving vehicle. The 3 repeated injections of PEG-r-crisantaspase with an interval of 7 days delayed leukemia development for 17 days. All control mice receiving the vehicle had to be sacrificed at day 44 after leukemia inoculation, whereas of 28 mice receiving PEG-r-crisantaspase, 5 reached this endpoint at day 51 and the others at day 56. PEG-r-crisantaspase significantly improved the survival of leukemia-bearing mice for 7 to 12 days, regardless of the dose tested. Conclusions:These data provide evidence that PEG-r-crisantaspase has similar in vitro cytotoxic effect to n-crisantaspase on leukemia and lymphoma cell lines and significantly reduces the expansion of leukemic cells in leukemia-bearing mice, prolonging the survival of the animals. These results, together with preclinical PK/PD and immunogenicity data, support the clinical development of PEG-r-crisantaspase. A phase I dose escalation study in adult patients with relapsed or refractory hematological malignancies has been recently initiated.Research support from Alizé Pharma, the European Regional Development Fund (ERDF) and Grand Lyon. Disclosures:Allas:Alizé pharma: Employment. Sahakian:Alizé pharma: Employment. Julien:Alizé pharma: Employment. Abribat:Alizé pharma: Employment.

CRLF2 Rearrangement Is Not Associated with Activated mTOR Pathway in NOD/SCID/huALL Xenograft Mouse Model.

AbstractAbstract 2518B cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease and frequently associated with genetic alterations. However about one quarter of ALL patients lack characteristic chromosomal rearrangements representing a subset of leukemia not well understood. Expression of cytokine receptor-like factor 2 (CRLF2) has recently been shown to be up-regulated as well as mutated in BCP-ALL patients, including Down syndrome (DS-ALL) patients, lacking recurring chromosomal translocations. Several alterations in CRLF2 resulting in its overexpression have been described: a focal ∼320 kilobase interstitial deletion of the pseudo-autosomal region of the sex chromosomes (Xp22.23 or Yp11.32) that creates a fusion of CRLF2 to the G-protein-coupled purinergic receptor P2RY8 gene (P2RY8-CRLF2), translocation of CRLF2 with the IGH@ locus of chromosome 14, and a point mutation in CRLF2 resulting in a phenylalanine-to-cysteine substitution at amino acid 232 (F232C). Recently it has been shown that patients with CRLF2-P2RY8 fusion gene are associated with poor prognosis although they were initially stratified into intermediate risk (IR) group by minimal residual disease (MRD) criteria. This emphasizes the need to identify the mechanism of CRLF2 disregulation in leukemia thereby providing new therapeutic strategies.We have characterized leukemia samples for the presence of CRLF2 expression at the protein level by FACS, at transcript level by RT-PCR, mutations in CRLF2 and JAK2 by next generation amplicon sequencing and CRLF2-P2RY8 fusion gene and IGH@ translocation by RT-PCR. Interestingly, in our recent study we have observed activated mTOR pathway in a subset of B-ALL xenograft leukemia without recurring genetic alterations that were sensitive to mTOR inhibition ex vivo and in vivo. In view of the recently suggested interconnection between TSLP/CRLF2 and PI3K/mTOR signaling networks we analyzed the mTOR pathway activation in CRLF2 rearranged ALL.In this study we functionally address mTOR pathway activation in xenograft ALL samples with CRLF2 rearrangements and without any known chromosomal rearrangements ex vivo. We have adopted the NOD/SCID/huALL xenotransplant mouse model for this study. Xenograft leukemia cells with CRLF2 rearrangements and without recurring genetic alterations (CRLF2 rearrangements, n=4; CRLF2 wild type, n=11) were re-transplanted onto NOD/SCID mice and ALL cells were harvested at disease onset from leukemia bearing mice. The mTOR pathway activation was analyzed by flow cytometry assessing phosphorylation of ribosomal protein S6 (P-S6), a molecule downstream of the mTOR pathway. No significant difference in basal P-S6 level was found between CRLF2 rearranged and CRLF2 wild type leukemia. Moreover, reduction of the mTOR pathway activation upon mTOR inhibition by rapamycin and dual PI3K-mTOR inhibitor BEZ235 was analyzed. No significant reduction of the mTOR activity was observed in CRLF2 rearranged leukemia compared to CRLF2 wild type leukemia. Since JAK2 mutations have been associated with CRLF2 rearrangements, we also analyzed the JAK-STAT5 pathway assessing P-STAT5 ex vivo. Only one patient derived xenograft ALL sample carried a JAK2 mutation however no differential activation of JAK-STAT5 pathway was observed between JAK2wt/CRLF2r and JAK2mut/CRLF2r samples ex vivo. Also no significant difference in basal P-STAT5 was observed between CRLF2 rearranged and CRLF2 wild type leukemia. Additionally we checked the effect of mTOR inhibition on P-STAT5. No significant reduction in P-STAT5 level upon mTOR inhibition was observed pointing P-STAT5 activation independent of mTOR activation.In this study we have shown that CRLF2 rearranged leukemia is not associated with activated mTOR pathway and also not sensitive to mTOR inhibition either by rapamycin or dual PI3K-mTOR inhibitor NVP-BEZ235. In CRLF2 rearranged and CRLF2 wild type leukemia JAK-STAT5 pathway did not show differential activation and was unaffected by mTOR inhibition. It remains to be clarified whether both pathways could be activated with appropriate stimuli as was demonstrated for diagnostic specimens. To fully understand the exact mechanism of CRLF2 rearranged leukemia development and providing new therapeutic strategies, study the interlinks with other cell-signaling pathways is mandatory. Disclosures:No relevant conflicts of interest to declare.

Tissue Distribution of a Minor Antigen, but Not PD-1 Expression, Predicts the Antileukemia Efficacy of Adoptively Transferred, Antigen-Specific T-Cells in a Preclinical Model of Allogeneic Transplant

Abstract 456The clinical success of T-cell directed allogeneic immunotherapy for hematologic malignancy relies on the selection of antigens that elicit effective antitumor responses. While it has been demonstrated that vaccination against a single minor histocompability antigen (miHA) can cure solid and hematologic tumors, we have observed that alloreactivity produced by broad expression of miHA can reduce antigen-specific, solid tumor vaccine responses. In this study, we hypothesized that shared expression of a miHA would diminish the antileukemia potency of adoptively transferred, miHA-specific CD8 T-cells.We developed a murine allotransplant model that allowed us to identify and analyze HY-specific alloreactive CD8 T-cells after adoptive transfer using congenic markers. Following lethal irradiation, T-cell depleted female marrow was transplanted into allogeneic male [F B6(CD45.1+) > M B6(CD45.1+)= F>M] and syngeneic female [F B6(CD45.1+) > F B6(CD45.1+) = F>F] recipients. CD8 T cells (CD45.2+) specific for the Class I immunodominant HY peptide (HY-CD8) were then adoptively transferred. Using this method, we have generated clinically significant, nonlethal GVHD in male recipients and have demonstrated that, despite robust expansion of HY-CD8, these T cells are unable to mediate effective antitumor responses to an HY-expressing epithelial tumor (MB49). This phenomenon cannot be overcome with dendritic cell vaccination and is associated with high levels of PD-1 expression on HY-CD8.In the present study, we first determined whether tissue restriction of HY expression could improve HY-CD8 responses. [M>F] bone marrow chimeras were created to restrict HY expression to hematopoietic tissues, followed by adoptive transfer of HY-CD8 and MB49 subcutaneous challenge. In this setting, immunity to MB49 was preserved (55% +/− 7.4% tumor-free survival) without clinical signs of GVHD (p<0.0001) and maintenance of male marrow engraftment. As expected, HY-CD8 recovered from the bone marrow of tumor-free survivors maintained the ability to proliferate to HY peptide ex vivo by CFSE dilution. Unexpectedly, nearly 100% of HY-CD8 recovered from secondary lymphoid tissues of all [M>F] recipients expressed PD-1, regardless of whether they controlled MB49 in vivo. Syngeneic [F>F] recipients controlled MB49 equally well, (62 +/− 5.3% tumor-free survival), but neither tumor-bearing nor tumor-free survivors expressed PD-1 on recovered HY-CD8.Next, we determined whether hematopoietic restriction of HY could also preserve antileukemia responses. An immunogenic, HY-expressing, GFP+ murine pre-B cell leukemia was intravenously injected at the time of transplant and followed by HY-CD8 adoptive transfer. Unlike the solid tumor model, hematopoietic restriction of HY resulted in 100% leukemia-associated mortality (GFP+ marrow leukemia burden 64.6 +/− 9.7%) at a rate similar to [M>F] recipients that did not receive HY-CD8. HY-CD8 recovered from bone marrow of leukemia-bearing mice did not express PD-1. In contrast, [F>M] allogeneic recipients had 82.1 +/− 5.8% leukemia-free survival with 100% of HY-CD8 expressing PD-1. Similar to the solid tumor model, syngeneic [F>F] recipients had 62 +/− 5.2% survival, and no expression of PD-1 on HY-CD8.In summary, while adoptive transfer of CD8 T cells specific for a hematopoietically restricted minor antigen improved solid tumor immunity, it did not preserve antileukemia responses. Effective antileukemia responses were generated when the antigen was redistributed to nonhematopoietic tissues. These data suggest that the tissue location of an endogenous minor antigen relative to the tumor can directly impact the function of adoptive T-cells targeting that antigen. Further, PD-1 expression on antigen specific CD8 does not correlate with antileukemia efficacy, suggesting that PD-1 biology may be uniquely modulated by the alloimmune environment. Disclosures:No relevant conflicts of interest to declare.

The Small Molecule Inhibitor of VLA4 TBC3486 Sensitizes Resistant ALL to Chemotherapy

AbstractAbstract 1500Significant progress notwithstanding, drug resistant acute lymphoblastic leukemia (ALL) remains a therapeutic challenge, as well as acute and long-term off-target toxicity of anti-ALL therapies can be dose-limiting or debilitating. Therefore, the development of more targeted therapies is desirable. We recently provided evidence that chemotherapy resistance of ALL cells can be partly overcome by interfering with the function of VLA4, the alpha4beta1 integrin, in vivo. In those studies, we used the anti-functional antibody Natalizumab. We extended our studies to an alternative VLA4 inhibitor, the novel non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicate that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of VLA-4. As such, the compound has been shown to be efficacious in VLA-4 dependent models of inflammatory and autoimmune disease. The potential usefulness of this novel inhibitor in leukemia treatment was tested in our established in vitro and in vivo assays. LAX7R cells, primary pre-B-ALL with a normal karyotype from a patient with an early relapse, were used throughout for the studies reported here. LAX7R cells were treated with 25μM TBC3486 or THI0012 control, the inactive enantiomer of TBC3486, and seeded onto plates coated with human VCAM-1. Adhesion, scored after 2 days, was significantly inhibited by TBC3486 compared to control treated cells (7.9%±4.0 vs 95.4%±8.0; p=0.003). Proliferation rate and cell viability were unaffected by the treatments. In a co-culture system of LAX7R cells with OP9 stroma cells, which we use as an in vitro model of stroma-mediated chemotherapy resistance, we assessed differential effects of VDL (Vincristine, Dexamethasone, L-Asparaginase) on leukemia cell survival in the presence or absence of TBC3486. Stromal adhesion significantly protected LAX7R cells against VDL chemotherapy; this effect was significantly attenuated by TBC3486 compared to the control as determined by Trypan blue exclusion of dead cells (Cell viability of 39.9%±5.1 vs. 57.2±1.8; p=0.02). After these encouraging observations, we next evaluated the benefit of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R cells were lentivirally labelled with luciferase for in vivo tracking and injected into NOD/SCID hosts. Three days after leukemia cell transfer, mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2 weeks (intraperitoneally), with or without VDL chemotherapy. This experiment is in progress, but already survival of leukemia-bearing mice was significantly prolonged, from a median survival time (MST) for control mice of 33 days post-leukemia injection to a MST of 47 days post-leukemia injection for TBC3486 treated mice (p=0.02). Similarly, bioluminescence imaging revealed a marked delay of leukemia cell dissemination (p<0.0001). Taken together, our data demonstrate that small molecule inhibition of VLA4 using the novel TBC3486 is a suitable approach for targeting of chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant small molecule inhibition of integrins to overcome relapse of ALL. Disclosures:No relevant conflicts of interest to declare.

Bortezomib suppresses the growth of leukemia cells with Notch1 overexpression in vivo and in vitro

Bortezomib has been widely used in the treatment of various cancers; however, its exact mechanisms of action are not fully understood, particularly in acute T lymphoblast leukemia (T-ALL). Here, we visualize the anti-leukemia effect of bortezomib in both human T-ALL cell line and animal models. In vitro study, a human T-ALL cell line bearing Notch1 mutations, MOLT-4, was treated with bortezomib. At clinically achievable concentrations, bortezomib inhibited cell growth by inducing G1 phase arrest and apoptosis with a dose-dependent manner. A murine tumor xenograft model was achieved by subcutaneous injection of MOLT-4 cells for in vivo study. Administration of bortezomib significantly reduced tumor mass volume when compared with controls. Of note, bortezomib inhibited growth of leukemia cells in a Notch1-induced murine T-ALL model, and the life span of leukemia-bearing mice was markedly increased. Further studies revealed that bortezomib led to inhibited expression of Notch1 target genes. Taken together, our results demonstrate that bortezomib shows significant anti-leukemia effect in T-ALL bearing Notch1 mutations in vitro and in vivo. The present study provides evidence that bortezomib might be a candidate therapeutic reagent in the treatment of T-ALL.

Plasmacytoid dendritic cell enrichment of bone marrow allografts enhances GvL activity by facilitating donor T cell engraftment and Th1 polarization (126.25)

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) can cure patients with relapsed acute leukemia via a graft vs. leukemia (GvL) effect, but is complicated by graft vs. host disease (GvHD). Host dendritic cells (DC) following chemotherapy initiate GvHD in mouse models of allo-HSCT, but the role of donor DC is unclear. We have previously shown that transplanting purified plasmacytoid DC (pDC) along with HSC and T cells leads to Th1 donor T cell polarization and improved GvL activity. The goals of this study were: 1) To determine whether passive enrichment of pDC by depleting myeloid DC (mDC) from a bone marrow (BM) graft along with a fixed number of donor T cells would increase Th1 polarization and improve survival in leukemia-bearing mice, and 2) determine the role of IL-12 produced by donor pDC. Lethally irradiated B10.BR or FVB mice received BM and T cells from B6 or IL-12 KO B6 donors. Depletion of mDC from BM was achieved by FACS. Recipients of mDC-depleted BM had increased serum IFN-γ and increased donor T-cell proliferation and engraftment. Transplanting mDC-depleted BM enhanced survival without increasing GvHD in leukemia-bearing mice compared with undepleted BM. Enhanced proliferation and engraftment of donor T cells was not seen with mDC-depleted BM from IL-12 KO donors. These data show that pDC enrichment of BM allografts enhances donor T-cell Th1 polarization, engraftment, and GvL activity. The immune effects of mDC-depletion appear to be IL-12 dependent.

Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody

BackgroundThe assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents.MethodsTwo mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells.ResultsThe probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice.ConclusionsA mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.

In VitroandIn VivoAntitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL againstCD33+Acute Myeloid Leukemia

Genetic engineering of T cells with chimeric T-cell receptors (CARs) is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV-) specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34+ hematopoietic progenitors. Moreover, after intravenous administration into CD33+ human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

Predicting Clinical Dose-Exposure and Exposure-Response Relationships of Pan-Antiapoptotic BCL-2 Family Inhibitor Obatoclax in MLL Rearranged Infant Leukemias From Preclinical Disease Models and Adult Experience

Abstract 2580Challenges in developing new agents for infant MLL leukemia are compounded by rarity of cases, inherent drug resistance and unique vulnerabilities of infants to treatment-related morbidity and mortality. An additional limitation is the extent to which preclinical data and adult clinical experience can predict clinical dose-exposure and exposure-response relationships and guide dosing strategies (dose, duration, combination, schedule) and study designs (sample size, sampling scheme). Pharmacometric approaches are increasingly being used to quantify drug action and clinical performance. These require a continuum of models with hypotheses and assumptions and pharmacologic rationale to be challenged by targeted experimentation and subsequent model refinement. Key assumptions and experimental conditions are reassessed to optimize experimental designs and test evolving understanding about drug behavior and disease biology. Here we establish “proof-of-concept” quantitative pharmacology relationships between data derived from in vitro obatoclax activity in cell lines and patient samples, historical adult experience and a preclinical diseased mouse model to optimize strategies to treat infant leukemia.Obatoclax in vitro activity was quantified by MTT assays in MLL-rearranged cell lines and primary infant ALL and bilineal MLL leukemia samples. Obatoclax biodistribution was characterized in MLL leukemia-bearing mice to evaluate its potential to achieve target exposures in infants with leukemia by the following steps: 1) A nonlinear mixed-effect model based on existing obatoclax toxicokinetic data in healthy Balb/C mice guided design of a pilot study in diseased NOD-scid-IL-2Rgnull (NSG) mice where target exposure requirements were based on the AUC24h associated with peak oligonucleosomal DNA release as an apoptosis biomarker in an adult Phase I CLL trial (O'Brien 2008) and single-agent MTT assays of obatoclax in MLL cell lines and primary infant leukemia samples. 2) NSG MLL-ENL bilineal leukemia bearing mice with established xenografts received a single IV bolus dose of obatoclax (1.2 or 4.8 mg/kg; 100% or 400% of predicted target exposure). 3) Obatoclax was measured in the plasma, spleen, liver, kidney and brain for 24 h after drug administration (courtesy GeminX). A physiologically-based pharmacokinetic model (PBPK) model was developed to predict exposures in mouse target tissues based on obatoclax physicochemical properties and then scaled to a human infant.Preclinical in vitro experiments suggested IC50 values between 13 and 918 nM in primary MLL leukemia cells. Population mean (% RSE) parameter estimates for plasma clearance (CL), intercompartmental clearance (Q), plasma distribution volume (Vd) and peripheral distribution volume (V2) were 5.1 (10.1) L/h/kg, 1.78 (37) L/h/kg, 9.15 (10.2) L/kg and 17.3 (23.8) L/kg, respectively. PK parameters were close to those predicted from healthy Balb/C mice except that Cmax was lower, possibly suggesting a larger Vd in mice with disease. The half-life was almost 4-fold higher suggesting slower elimination due to mouse strain or disease burden. There was sustained drug retention in spleen, liver, kidney and brain. Brain:plasma were ∼2-10:1, higher than the <1:1 typical of most drugs, indicating excellent CNS penetration. The PBPK model for obatoclax in the mouse compared well with observed biodistribution in diseased NSG mouse tissues (Fig 1). Though brain penetration was good, deviations between observed and PBPK-simulated exposure values may suggest involvement of transporters. PBPK simulation of obatoclax administration to a 1 yr, 10 kg human infant suggested that adequate drug exposure to target organs should be achievable at clinically relevant doses (Fig 2).This work links models that define obatoclax in vitro exposure targets with in vivo exposure obtained in leukemia bearing mice. Predictability of these models relative to infant leukemia will be assessed following completion of the ongoing COG phase I obatoclax trial in relapsed/refractory pediatric cancers (ADVL0816). Our analyses imply that projecting outcome in treating infant MLL disease with obatoclax will be possible once these models are further refined. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.