scholarly journals A p53-MDM2 Interaction Inhibitor, DS-5272, Inhibits the Development of MLL-Fusion Leukemia with the Assistance of Tumor Immunity

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 796-796
Author(s):  
Susumu Goyama ◽  
Yasutaka Hayashi ◽  
XiaoXiao Liu ◽  
Shiori Shikata ◽  
Yosuke Tanaka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Tumor Immunity ◽  
P53 Protein ◽  
Leukemia Cells ◽  
Mouse Leukemia ◽  
Nsg Mice ◽  
P53 Activation

Abstract MLL-fusion leukemia is an aggressive form of leukemia carrying chimeric fusion of the MLL gene. Adverse therapy response of MLL-fusion leukemia is partly associated with the attenuated p53 response. The principal cellular antagonist of p53 is an E3 ubiquitin ligase MDM2. MDM2 binds to p53 and induces proteasomal degradation to downregulate p53 protein level. Therefore, targeting of p53-MDM2 interaction to reactivate p53 function is an attractive therapeutic approach for MLL-fusion leukemia. In this study, we assessed the effect of an orally active inhibitor of p53-MDM2 interaction, DS-5272, in a mouse leukemia model driven by MLL-AF9. DS-5272 inhibited in vitro growth of mouse leukemia cells transformed by MLL-AF9 with the IC50 value in the nanomolar range. A single administration of DS-5272 in vivo upregulated expression of p53 protein as well as its target genes, and induced cell cycle arrest, apoptosis, and differentiation of MLL-AF9 cells. Multiple oral doseadministration of DS-5272 (3 times per week) caused nearly complete tumor regressions of MLL-AF9 leukemia cells that were sustained well beyond the drug administration period with tolerable toxicity. In contrast, DS-5272 showed little effect on p53-deficient bone marrow cells transformed by MLL-AF9 both in vitro and in vivo, confirming that the antileukemic effect of DS-5272 is mediated by p53 activation. Despite the remarkable effect of DS-5272 against MLL-AF9 leukemia with wild-type p53, all mice eventually developed leukemia after a long latency, indicating the existence of leukemia stem cells (LSCs) that are resistant to p53 activation. The therapy-resistant LSCs were relatively enriched within the bone marrow (BM) endosteal region where osteoblasts reside. In contrast, LSCs in the BM central portion were eliminated almost completely by the DS-5272 treatment. Thus, LSCs within the osteoblast-rich area appear to be protected from p53-induced cell death. RNA-Seq analyses revealed the upregulation of inflammation- and interferon-associated genes in MLL-AF9 leukemia cells treated with DS-5272. Furthermore, DS-5272 treatment induced upregulation of PD-L1, a well-known suppressor of tumor immunity, in MLL-AF9 cells. These expression changes suggest that p53 activation triggered an immune-inflammatory response that led to leukemia regression. Interestingly, LSCs reside in the BM endosteal region expressed higher level of PD-L1 compared with those in the BM central portion, which may account for their low sensitivity to DS-5272. To examine the potential contribution of tumor immunity to the DS-5272-mediated suppression of leukemic growth, we next assessed the in vivo effect of DS-5272 on MLL-AF9 leukemia using immunodeficient NOD.Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice as recipients. Strikingly, the antileukemia effect of DS-5272 was markedly attenuated in NSG mice, indicating the important role of tumor immunity to enhance the efficacy of DS-5272. We then depleted PD-L1 in MLL-AF9 cells using the CRISPR/Cas9 system, and found that PD-L1-depleted MLL-AF9 cells become more sensitive to DS-5272 treatment. These data suggest that DS-5272 inhibits the development of MLL-AF9 leukemia with the assistance of tumor immunity, and its therapeutic efficacy is enhanced by PD-L1 inhibition in MLL-AF9 cells. In summary, our study demonstrated the potent antitumor activity of a p53-MDM2 interaction inhibitor, DS-5272, against MLL-fusion leukemia with intact p53. LSCs reside within the BM endosteal region express high level of PD-L1 and are relatively resistant to DS-5272 treatment. Combining p53-MDM2 inhibitors with the immune checkpoint inhibitors may result in synergistic enhancement of cell death and improved efficacy in the treatment of myeloid leukemia. Disclosures Kitamura: Daiichi Sankyo: Research Funding.

Proteomic, Gene Expression, and Micro-RNA Analysis Of Bone Marrow Mesenchymal Stromal Cells In Acute Myeloid Leukemia Identifies Pro-Inflammatory, Pro-Survival Signatures In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3685-3685
Author(s):  
Michael Andreeff ◽  
Rui-yu Wang ◽  
Richard E. Davis ◽  
Rodrigo Jacamo ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Micro Rna ◽  
Leukemia Cells ◽  
Rna Analysis ◽  
Nsg Mice ◽  
Acute Myeloid

Abstract The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) generates resistance signals that protect AML cells/stem cells from chemotherapy. The mechanisms how the BME might support leukemia cell survival are unclear but elucidation of this process could prove useful for therapy design. Here we report new insights specific to stroma functionality in AML. A series of novel experimental approaches were developed including : 1) nanostring micro-RNA and proteomic analysis using reverse-phase protein arrays (RPPAs) of MSC derived from AML patients and normal donors; 2) genome-wide RNA analysis of FACS-sorted MSCs using Illumina arrays of genetically-defined human and murine AML cell lines/primary AML samples after co-culture with normal MSC in vitro; 3) in vivo interaction between genetically-defined murine AML and stromal cells in syngenic C57BL/6J mice, followed by harvesting and FACS-isolation of specific MSC after leukemia engraftment; 4) use of genetically-modified human MSC in vivo in our ectopic humanized bone marrow model in NSG mice (Blood 2012 : 119,4971), followed by bioluminescence growth and homing analysis of human leukemia cells. This model allows the study of in vivo effects of altered MSC on human AML development. 1) Proteomic and transcriptomic analysis of primary MSC from AML patients (n = 106) and normal MSC (n = 71) by RPPA using validated mAbs to 150 proteins and phospho-proteins demonstrated major differences by hierarchical clustering analysis: GSKA, STAT1, PDK1, PP2A, CDKN1A, CDK4, and STAT5AB were significantly over-expressed in AML- vs. normal MSC, while STMN1, SIRT1, SMAD1, SMAD4, HSP90 and EIF2S1 were under-expressed. Differences were observed between MSC from newly diagnosed vs. relapsed AML-MSC. Nanostring analysis of 38 AML-MSC and 24 normal MSC identified differential expression of numerous miRs, a select group of which has been validated so far by qRT-PCR. AML MSC express reduced levels of let-7g, let-7c, miR 21 and miR93, and elevated levels of miR410 compared to normal MSC. Pathways were identified in MSC that might contribute to leukemia survival. 2) Analysis OCI-AML3 cells co-cultured with normal -MSC revealed upregulation of a variety of genes in MSC encoding cytokines and chemokines and gene set enrichment analysis (GSEA) identified activation of NFkB in MSC as a potential cause of these changes. When the ectopic humanized bone marrow model system in NSG mice was used, suppression of NFkB in MSC resulted in a ∼ 50% reduction of AML burden. When murine MSC cultured with wt p53 MLL/ENL-Luc-FLT3-ITD cells were compared to isogenic cells with deleted p53, striking differences were seen in the MSC transcriptome: 429 differentially expressed genes were identified that distinguished co-cultures with p53wt and p53-/- cells, suggesting that AML cells may communicate signals to their microenvironment in a p53-dependent manner. GSEA identified NFkB and HIF-1a as targets, data were confirmed independently, and HIF-1a knockout MSC were found to be inhospitable for AML in the ectopic in vivo model. 3) These syngenic cells were introduced into B57BL/6J mice and MSC were isolated after leukemia engraftment: 147 genes were consistently upregulated and 236 genes downregulated in MSC by their interactions with AML in vivo. Upregulated genes included CTGF, CXCL12, genes related to complement (C4A, C4B, Serpin G1), and IGFBP5, an inhibitor of osteoblast differentiation. Identification of CXCL12 was intriguing as Link's group recently reported the critical role of CXCL12 produced by early MSC in normal hematopoiesis (Nature 2013 : 495,227). Both AML-ETO and MLL-ENL leukemias caused upregulation of CTGF, metalloproteinases, adhesion molecules, and NFkB-related genes in vivo. IPA analysis showed responses in BM-MSC associated with inflammation, cellular movement, cell-cell signaling, cellular growth and proliferation and immune cell trafficking. Conclusion AML cells induce changes in MSC, in short term co-cultures in vitro, or in syngenic systems in vivo, that are consistent with pro-survival, anti-apoptotic, and growth-stimulatory signals that mimic inflammatory responses. Large-scale analysis of primary AML-derived MSC confirms and extends this data. Results facilitate the development of therapeutic strategies to render the BM microenvironment inhospitable to leukemia cells but supportive of normal hematopoiesis. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1224-1224
Author(s):  
Jerry C. Cheng ◽  
Dejah Judelson ◽  
Kentaro Kinjo ◽  
Jenny Chang ◽  
Elliot Landaw ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Leukemia Cells ◽  
Mouse Bone Marrow ◽  
T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 893-893
Author(s):  
Po Yee Mak ◽  
Duncan H Mak ◽  
Yuexi Shi ◽  
Vivian Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Apoptosis Resistance ◽  
Control Mscs ◽  
Extrinsic Apoptosis ◽  
Induced Apoptosis ◽  
And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3090-3090 ◽  
Author(s):  
Folashade Otegbeye ◽  
Nathan Mackowski ◽  
Evelyn Ojo ◽  
Marcos De Lima ◽  
David N. Wald
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Leukemia Cells ◽  
Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

scholarly journals BST-236, a Novel Cytarabine Prodrug, Is Safer and As Effective As Cytarabine in In Vivo Leukemia Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1451-1451 ◽  
Author(s):  
Shoshi Tessler ◽  
Inbal Mishalian ◽  
Ronny Peri-Naor ◽  
Stela Gengrinovitch ◽  
Raphael Mayer ◽  
...  
Keyword(s):  
Weight Loss ◽  
Cell Death ◽  
Systemic Toxicity ◽  
In Vivo Studies ◽  
Leukemia Cells ◽  
Severe Toxicity ◽  
Unfit Patients ◽  
Kinetics Of

Abstract Introduction: Acute myeloid leukemia (AML) is associated with poor outcomes in older and medically unfit patients, largely due to the severe toxicity associated with cytarabine treatment, which precludes the administration of effective cytarabine doses. BST-236 is a prodrug of cytarabine, inactive in its prodrug form and designed to deliver cytarabine to leukemia cells with reduced systemic toxicity, thus to enable delivery of high cytarabine doses to leukemia cells with relative sparing of normal tissues. Results: BST-236 is a conjugate of cytarabine and asparagine (Figure 1). It was demonstrated that BST-236 is inactive as an intact prodrug and that its activity is exerted by release of cytarabine via non-enzymatic hydrolysis. Unlike free cytarabine, the bound cytarabine in BST-236 is not phosphorylated into its active metabolite Ara-CTP and it is protected by the asparagine residue from deamination into its inactive form Ara-U. In vitro studies demonstrate that BST-236 enters into leukemia cells, accompanied by cellular accumulation of free cytarabine, which is released from BST-236 (Figure 2). Like cytarabine, treatment with BST-236 result in induction of cell death of various leukemia cell lines via apoptosis, an activity which is dependent on the human equilibrative nucleoside transporter 1 (hENT1). The in vitro kinetics of BST-236-induced toxicity were found to be delayed compared to administration of free cytarabine, correlating with an observed delayed cellular availability of cytarabine. In vivo studies in mice and dogs demonstrate that BST-236 concentrations in the plasma are dose-proportional, with a prodrug-typical profile and only ~5% of free cytarabine present in the plasma. The maximal tolerated dose of BST-236 was found to be several-fold higher than reported for cytarabine, with mainly hematological effects and no unexpected toxicities. In vivo head-to-head studies in human leukemia mouse models with equimolar doses of cytarabine and BST-236 demonstrate similar efficacy of complete elimination of the leukemia cells in the bone marrow, spleen, and peripheral blood by both molecules (Figure 3A). However, while cytarabine treatment was associated with significant toxicity including weight loss, dramatic reduction in spleen size and number of mouse spleen cells, and delayed normal murine white blood cell recovery, equimolar BST-236 doses enabled spleen and BM recovery with minimal weight loss and no observed clinical signs (Figure 3B, 3C). Summary: in vitro and in vivo studies demonstrate that BST-236 is a prodrug of cytarabine, which enables the delivery of cytarabine to target cells, resulting in elimination of the leukemia with reduced systemic toxicity compared to free cytarabine. The data also suggest that while the mechanism of cell death induced by BST-236 and cytarabine is similar, the observed differential kinetics of the delivery of cytarabine by BST-236 and its metabolism may explain its reduced systemic toxicity. Our nonclinical findings are in line with the clinical results of the BST-236 Phase 1/2 study (ASH 2017 abstract no 893, manuscript in preparation) and suggest that BST-236 may enable delivery of high cytarabine doses to older and medically-unfit patients who currently cannot benefit from an effective cytarabine therapy. This suggestion is to be confirmed by an ongoing Phase 2b study. Disclosures Tessler: Biosight: Employment. Gengrinovitch:Biosight: Employment. Ben Yakar:Biosight: Employment. Peled:Cellect Biotherapeutics Ltd: Consultancy. Flaishon:Biosight: Employment.

scholarly journals Human Cytokine-Induced Memory-like NK Cells Exhibit in Vivo Anti-Leukemia Activity in Xenografted NSG Mice and in Patients with Acute Myeloid Leukemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 101-101
Author(s):  
Rizwan Romee ◽  
Rosario Maximillian ◽  
Melissa M Berrien-Elliott ◽  
Julia A Wagner ◽  
Brea A Jewell ◽  
...  
Keyword(s):  
Nk Cells ◽  
Myeloid Leukemia ◽  
Low Dose ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Cell Frequency ◽  
Nsg Mice ◽  
Post Infusion

Abstract Natural killer (NK) cells mediate anti-AML responses and previously published clinical trials of adoptive allogeneic NK cell therapy provide proof-of-principle that NK cells may eliminate leukemia cells in patients. However, complete remissions occur in 30-50% of patients with active AML and are typically of limited duration. Thus, improvements are needed for this promising cellular immunotherapy strategy. Following paradigm-shifting studies in mice, it was established that human NK cells exhibit an innate 'memory-like' responses following a brief, combined pre-activation with IL-12, -15, and -18 (Romee R et. al., Blood, 2012). These long-lived memory-like NK cells have an enhanced ability to produce IFN-g in response to restimulation with cytokines or activating receptor ligation, even following extensive proliferation. We hypothesized that memory-like NK cells exhibit enhanced responses to myeloid leukemia. Compared to control NK cells from the same donor, IL-12/15/18-induced memory-like NK cells produced significantly increased IFN-g upon co-culture with primary AML blasts in vitro (P<0.001), following 7 days of rest in low dose IL-15 vitro. In addition, memory-like NK cells had increased granzyme B expression (P<0.01), and enhanced killing of K562 leukemia targets in vitro (P<0.05). Utilizing an in vivo xenograft model of human NK cells in NSG mice (Leong J et. al., BBMT, 2014), IL-12/15/18-induced memory-like NK cells that differentiated in NSG mice for 7 days exhibited increased IFN-g responses after ex vivo re-stimulation with K562 leukemia, confirming their memory-like functionality (P<0.05). To test in vivo responses to human leukemia in this model, luciferase-expressing K562 cells were engrafted into NSG mice (1x106/mouse, IV), and on day 3, groups of mice were injected with IL-12/15/18-pre-activated or control NK cells from the same donor (4x106/mouse). Mice treated with a single dose of memory-like NK cells exhibited significantly improved in vivo leukemia control measured by whole mouse bioluminescent imaging (P=0.03), as well as overall survival (P<0.05), compared to mice treated with control or no NK cells. Based on these pre-clinical findings, we initiated a first-in-human clinical trial of HLA-haploidentical IL-12/15/18-induced memory-like NK cells in patients with AML (NCT01898793). Relapsed/refractory (rel/ref) AML patients receive lymphodepleting non-myeloablative flu/cy conditioning, infusion of a single dose of CD56+CD3- memory-like donor NK cells, followed by two weeks of low dose rhIL-2. Three patients were treated at dose level 1 (0.5x106 cells/kg) and two patients treated at dose level 2 (1.0x106/kg) with no DLTs observed, and accrual continues. Correlative analyses utilizing donor-specific HLA mAbs allow tracking of donor memory-like NK cell frequency and function following adoptive transfer. Donor memory-like NK cells were detectable in the PB and BM of all tested patients with informative HLA (4/5), peak in frequency at 7-8 days post-infusion, and contract after 14-21 days as expected following recipient T cell recovery (Figure). Memory-like NK cells exhibit significantly increased Ki67%+ as a marker of proliferation at day 7 [97.8+1.0% (donor) vs. 21.6+5.5% (recipient), mean+SEM, P<0.001]. Moreover, functional analyses of NK cells at days 7-8 post-infusion reveal increased numbers of donor IFN-g+ NK cells following restimulation with K562 leukemia cells in the same blood [1009+590 (donor) vs. 8+3 (recipient) IFN-g+ NK cells] or BM [686+423 (donor) vs. 4+2 (recipient) IFN-g+ NK cells] samples. Two of four evaluable patients treated with memory-like NK cells had leukemia free BM and PB at days 14 post-therapy, which correlated with BM NK cell frequency and IFN-g production (Figure). CIML007 had rel/ref AML with 48% BM blasts pre-therapy, and had no evidence of leukemia on day 14, 28, and 100 BM biopsies, and has an ongoing complete remission more than 100 days after this therapy. CIML009 had 80% BM blasts pre-therapy, and had no evidence of leukemia on day 14 BM biopsy post-infusion. Thus, human IL-12/15/18-induced memory-like NK cells expand and have enhanced anti-AML function following adoptive transfer in patients, thereby constituting a promising translational innovation for immunotherapy of AML. Figure 1. Figure 1. Disclosures Fehniger: Celgene: Research Funding.

Targeting apoptosis and autophagy by a novel bcl-2 inhibitor, GX15-070, in neuroblastoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 10048-10048
Author(s):  
Herve Sartelet ◽  
Sonia Cournoyer ◽  
Anissa Addioui ◽  
Assila Belounis ◽  
Mona Beaunoyer ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Survival ◽  
Annexin V ◽  
High Sensitivity ◽  
Western Blots ◽  
Pediatric Tumor ◽  
Nsg Mice ◽  
Apoptotic Protein

10048 Background: Neuroblastoma (NB) is a frequent pediatric tumor with poor prognosis. The disregulation of the anti-apoptotic protein Bcl-2 is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate the in vitro and in vivo therapeutic efficiency of GX 15-070, a Bcl-2 inhibitor, used alone and in combination with conventional drugs used in the treatment of NB and hydroxychloroquine (HCQ), a known autophagy inhibitor. Methods: Using 6 NB cell lines, cell viability (MTT) assays were done at progressively increased concentrations of GX 15-070 alone or in combination with cisplatin or with Z-VAD-FMK, a broad-spectrum caspase inhibitor. Apoptosis was tested by evaluating the cleavage of caspase 3 by western blots (WB) and the Annexin V/7-AAD staining studied by FACS. To assess if autophagy was modified by GX 15-070, the cleavage of LC3 protein was tested by WB and cell survival were tested with combination of GX 15-070 and HCQ. To verify the anti-tumor activity in vivo of GX 15-070, orthotopic injections were made on NSG mice treated with GX 15-070 alone and in combination with HCQ. Results: It was observed a high sensitivity of the NB cells to GX 15-070 with increase of cell death and a potential synergistic of this molecule when it’s combined with cisplatin or HCQ. This cell death was due to apoptosis and may also be inhibited by Z-VAD-FMK. GX 15-070 alone or associated to cisplatin increased the autophagy. The in vivo study showed that GX 15-070 treatment used alone or in combination with HCQ significantly decreased the size of the tumor. Conclusions: Our results support the interest of GX 15-070 in the treatment of NB alone or in combination with classical drugs. Our studies also support that activation of apoptosis associated with inhibition of autophagy have a synergistic potential against tumoral progression and must have to be considered in further mechanistic studies for the optimization of more efficient combined therapies in the treatment of NB.

Abstract 130: Ox-LDL Impairs The Survival Of Bone Marrow Stem Cells Partially Through Membrane Damage Independent Of ROS Production In Vitro

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Xin Li ◽  
Yuan Xiao ◽  
Yuqi Cui ◽  
Hua Zhu ◽  
Chandrakala A Narasimhulu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Death ◽  
Membrane Damage ◽  
Final Concentration ◽  
Bone Marrow Stem Cells ◽  
Cell Membrane Damage ◽  
Cell Based Therapy

Aims: cell-based therapy with bone marrow stem cells (MSCs) remains a viable option for tissue repair and regeneration. One of the major challenges for cell-based therapy is the limited survival of the cells after in vivo administration. The exact mechanism(s) for impaired in vivo survival of the implanted MSCs remains to be defined. Oxidized low-density lipid protein (ox-LDL) is a natural product in human blood, and the major contributor to the development of atherosclerosis. The present study was to investigate the effect of ox-LDL on the survival of bone marrow stem cells and the mechanisms in vitro. Methods and Results: Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL (with the final concentration of 10 and 20 ug/ml) for up to 48 hours. Exposure to ox-LDL resulted in significant cell death and apoptosis of MAPCs in association with a significant increase in LDH release in the conditioned media in a dose- and time-dependent manner, indicating significant cell membrane damage. The membrane damage was further confirmed with the rapid entry of the small fluorescent dye FM1-43 as detected using confocal microscope. Ox-LDL generated a significant amount of reactive oxygen species (ROS) in the culture system as measured with electron paramagnetic resonance spectroscopy. The antioxidant N-acetylcysteine (NAC, 0.1 mM) completely inhibited the production of ROS from ox-LDL. However, it didn’t prevent ox-LDL-induced cell death or apoptosis. However, pre-treatment of the cells with the specific membrane protective recombinant human MG53 protein (rhMG53)(66 ug/ml, final concentration) significantly, reduced LDH release and the entry of FM1-43 dye into the cells exposed to ox-LDL. Conclusion: Ox-LDL enhanced cell death and apoptosis of MAPCs with a mechanism independent of ROS generation in vitro. Ox-LDL impaired the survival of MAPCs partially through cell membrane damage in vitro.

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