Toll-Like Receptor Agonists Induce Immunogeneicity and Apoptosis of Acute Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 160-160
Author(s):  
Maki Shindo ◽  
Xueqing Liang ◽  
Zhimei Wang ◽  
Jeffery S. Miller ◽  
Martin Carroll ◽  
...  
Keyword(s):  
T Cell ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Toll Like Receptor ◽  
Tlr Agonists ◽  
Tlr7 Agonist ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a common form of acute leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improve cure rates in AML patients. Toll-like receptor (TLR) agonists have been shown to elicit anti-leukemia effects in murine AML models. However, TLR expression profile of human AML cells is unknown. We analyzed TLR1-10 mRNA expression in purified AML cells from 41 patients with different AML subtypes (M0, M1, M2, M3, M4, or M5; n > 5 per group) by real-time RT-PCR. The majority of AML samples expressed high level of TLR2, 4, 7, 8, low level of TLR1, 5, 9, 10, and undetectable level of TLR3. Significant higher TLR4 and TLR7 expressions were detected on M4 and M5 subtypes of AML cells. Triggering TLR4 or TLR7 with specific TLR agonists (Monophosphoryl Lipid A or Imiquimod) significantly increased the surface expression of molecules essential for T cell activation (CD54, CD80, CD86) on AML M4/M5 cells and enhanced T-cell mediated proliferative responses against AML cells. Thus, TLR signaling enhances the immunogenicity of AML M4/M5 cells and makes them more suitable targets for T cell mediated attack. Most importantly, TLR7 agonist strongly induced apoptotic death of primary AML M4/M5 cells and inhibited the growth of TLR7-expressing AML cell lines (U937, HL-60, KG-1) in culture in a drug dose dependent manner. The addition of TLR7 agonist at 10 ug/ml fully induced apoptosis of AML cells and inhibited the growth of AML cell lines, as confirmed by viable cell counts and TMRE staining. Intracellular staining demonstrated that TLR7 agonist treatment significantly down-regulated the signal transducer and activator of transcription (STAT)3 and STAT5 protein expression in AML cells. These results suggest that TLR7 agonist-induced apoptosis of AML cells is likely via inhibition of STAT3 and/or STAT5 signaling pathway. To study the in vivo effects of TLR7 agonist against human AML cells, primary AML M4/M5 cells or a monocytic AML cell line (U937) were injected i.p. into NOD-SCID IL2Rgamma<null> mice with or without subsequent TLR7 agonist treatment. Mice receiving TLR7 agonist treatment (1 mg/kg daily i.p. infusion for 5 days) significantly reduced tumor burden with substantially lower numbers of engrafted leukemia cells detected in these xenograft mice. Flow cytometry results confirmed that residual AML cells recovered from mice treated with TLR7 agonist were apoptotic with down-regulated expression of TMRE and STAT3/STAT5, confirming previous in vitro findings that TLR7 agonist-treated AML cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established human AML is being investigated using these xenograft mouse models. In summary, our results provide the first report of TLR expression profile of human AML cells and demonstrate that TLR targeting of AML cells can increase the immunogeneicity of leukemia cells and directly induce AML apoptosis in vitro and in vivo, providing new insights into the biology and therapy of AML.

Toll-Like Receptor-7 Agonist Directly Inhibits the Growth and Induces Apoptosis of MLL-AF9 Leukemia Cells in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2989-2989
Author(s):  
Xueqing Liang ◽  
Ashish Kumar ◽  
Zhimei Wang ◽  
Brenda Weigel ◽  
Bruce R Blazar ◽  
...  
Keyword(s):  
Lethal Dose ◽  
Poly I:C ◽  
Leukemia Cells ◽  
Toll Like Receptor ◽  
Intracellular Staining ◽  
Tlr Agonists ◽  
Tlr7 Agonist ◽  
Regulated Expression

Abstract Leukemia involving rearrangements of the MLL gene is resistant to standard therapies and is often a fatal disease. MLL gene-rearrangements are commonly associated with infant-leukemia and secondary leukemias. New therapeutic strategies are needed to achieve longer survival and improve cure rates in patients with these and other refractory leukemias. Toll-like receptor (TLR) agonists are known as potent immune stimulatory agents that can elicit host anti-tumor effects in murine tumor models. We hypothesized that targeting TLRs expressed on leukemia cells with TLR agonists may have direct antitumor effects against leukemia cells. In this study, we investigated the effects of TLR agonists specific for TLR3, 4, 7, and 9, (i.e., polyinosine-polycytidylic acid (Poly(I:C)), monophosphoryl lipid A (MPL), imiquimod (IMQ), and CpG oligodeoxynucleotides (CpG ODN)), in MLL-AF9 knock-in mice that develop myeloid leukemia akin to human MLL-AF9 disease. In contrast to Poly(I:C), MPL, and CpG ODN, treatment of MLLAF9 cells with TLR7 agonist IMQ significantly increased the surface expression of CD40, CD54, CD80, and CD86 on MLL-AF9 cells in vitro. TLR7 mRNA and protein expression in MLL-AF9 cells were confirmed by real-time RT-PCR and intracellular staining/FACS analysis. Most importantly, TLR7 agonist strongly inhibited the in vitro MLL-AF9 cells in a drug dose- and treatment time-dependent manner. Whereas MLL-AF9 cells proliferated rapidly in culture with more than 40-fold increase of cell number in 5 days, the addition of IMQ at 5 mcg/ml fully inhibited the growth and induced profound apoptosis of MLL-AF9 cells with less than 2% of leukemia cells left at day 5 of culture. IMQ-mediated apoptotic death of MLL-AF9 was confirmed by viable cell counts, TMRE staining, Western blots and intracellular staining of the cleavage of caspases and PARP. Preincubation of MLLAF9 cells with caspases 8 and 10 inhibitors effectively blocked IMQ-induced apoptosis and sustained the proliferation of leukemia cells in cultures. To further determine the intracellular pathways engaged by IMQ, microarray gene expression profiles of 24-hour IMQ-treated vs. untreated MLL-AF9 cells were compared. Gene Set Enrichment Analysis (GSEA) showed that IMQ treatment resulted in up-regulated expression of a set of proapoptotic genes (e.g., p53, Bax, caspase 8, Apaf-1, etc) involved in apoptotic pathways. To determine whether IMQ pre-treatment of MLL-AF9 cells would prolong survival due to an apoptotic effect, cohorts of NOD-scid IL2Rgamma mice were i.p. injected with a lethal dose of MLL-AF9 cells with or without pre-incubation with IMQ. Mice receiving 5×106 untreated MLL-AF9 cells resulted in uniform lethality in 4 weeks. In contrast, mice receiving the same lethal dose of MLL-AF9 cells pretreated with IMQ had a significant prolonged survival, confirming in vitro findings that IMQ-treated MLL-AF9 cells undergo apoptosis. Administration of IMQ (daily i.p. injection at 1 mg/kg for 5 days) strongly inhibited leukemia cells growth and significantly prolonged the survival time of MLLAF9 mice. Flow cytometry results confirmed that residual MLL-AF9 cells recovered from IMQ treated mice were apoptotic and had up-regulated expression of cleaved caspases and PARP. In summary, our results demonstrate that TLR7 targeting of MLL-AF9 cells can directly induce apoptosis of MLL-AF9 cells in vitro and in vivo, providing new insights into the TLR-targeted therapy of refractory or relapsed leukemias.

scholarly journals Checkpoint Blockade in Combination with CD33 Chimeric Antigen Receptor T Cell Therapy and Hypomethylating Agent Against Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1383-1383 ◽  
Author(s):  
Tongyuan Xue ◽  
Marissa Del Real ◽  
Emanuela Marcucci ◽  
Candida Toribio ◽  
Sonia Maryam Setayesh ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
T Cell Therapy ◽  
Programmed Death ◽  
Car T ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The cure rate for primary AML patients is only 35% and decreases with age. Novel and effective immunotherapies for patients with relapsed and/or refractory (r/r) AML remain an urgent unmet need. CD33 is an attractive immunotherapeutic target for myeloid malignancies given its expression on more than 85% of AML patient samples. We therefore set out to design and test CD33 chimeric antigen receptor (CD33CAR) T cells preclinically as a single agent and in combinational therapy. To assess antileukemic responses of CD33CAR T cells in vitro and in vivo, we enriched CD4/CD8 T cells from peripheral blood mononuclear cells (PBMCs) and genetically modified them to express a second-generation CD33CAR. CD33CAR T cells exhibited potent antigen dependent CD107a degranulation, IFN-γ production and killing activities against AML cells in vitro. Using a NOD-SCID-IL2Rgnull (NSG) xenograft model engrafted with MOLM-14-ffluc, a CD33 expressing AML cell line transduced with lentivirus carrying firefly luciferase (ffluc) and enhanced green fluorescent protein (eGFP), 3 million CD33CAR or mock T cells were introduced intravenously. CD33 CAR T cell-treated group displayed 98.2% leukemic regression 4 days post CAR T infusion, and 99.6% reduction on day 31. Bioluminescent imaging (BLI) and Kaplan-Meier analysis demonstrated that CD33CAR T cells significantly decreased leukemic burden and prolonged overall survival compared to mock T cells in vivo. Decitabine, a DNA hypomethylating agent (HMA), is a main therapeutic agent for treating AML. We observed HMA treatment led to increased CD33 expression on MOLM-14 cells in vitro. We hypothesized that decitabine can potentiate CD33CAR T cell-mediated AML killing by increasing CD33 expression. MOLM-14 cells were treated with either decitabine alone, CD33CAR T cells alone, or sequential treatment using various concentrations of decitabine or DMSO followed by CD33CAR or mock T cells in an E:T ratio of 1:100. We determined the target specific killing activities in each group using flow cytometric based analysis 48 and 96 hours later. The decitabine followed by CD33CAR T cells treatment reproducibly resulted in the most robust antileukemic activity with 80.6% MOLM-14 cells killed. In comparison, CD33CAR T cells or decitabine monotherapy resulted in 11.5% and 50.9% killing, respectively. In vivo testing of the combinational effects of decitabine and CD33CAR T cells are underway and will be updated at the meeting. Finally, checkpoint blockade targeting programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) has shown survival benefits, particularly in combination with HMA, for patients with r/r AML (Daver et al. 2019). We observed elevated PD-L1 expression on residual AML blasts that survived the treatment with decitabine in combination with CD33CAR T cells. Therefore, we hypothesized that blockade of PD-1/PD-L1 interaction might further improve the antileukemic effect of CD33CAR T cells against AML cells post antigen induction by decitabine. MOLM-14 cells were treated with decitabine for 2 days and CD33CAR T cells were added in an E:T ratio of 1:75. Anti-PD-1 or IgG4 antibody was added to the culture at various concentrations. The most robust CD33 specific killing was seen in the culture with anti-PD-1 antibody added. Further characterization are underway and will be presented. Taken together, our preclinical findings have demonstrated the potency of the CD33CAR T cell therapy and ways to optimize its efficacy. Our results support clinical translation of CD33CAR T cells for patients with AML. Disclosures Budde: F. Hoffmann-La Roche Ltd: Consultancy.

Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2846-2846 ◽  
Author(s):  
Nan-Nan Zhang ◽  
Lei Chen ◽  
Wu Zhang ◽  
Xian-Yang Li ◽  
Lin-Jia Jiang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Terminal Part ◽  
Granulocytic Differentiation ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.

scholarly journals Arginine deprivation using pegylated arginine deiminase has activity against primary acute myeloid leukemia cells in vivo

Blood ◽  
2015 ◽  
Vol 125 (26) ◽  
pp. 4060-4068 ◽  
Author(s):  
Farideh Miraki-Moud ◽  
Essam Ghazaly ◽  
Linda Ariza-McNaughton ◽  
Katharine A. Hodby ◽  
Andrew Clear ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Arginine Deiminase ◽  
Leukemia Cells ◽  
Arginine Deprivation ◽  
Key Points ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Key Points Most AMLs lack ASS1, which allows synthesis of arginine, and so depend on exogenous sources. Depletion of arginine via ADI-PEG 20 reduces the burden of primary AML in vivo and in vitro.

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.

Disulfiram, an clinically used anti-alcoholism drug has the potential to target acute myeloid leukemia cells in a copper-dependent manner in vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5040-5040
Author(s):  
Bing Xu ◽  
Rongwei Li ◽  
Huijuan Dong ◽  
Feili Chen ◽  
Yuejian Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Specific Antigen ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Control Group ◽  
Acute Myeloid

Abstract Background Disulfiram(DS), an old drug clinically used for alcoholism, was reported to have antitumor effects, recent studies have found that Copper(Cu) can significantly enhance the DS-induced cell death in vitro in a variety of tumor cells. Our previous studies also demonstrated that disulfiram/copper (DS/Cu) couldtarget human leukemia cell lines(like KG1α,Molt4) through the activation of JNK, in vitro. However, there is few report about the ability of DS/Cu in killing cancer cells in vivo. Aims This study aims to explore the effect of DS/Cu on acute myeloid leukemia cell line KG1αin vivo and clarify the underlining mechanism. Methods 6-8 week old female NOD/SCID mice were sublethally irradiated with 2Gy X-ray the day before transplantation, followed by intravenous injection of KG1α cells (1×107 cells) suspended in 0.2 mL of PBS. 5 weeks after transplantation mice were randomly divided into three treatment groups: vehicle (0.9% saline), a combination of DS and Cu daily for 2 weeks, Ara-C alone twice before killing. Mice were sacrificed after 2 weeks treatment with tissues of spleen, liver, bone marrow being observed using histopathology method to detect the invasion of leukemia. The DS/Cu-induced p-c-jun activation was also examined by western blot using tissues of spleen, liver, bone marrow. Statistical analysis was carried out with one-way ANOVA to assess statistical significance (*p < 0.05). Results 4 weeks after transplantation, mice were dispirited with low appetite, down-bent gait, wrinkled fur, slow move, just like suffered from leukemia. What’s more, immature blasts like morphology similar to KG1α were found in the peripheral blood of the mice(11%±3.41). All the mice were sacrificed after 2 weeks treatment, mice in control group were observed with slightly larger spleen and liver with the morphology of invasion of leukemia such as a granular appearance than the other two groups. Histopathology examination showed that leukemia cells infiltrate liver, spleen and bone marrow, and the immunohistochemistry examination found that the leukemia cells in spleen, liver and bone marrow expressed human specific antigen CD45 with the highest expression level in the control group. Moreover, solid tumor could be observed in the peritoneal cavity of two mice in the control group with expression of human specific antigen CD45detected by immunohistochemistry examination. Western blot in this study showed DS/Cu complex induced phosphorylation of c-Jun expression in the spleen, liver and bone marrow. Conclusion DS/Cu complex could effectively target the acute myeloid leukemia cells in the acute leukemia NOD/SCID mice while inhibiting the invasion of leukemia to some extent, and the activation of JNK might play a functional role in DS/Cu mediated antileukemic effects. Disclosures: No relevant conflicts of interest to declare.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3790-3790 ◽  
Author(s):  
Weiguo Zhang ◽  
Nalini Patel ◽  
William E. Fogler ◽  
John L. Magnani ◽  
Michael Andreeff
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Acute Myeloid ◽  
Cells Cultured

Abstract Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008). The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively. The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. Figure 1. Figure 1. Disclosures Zhang: Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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