Acute Myelogenous Leukemia Blast Cells Research Articles

Suppression of mTOR Signaling Attenuates Apoptotic Effects Induced By the NEDD8-Activating Enzyme Inhibitor Pevonedistat (MLN4924) in Acute Myeloid Leukemia Cells

Acute myelogenous leukemia (AML) is a heterogeneous disease and often relapses after standard chemotherapy. Therefore, novel therapies for AML are needed. Recently, the NEDD8 conjugation pathway and the mammalian target of rapamycin (mTOR) signaling pathway have emerged as important regulatory pathways for cancer therapy. Since some components of the mTORC1/2 pathways such as Deptor are neddylation targets, we postulated that combined exposure to NEDD8-activating enzyme (NAE) inhibitor pevonedistat and the dual mTORC1/2 inhibitor sapanisertib (TAK-228) might have additive or synergistic effects in AML.To test pevonedistat and sapanisertib effects on AML cells, we used the agents alone or in combination followed by assessment of proliferation, apoptosis and signal transduction in human AML cell lines as well as in primary cells. By AnnexinV/7AAD flow cytometry analysis, we found that pevonedistat significantly induced AML cell death and apoptosis in a time- and dose-dependent manner. Western blot showed that poly (ADP-ribose) polymerase (PARP) and caspases 3/8/9 were cleaved, indicating that pevonedistat-treated cells were actively undergoing apoptosis. Among the six AML cell lines (HL-60, KG1a, Molm-13, MV4-11, THP-1 and U937) tested, MV4-11 was the most sensitive cell line (IC50 <25nM) and KG1a was the least sensitive cell line (IC50 >1000nM) after 48 hours of exposure. The average IC50 on primary AML blast cells (5 primary samples) was 400nM at 48 hours. Western blot analysis showed that the neddylated cullin levels were markedly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. We also found that pevonedistat induced morphologic changes and increased CD11b expression in Molm-13 and MV4-11 cells, indicating cell differentiation.The effects of pevonedistat on AML cells could be attenuated by pre- or co-treatment with the mTORC1/2 inhibitor sapanisertib. We found that sapanisertib alone induced limited cell death in most AML cell lines and primary AML patient blasts. The results from XTT assay and flow cytometry analysis indicated that sapanisertib alone significantly decreased cell size, arrested cells in G0-G1 phase and reduced protein content of AML cells but did not induce robust apoptosis. This suggested that through inactivating mTOR signal pathway, sapanisertib suppressed cell activity and induced a dormant/quiescent status and thereby attenuated the apoptotic effects of pevonedistat. With concurrent pevonedistat and sapanisertib exposure, the expression of apoptotic mediators such as cleaved PARP and caspases 3 were decreased compare to pevonedistat treatment alone (See Figure).To confirm the role of mTOR inhibition on pevonedistat-induced AML apoptosis, we targeted mTORC1/2 by knocking down mTOR expression with small hairpin RNAs (shRNA). Downregulation of mTOR expression mirrored the effects of sapanisertib. mTOR inhibition by shRNAs resulted in decreased AML cell size and arrest of cells in G0-G1 phase. In addition, pevonedistat-induced cell death was significantly impeded in the cells infected with mTOR shRNA lentivirus compared to scrambled shRNA (p<0.01). Therefore, we validated that mTOR signal inactivation protects AML cells from NAE inhibitor pevonedistat-induced apoptosis using both pharmaceutical and genetic approaches. To further study the mechanism behind mTOR inhibition inducing resistance to pevonedistat, we tested rapamycin, an mTORC1 inhibitor. Concurrent exposure to rapamycin did not significantly alter the effects of pevonedistat and did not change cell size and cell cycle status. In addition, rapamycin did not inhibit the phosphorylation of 4E-BP1, suggesting that mTORC1 inhibition alone may be insufficient to inhibit the effects of pevonedistat, and suggesting a role for mTORC2.Taken together, our data demonstrate that the NEDDylation pathway inhibitor pevonedistat has strong potential as a therapeutic agent for AML therapy via apoptosis induction. However, when AML cells are arrested in G0-G1 with reduced proliferation rates by dual mTORC1/2-inhibition, the ability of pevonedistat to induce apoptosis is suppressed. Whether this is due solely to the cytostatic effects of dual mTOR inhibitors or also involves other mechanisms remains uncertain. While both sapanisertib and pevonedistat have clear anti-leukemia effects, their concurrent use may inhibit NAE inhibitor-induced apoptosis. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

MEN1112/OBT357, an Anti Bst1/CD157 Humanized Antibody Inducing Acute Myelogenous Leukemia (AML) Blast Depletion in an Autologous Ex Vivo Assay: A Potential New Targeted Therapy for AML

Acute myeloid leukemia (AML) is a disease with a poor outcome and novel approaches are needed to improve survival and decrease toxicity of current therapies. Bst1/CD157 is a protein belonging to the ADP-ribosyl-cyclase family expressed on monocytes and neutrophils. This antigen was shown to be also expressed in peripheral blood (PB) and bone marrow (BM) blasts of acute myeloid leukemia (AML) patients either at primary diagnosis or at relapse(1,2,3). MEN1112/OBT357 is a humanized, de-fucosylated antibody targeting Bst1/CD157 with high affinity and developed to generate antibody dependent cell-mediated cytotoxicity (ADCC) response against AML blasts. Peripheral blood (PB) and bone marrow (BM) samples of 38 AML patients (29 at diagnosis, 6 at relapse, 3 resistant), have been analyzed for the expression of Bst1/CD157 on AML blast cells by fluorescence-activated cell sorting (FACS) using a PE conjugated form of MEN1112/OBT357. Bst1/CD157 expression has been confirmed in 91% and 96% of PB and BM AML samples, respectively. Furthermore, statistical analysis demonstrated that monocyte-oriented blasts are characterized by a brighter expression of Bst1/CD157 compared to blasts of non-monocytic lineage. The efficacy of MEN1112/OBT357 in depleting AML blasts was evaluated through FACS analysis in an autologous ex vivo assay performed on whole blood. The assay was set up using blood from healthy donors exposed to 10 μg/ml Rituximab for 18 hours to induce B cell depletion. In the same conditions, the ability of 10 μg/ml MEN1112/OBT357 to induce blasts depletion was tested.In whole PB,MEN1112/OBT357 was able to deplete AML blasts in 15/32 evaluable cases (46%). In BM, MEN1112/OBT357 induced blast depletion in 9/24 evaluable cases (36%). Interestingly, higher depletion rate was observed in relapse/refractory patients. When CD16A-158Phe/Val polymorphisms were analyzed utilizing a sequence based typing (SBT) assay, it was demonstrated that AML blast depletion was independent by FcRg polymorphism. Furthermore, no significant shedding of Bst1/CD157 antigen was observed in sera from AML patients, compared to the sera from patients with other hematologic diseases or healthy donors.In summary, we confirmed the frequent expression of Bst1/CD157 on blasts from AML patients, with the brightest pattern of positivity observed in cases belonging to monocytic differentiation lineage. MEN1112/OBT357 also induced a promising ADCC against AML blasts in an autologous setting, which is independent from FcR g phenotype. Since in vivo the exposure of AML blasts to MEN1112/OBT357 largely exceeds the incubation time of the depletion assay, we expect a further improvement of its anti-leukemic effect in the clinical setting. Based on these results, a phase I study in patients with relapsed or refractory AML has been initiated in December 2014. DisclosuresBellarosa:Menarini Ricerche: Employment. Bressan:Menarini Ricerche: Employment. Wilson:Oxford Biotherapeutics: Employment. Manzini:Menarini Ricerche: Employment. Capriati:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche: Employment.

HSP70 Inhibitor, YK5, Synergizes with Chemotherapeutic Agents and Prevents Chemoresistance in Acute Myelogenous Leukemia (AML).

Abstract 2476Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance and poor prognosis. YK5, a novel inhibitor of tumor-HSP70, has been shown to induce potent cell death in AML blast, progenitor, and stem cell populations with minimal effects in normal hematopoietic cells. Due to the role of HSP70 in drug resistance, we examined the effect of combining YK5 with other chemotherapeutic agents, including arsenic trioxide, cytarabine, suberoylanilide hydroxamic acid (SAHA) and PU-H71, a novel tumor-specific HSP90 inhibitor.We tested the ability of YK5 to synergize with either AsO3, AraC, SAHA, or PU-H71 in primary AML samples. Using multiparameter flow cytometry to measure viability after 48 hours of treatment, we found that combining 1μM YK5 with either 500nM AsO3 or PU-H71 resulted in a significant increase in cell death when compared to either agent alone (n=9, mean viability: 51.8, 67.2, and 13.4% for AsO3, YK5, and AsO3/YK5, respectively, P = 0.0018; mean viability: 57.1 and 20.8% for PU-H71 and PU-H71/YK5, respectively, P = 0.0029). A synergistic relationship between YK5 and both AsO3 and PU-H71 was found in all nine primary samples (combination indexes 0.29 – 0.76 with YK5/AsO3, 0.33 – 0.83 with YK5/PU-H71). In contrast, the combination of YK5 with either AsO3 or PU-H71 in CD34+ cord blood mononuclear cells did not result in a significant increase in cell death when compared to either agent alone (mean viability: 42.4, 72.4, and 37.2% for AsO3, YK5, and AsO3/YK5, respectively; mean viability: 61.1 and 51.1% for PU-H71 and PU-H71/YK5, respectively). YK5 in combination with either AraC or SAHA, however, did not result in a significant increase in cell death when compared to either drug alone, with an additive effect being demonstrated with a 1:1 YK5 to AraC/SAHA drug ratio (Mean CI = 0.9918).To determine the mechanism of the observed synergistic activity, intracellular HSP70 and active caspase-3, a client of HSP70, were measured using flow cytometry. Both AsO3 and PU-H71 significantly increased intracellular HSP70 and caspase-3 (Mean fold change = 18.3, 21.0 of HSP70 and 9.9, 8.3 of Caspase-3 for AsO3 and PU-H71 treatment, respectively), while treatment with AraC or SAHA resulted in no change in HSP70 levels. Furthermore, quantitative PCR revealed that treatment with either AsO3 or PU-H71 strongly upregulated HSPA1A and HSPA6, the main stress-inducible isoforms of HSP70 (Mean fold change = 15.9, 14.1 of HSPA1A, and 20.8, 23.4 of HSPA6 for AsO3 and PU-H71 treatment, respectively). AraC and SAHA had no significant upregulation of these genes. We have previously shown that increased levels of HSPA1A correlate with sensitivity to HSP70 inhibition via YK5.To further explore the mechanism of this observed synergy, flow cytometry was used to measure the levels of reactive oxygen species (ROS). Treatment with AsO3, PU-H71, AraC, or SAHA resulted in a significant increase in ROS (Mean fold change = 2.75, 1.92, 2.89, 1.67, respectively). Quantitative PCR also confirmed the activation of the oxidative stress response by the upregulation of heme oxygenase 1 (HMOX1) by treatment with these drugs (Mean fold change = 10.9, 8.7, 11.2, 7.7, respectively). YK5, however, did not induce ROS or upregulate HMOX1. Interestingly, pretreatment with NAC in primary AML samples (n=4) resulted in no protection from YK5 synergistic effect when combined with either AsO3 or PU-H71. These results suggest that YK5 synergizes with AsO3 and PU-H71 due to the increase in intracellular HSP70 caused by these drugs. This synergy is most likely due to the activation of the heat shock response and independent of the production of ROS due to drug treatment.In summary, we have found that the novel tumor-HSP70 inhibitor YK5 can synergize with AsO3 and PU-H71 in primary human AML, and that the basis of this synergism is due to the increase in intracellular HSP70 caused by these chemotherapeutic agents. HSP70 inhibition represents a novel approach in AML treatment and can be particularly significant to drug-resistant patients when combined with other chemotherapy. Disclosures:Roboz:Astex Pharmaceuticals: Research Funding.

Elevated calpain activity in acute myelogenous leukemia correlates with decreased calpastatin expression

Calpains are intracellular cysteine proteases that have crucial roles in many physiological and pathological processes. Elevated calpain activity has been associated with many pathological states. Calpain inhibition can be protective or lethal depending on the context. Previous work has shown that c-myc transformation regulates calpain activity by suppressing calpastatin, the endogenous negative regulator of calpain. Here, we have investigated calpain activity in primary acute myelogenous leukemia (AML) blast cells. Calpain activity was heterogeneous and greatly elevated over a wide range in AML blast cells, with no correlation to FAB classification. Activity was particularly elevated in the CD34+CD38− enriched fraction compared with the CD34+CD38+ fraction. Treatment of the cells with the specific calpain inhibitor, PD150606, induced significant apoptosis in AML blast cells but not in normal equivalent cells. Sensitivity to calpain inhibition correlated with calpain activity and preferentially targeted CD34+CD38− cells. There was no correlation between calpain activity and p-ERK levels, suggesting the ras pathway may not be a major contributor to calpain activity in AML. A significant negative correlation existed between calpain activity and calpastatin, suggesting calpastatin is the major regulator of activity in these cells. Analysis of previously published microarray data from a variety of AML patients demonstrated a significant negative correlation between calpastatin and c-myc expression. Patients who achieved a complete remission had significantly lower calpain activity than those who had no response to treatment. Taken together, these results demonstrate elevated calpain activity in AML, anti-leukemic activity of calpain inhibition and prognostic potential of calpain activity measurement.

FLT3-ITD+ AML Blast, Progenitor and Stem Cell Populations Demonstrate Higher Sensitivity to the Hsp90 Inhibitor PU-H71,

Abstract 3500Hsp90, one of the best-characterized molecular chaperones, plays indispensable roles in folding and assembly, intracellular transport, stabilization, and degradation of proteins, and therefore, facilitating cell signaling. Hsp90 is also involved in tumorigenesis by stabilizing oncogenic client proteins. Thus, Hsp90 inhibition has been considered a promising therapeutic strategy for different types of cancer including leukemia. PU-H71 is a novel HSP90 inhibitor with high specificity for oncogenic Hsp90. We investigated the effect of PU-H71 in acute myelogenous leukemia (AML), in particularly, AML stem cells (AML-SCs) that are known to give rise to AML blasts, are refractory to conventional therapies, and thus likely to account for AML relapses.The effect of PU-H71 was evaluated using a panel of 12 leukemia cell lines. Among the 12 leukemia cell lines tested, MOLM-13 and MV4-11 cells were the most sensitive (LD50s 253 nM and 120 nM respectively). Both MV4-11 and MOLM-13 carry FLT3-ITD mutation (occurring in ∼40% AML cases) and MLL translocations (occurring in ∼20% AML cases). Both MLL and FLT3 have been reported as client proteins of Hsp90. However, other leukemia cell lines with MLL rearrangements such as THP-1 and a MLL-ENL cell line derived from MLL-ENL transformed human CD34+ cord blood cells exhibited resistance to PU-H71 treatment (LD50 > 2 μM). The data suggested that FLT3-ITD+ AML samples may display higher sensitivity to Hsp90 inhibition.To confirm the higher sensitivity of FLT3-ITD+ AML cells to Hsp90 targeted therapy, 15 primary AML patient samples (8 FLT3-ITD mutants and 7 wild type FLT3) were treated with increasing concentrations of PU-H71. Cell viability on different cell populations was evaluated using multiparameter flow cytometry at 48 hours after treatment with PU-H71. The average LD50 of PU-H71 in FLT3-ITD+ AML cells was 492 nM (95% CI, 127.636 – 856.364). In contrast, the average LD50 in AML samples with wild type FLT3 was 2.795 μM (95% CI, 1.058 – 4.532). The near 6-fold difference between LD50s for PU-H71 was significant (p=0.0068). Importantly PU-H71 also killed FLT3-ITD+ AML stem and progenitor cells more effectively. Furthermore, PU-H71 treatment decreased the ability to form colonies in FLT3-ITD+ AML specimens more effectively than FLT3 WT AMLs (97.6% and 79.3% decrease relative to control respectively; N=3; P=0.0236). Importantly, PU-H71 had minor toxicity to normal blood mononuclear cells and normal cord blood hematopoietic stem cells.FLT3-ITD+ cell lines and primary AML cells treated with 0.5 μM PU-H71 showed a substantial decrease of phosphorylated forms of Erk1/2, JNK, AKT, p70RSK, NF-κB(p65) and Stat5, which was observed within 4 hours post PU-H71 treatment, whereas the phosphorylation levels of MAPK p38 remained unaffected. Immunoblotting and phosphoflow assays corroborated the inhibition of AKT and Stat5 signaling by PU-H71 in stem and progenitor populations.In summary, FLT3-ITD+ AML cells display a stronger response to PU-H71, suggesting that the FLT3-ITD mutation results in a higher dependency on Hsp90 to stabilize the aberrant signaling elicited by constitutive activation of FLT3. Our data suggests that PU-H71 represents a novel therapy for FLT3-ITD+ AML patients with the potential to ablate AML-SCs. Disclosures:Roboz:EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

AML-Derived Microvesicles as Mediators of Cell-Cell Signaling In the Leukemic Microenvironment

AbstractAbstract 1700Directed signaling between acute myelogenous leukemia (AML) cells and the bone marrow microenvironment plays an important role in leukemia cell survival. How leukemic blasts transform the homeostatic bone marrow microenvironment is not fully explained by known ligand-receptor interactions. Microvesicles (MV) are membrane-enclosed compartments that, depending on cell specific pathways, range in diameter from 50–100 nm (exosomes) to 100–1000 nm (microparticles) and are constitutively released into circulation in many tissues. Biogenesis has been found to be upregulated in a range of cancers. We hypothesized that MV-mediated transfer of signaling components by AML blasts may contribute to intercellular signaling in the leukemic niche. In an initial screen, we characterized MV that we recovered from the culture supernatant of multiple AML-derived cell lines, as well as AML cells from patients. Live-cell imaging of primary AML blasts using the exosomal pathway-specific dye N-Rh-PE revealed that at least a portion of these vesicles could be categorized as exosomes. Consistent with the non-random incorporation of molecular cargo observed in recent studies with solid tumors, we found the protein and RNA content of MV to be distinct from cellular lysates. In a focused, candidate target approach, our experiments showed that MV contain mRNA transcripts encoding the broadly relevant AML prognostic markers FLT3-ITD, NPM1, and IGF-1R. In both cell lines and cultured primary AML cells, insulin receptor (IR) and IGF-1R mRNAs were enriched up to ∼400-fold over GAPDH in MV versus cellular fractions. The IGF-1R receptor tyrosine kinase (RTK) transduces growth and survival signals, and the IGF-I signaling pathway has recently been identified as a potentially important candidate for molecularly targeted drug therapy for AML. Corroborative evidence for a role of MV and IGF-I signaling in shaping the leukemic microenvironment comes from the detection of human IGF-1R mRNA in cDNA prepared from mouse stromal cells (OP9) co-cultured for 48 hours with primary AML cells across a cell impermeable transwell barrier. To determine if the mRNA is transferred in a biologically significant quantity, we developed a plasmid-based quantitative PCR method to measure and compare transferred transcripts to steady state IGF-1R mRNA levels in the AML cell line HL-60. Results indicated that 0.3 HL-60 cellular equivalents of the human IGF-1R transcript were detected per OP9 cell following co-culture with primary AML cells. These data imply that transfer occurs at a level that may be functionally relevant. To further explore the potential role of MV in AML-stroma signaling, experiments are underway with HL-60 cells stably transfected with a pcDNA vector encoding the human IGF1R with a C-terminal FLAG tag. These HL-60:IGF1R-FLAG cells release MV that contain the IGF1R-FLAG transcript and will serve as a model to explore the downstream intracellular RTK signaling. In conclusion, we report for the first time the production of MV containing protein and disease-specific transcripts by AML blast cells, the selective enrichment in MV of disease-relevant transcripts, and direct cell-cell transfer of leukemia cell mRNAs to bystander cells. Our findings underscore the potential role of MV in shaping the AML microenvironment through lateral transfer of RTK mRNA transcripts. We propose a role for MV during disease progression and highlight their potential as highly sensitive, cell-free, and minimally invasive AML biomarkers. Disclosures:No relevant conflicts of interest to declare.

Acute myelogenous leukemia blasts contribute to a bone marrow-stroma in in vivo NOD/SCID mouse system

Abstract Abstract 1621 Poster Board I-647 Aims: Cancer stem cell theory has been developed, and whose precise characteristics have been reported. However, there have been no reports on the differentiation of cancer stem cells into the environmental stromal cells. We reported previously that non-adherent acute myelogenous leukemia (AML) cells were differentiated into myofibroblasts to create a microenvironment for proliferation of AML blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system AML blast cells also convert to myofibroblasts to form stroma in vivo. Materials and Methods Bone marrow cells were collected from informed AML (M2) patients who had chromosomal translocation of RUNX1 and ETO, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, the non-adherent mononuclear cells were injected to the 3.0 Gray-irradiated non-obese diabetes (NOD)/SCID mouse intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Runx1 and ETO fusion transcript, and mice were sacrificed after chimeric mRNA was observed. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select AML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and Discussion When non-adherent AML (M2) blast cells were transplanted to NOD/SCID mice, cells were engrafted after 10 weeks. In murine bone marrow cells human stromal cells were identified, in which RUNX1 and ETO gene was fused with FISH analysis. When the parental AML blast cells were cultured on the expanded AML-derived myofibroblasts, AML cells grew extensively. These results indicate that AML cells can create their own microenvironment for proliferation in vivo. Disclosures No relevant conflicts of interest to declare.

Antineoplastic Mechanisms of Niclosamide in Acute Myelogenous Leukemia Stem Cells: Inactivation of the NF-κB Pathway and Generation of Reactive Oxygen Species

NF-kappaB may be a potential therapeutic target for acute myelogenous leukemia (AML) because NF-kappaB activation is found in primitive human AML blast cells. In this report, we initially discovered that the potent antineoplastic effect of niclosamide, a Food and Drug Administration-approved antihelminthic agent, was through inhibition of the NF-kappaB pathway in AML cells. Niclosamide inhibited the transcription and DNA binding of NF-kappaB. It blocked tumor necrosis factor-induced IkappaBalpha phosphorylation, translocation of p65, and expression of NF-kappaB-regulated genes. Niclosamide inhibited the steps TAK1-->IkappaB kinase (IKK) and IKK-->IkappaBalpha. Niclosamide also increased the levels of reactive oxygen species (ROS) in AML cells. Quenching ROS by the glutathione precursor N-acetylcysteine attenuated niclosamide-induced apoptosis. Our results together suggest that niclosamide inhibited the NF-kappaB pathway and increased ROS levels to induce apoptosis in AML cells. On translational study of the efficacy of niclosamide against AML, niclosamide killed progenitor/stem cells from AML patients but spared those from normal bone marrow. Niclosamide was synergistic with the frontline chemotherapeutic agents cytarabine, etoposide, and daunorubicin. It potently inhibited the growth of AML cells in vitro and in nude mice. Our results support further investigation of niclosamide in clinical trials of AML patients.

Acute Myelogenous Leukemia Blasts Contribute to a Bone Marrow-Stroma in In Vivo NOD/SCID Mouse System..

Abstract 1621Poster Board I-647Aims: Cancer stem cell theory has been developed, and whose precise characteristics have been reported. However, there have been no reports on the differentiation of cancer stem cells into the environmental stromal cells. We reported previously that non-adherent acute myelogenous leukemia (AML) cells were differentiated into myofibroblasts to create a microenvironment for proliferation of AML blasts in vitro. In this report we demonstrate that with severe combined immunodeficiency (SCID) mouse system AML blast cells also convert to myofibroblasts to form stroma in vivo. Materials and MethodsBone marrow cells were collected from informed AML (M2) patients who had chromosomal translocation of RUNX1 and ETO, from which mononuclear cells were separated with density-gradient sedimentation method. After discarded an adherent cell-fraction, the non-adherent mononuclear cells were injected to the 3.0 Gray-irradiated non-obese diabetes (NOD)/SCID mouse intravenously. For the inactivation of NK cells, anti-Asialo GM1 antibody was injected intra-peritoneally prior to the transplantation, and on each 11th day thereafter. Blood was collected to monitor Runx1 and ETO fusion transcript, and mice were sacrificed after chimeric mRNA was observed. Bone marrow cells were obtained, and sorted with anti-human CD133 antibody and -CD106 to select AML-derived human stromal myofibroblasts referred to the in vitro data. The isolated positive fraction was further cultured, and the biological and the molecular characteristics were analyzed. Results and DiscussionWhen non-adherent AML (M2) blast cells were transplanted to NOD/SCID mice, cells were engrafted after 10 weeks. In murine bone marrow cells human stromal cells were identified, in which RUNX1 and ETO gene was fused with FISH analysis. When the parental AML blast cells were cultured on the expanded AML-derived myofibroblasts, AML cells grew extensively. These results indicate that AML cells can create their own microenvironment for proliferation in vivo. DisclosuresNo relevant conflicts of interest to declare.

Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging.

The aim of this study was to assess the potential of store-operated Ca(2+) channel (SOC) antagonists as purging agents for leukemia cells. Clonogenic, limiting dilution, and nuclear condensation assays were used to evaluate SOC antagonist efficacy. SOC activity and endoplasmic reticulum Ca(2+) content were measured by flow cytometry. Murine bone marrow transplantation was used to determine purging efficacy and effects on hemopoietic reconstitution. Econazole (Ec) and ketotifen (Ke) were variably effective against human and murine leukemia cell lines after 24 hours of incubation. However, a 2-hour serum and bovine serum albumin-free treatment protocol with Ec was found to maximize differential sensitivity between leukemic cells and normal hemopoietic progenitors. Primary acute myelogenous leukemia blast cell viability was reduced 4.2 to 5.1 logs by 2-hour Ec treatment as measured by limiting dilution. An inverse relationship between endoplasmic reticulum Ca(2+) content and Ke sensitivity in leukemia and untransformed cells was observed. Nuclear condensation, an index of apoptosis, which occurred after 24-hour treatments with either Ec or Ke, was not observed after 2-hour serum- and bovine serum albumin-free Ec exposures; however, condensed nuclei were observed after an additional 10-hour incubation in growth medium without drug. Using bone marrow deliberately contaminated with 1% P815 cells, we showed that highly effective in vitro purging can be accomplished using Ec with no adverse effects on bone marrow reconstitution in mice. These studies suggest that SOC antagonists have potential as purging agents for residual leukemia cells present in bone marrow in the context of high-dose chemotherapy and autologous transplantation for leukemia.

Prolonged administration of all-trans retinoic acid in combination with intensive chemotherapy and G-CSF for adult acute myelogenous leukemia: single-centre pilot study in different risk groups

An exploratory trial was conducted to evaluate toxicity and potential therapeutic role of all trans-retinoic acid (ATRA) given long-term together with chemotherapy and G-CSF to adult patients with acute myelogenous leukemia (AML). ATRA was administered orally at 45 mg/m(2)/day on days 1-14 and 25 mg/m(2)/day on days 15-28 of two standard cycles (idarubicin, etoposide, cytarabine, G-CSF) and of up to three high-dose courses (cytarabine, G-CSF). The results obtained in 19 patients enrolled in the ATRA trial were compared with those from 29 comparable cases treated with the same schedule without ATRA, according to patient risk class and an in vitro study. ATRA was administered for a median of 52 days to the patients selected for study who achieved a remission. ATRA-related toxicity was mostly non-severe apart from high incidence of headache in conjunction with high-dose cytarabine. Complete remission (CR) rate after cycle 1 (54%), kinetics of hematological recovery, postremission treatment realization, disease-free survival (DFS 37.5% at three years) and overall survival (30% at three years) were not different between ATRA-treated and untreated patients. The only significant prognostic factor was the patient risk class, as defined by cytogenetics and other clinical criteria: DFS rate was 57% at three years in standard-risk cases compared to only 19% in the high-risk group, with no influx by ATRA in either category. The in vitro study, in patients with a definite clinical response, failed to document any inhibitory or pro-apoptotic effect of ATRA on AML blast cells. As a consequence to these results, the pilot ATRA phase was closed. This study does not suggest a significant role for the present ATRA schedule as an adjunct to standard antileukemic therapy in adult AML.

Effect of rhGM-CSF on the Kinetics of Hematopoiesis in Long-term Marrow Cultures from Patients with Acute Myelogenous Leukemia

In the present study, we have assessed the effects of recombinant human Granulocyte-Macrophage Colony-Stimulating Factor (rhGM-CSF) in Dexter-type long-term marrow cultures (LTMC) from patients with acute myelogenous leukemia (AML). Addition of rhGM-CSF to AML LTMC resulted in a significant increase in the number of total nucleated cells (1.3-4.3-fold, as compared to untreated cultures). However, a simultaneous decrease in the numbers of myeloid progenitor cells (CFU) was observed. Interestingly, there was a selective stimulation of the growth of leukemic progenitors (AML-CFU). Indeed, whereas on day 0 these cells were detected in only 2 patients, between weeks 1 and 5 they were detected in 10 of the 14 patients included in the study. It is noteworthy that around 50% of the cells detected in the non-adherent fraction of rhGM-CSF-treated AML LTMC were blasts, whereas in untreated cultures, blasts corresponded to only 23% of the non-adherent cells, and the majority corresponded to cells of the monocyte-macrophage lineage. These results indicate that rhGM-CSF is a cytokine with a significant stimulatory activity for the in vitro growth of AML progenitor and blast cells, and, together with previous reports in the literature, suggest that the use of rhGM-CSF in clinical settings must be taken with caution since this cytokine, although beneficial in reducing the risk of infections after chemotherapy, may induce the reappearance of the disease after treatment. Further studies should be encouraged to understand in greater detail the effects of rhGM-CSF, and other cytokines, on the hematopoietic system of AML patients.

Interleukin-11 (IL-11) enhances clonal proliferation of acute myelogenous leukemia cells with strong expression of the IL-11 receptor alpha chain and signal transducing gp130.

We examined the effect of recombinant human interleukin (IL)-11 alone or in combination with various colony-stimulating factors (CSFs), including IL-3, granulocyte/macrophage (GM)-CSF, granulocyte (G)-CSF, stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO), on colony formation by leukemic progenitor cells (L-CFU) obtained from 33 patients with acute myelogenous leukemia (AML). Leukemic colony formation was found in approximately 70 to 80% of the patients in the presence of at least one of the above CSFs. Although IL-11 alone did not support L-CFU, the growth of these progenitors in the presence of other cytokines was enhanced by IL-11 in 16 out of 33 patients and it showed a synergistic action with G-CSF in 12 of them. This synergistic action occurred in seven out of nine M5 patients (French-American-British (FAB) classification). A single cell clone-sorting experiment clearly demonstrated that this synergistic effect was operative at the single progenitor cell level. The number of leukemic cells proliferating in the presence of G-CSF+IL-11 was significantly higher than in the presence of G-CSF alone, suggesting that IL-11 recruited dormant leukemic progenitors into the cell cycle. Flow cytometric analysis revealed that all types of AML blast cells (M0 approximately M6) ubiquitously expressed gp130, although the level of expression was significantly higher in M5 cells. In contrast, expression of the IL-11 receptor alpha chain (IL-11Ralpha) varied between FAB types. Blast cells obtained from M1, M3 and M5 patients showed higher levels of expression, with M5 cells showing the strongest expression. Interestingly, the leukemic progenitor cells for which proliferation was synergistically enhanced by IL-11 had significantly higher expression of both IL-11Ralpha and gp130. These results suggest that administration of IL-11 in vivo may stimulate the proliferation of leukemic progenitor cells, particularly M5 cells, in the presence of G-CSF, and that the responsiveness of L-CFU to IL-11 may be predicted by a simple receptor assay.

Expression of granulocyte colony-stimulating factor– and granulocyte-macrophage colony-stimulating factor–associated signal transduction proteins of the JAK/STAT pathway in normal granulopoiesis and in blast cells of acute myelogenous leukemia

Acute myelogenous leukemia (AML) is characterized by the malignant transformation of hematopoietic stem cells leading to dysregulated growth and differentiation of myeloid cells. Normally, proliferation and differentiation of myeloid cells are regulated by cytokines such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Abnormal signaling of the signal transduction pathway from the cytokine receptors via Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) might be involved in the pathogenesis of AML. We examined whether an abnormal expression of one of the four JAKs, STAT1, STAT3, STAT5, or the tyrosine phosphatase SHP-1, a negative regulator of this pathway, is associated with malignant transformation in AML. Analysis of the expression of proteins of the JAK/STAT pathway in normal myeloid cells at three stages of maturation revealed a strong expression of all proteins in CD34 + cells, whereas the level of the proteins was significantly lower in granulocytic precursors and mature neutrophils. Furthermore, during maturation the relation of the isoforms of STAT1 and STAT3 changed from predominantly α to predominantly β. Leukemic blast cells from 25 patients and 12 cell lines showed a high level of STAT proteins and SHP-1, whereas a deficiency of at least one of the four JAKs was found in 10 of 25 patients. In primary AML blast cells a deficiency of three JAKs was more common in patients with an abnormal karyotype. In addition, a lack of JAK2 and Tyk2 protein was strongly associated with the FAB M2 phenotype. The proliferation rate in response to GM-CSF available in a small number of patients appears to be related to the JAK2 expression. Our data suggest that the degree of expression of G-CSF/GM-CSF receptor-associated proteins of the JAK/STAT pathway in normal myeloid cells is related with their clonogenic potential. STAT3 appears to be involved in early differentiation. Similar to CD34 + cells, it is likely that the high levels of STATs and SHP-1 found in leukemic cells reflects their proliferative activity, whereas a lack of members of the JAK family might lead to an inability to proliferate in response to G-CSF/GM-CSF described in a considerable percentage of AML blasts.

Inhibition of P-Glycoprotein and Recovery of Drug Sensitivity of Human Acute Leukemic Blast Cells by Multidrug Resistance Gene (mdr1) Antisense Oligonucleotides

To overcome the problem of multidrug resistance, we investigated the effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS) in suppressing multidrug resistance gene (mdr1) expression in drug-resistant acute myelogenous leukemia (AML) blast cells and the K562 adriamycin-resistant cell line K562/ADM. The percentage of cells with the mdr1 gene product P-glycoprotein (P-gp) was decreased from 100% to 26% by 20 μmol/L MDR1-AS in the K562/ADM cells, and from 48.1% to 10.2% by 2.5 μmol/L MDR1-AS in the AML blast cells. Western blot analysis also showed a decrease in the amount of P-gp in the MDR1-AS–treated K562/ADM cells. This effect was specific to MDR1-AS, and not observed with sense or random control oligonucleotides. The expression of mdr1 mRNA in K562/ADM and AML blast cells treated with MDR1-AS was decreased compared with the random control. Intracellular rhodamine retention and [3H]daunorubicin also increased after antisense treatment. Chemosensitivity to daunorubicin increased in MDR1-AS–treated blast cells up to 5.9-fold in the K562/ADM cells and 3.0- to 6.4-fold in the AML blast cells. The expression of mdr1mRNA derived from colony cells decreased in the MDR1-AS–treated groups. No inhibitory effect of the oligonucleotides on normal bone marrow progenitors was observed. These findings suggest that MDR1-AS is useful to overcome multidrug resistance in the treatment of leukemia.

Inhibition of P-Glycoprotein and Recovery of Drug Sensitivity of Human Acute Leukemic Blast Cells by Multidrug Resistance Gene (mdr1) Antisense Oligonucleotides

AbstractTo overcome the problem of multidrug resistance, we investigated the effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS) in suppressing multidrug resistance gene (mdr1) expression in drug-resistant acute myelogenous leukemia (AML) blast cells and the K562 adriamycin-resistant cell line K562/ADM. The percentage of cells with the mdr1gene product P-glycoprotein (P-gp) was decreased from 100% to 26% by 20 μmol/L MDR1-AS in the K562/ADM cells, and from 48.1% to 10.2% by 2.5 μmol/L MDR1-AS in the AML blast cells. Western blot analysis also showed a decrease in the amount of P-gp in the MDR1-AS–treated K562/ADM cells. This effect was specific to MDR1-AS, and not observed with sense or random control oligonucleotides. The expression of mdr1mRNA in K562/ADM and AML blast cells treated with MDR1-AS was decreased compared with the random control. Intracellular rhodamine retention and [3H]daunorubicin also increased after antisense treatment. Chemosensitivity to daunorubicin increased in MDR1-AS–treated blast cells up to 5.9-fold in the K562/ADM cells and 3.0- to 6.4-fold in the AML blast cells. The expression of mdr1mRNA derived from colony cells decreased in the MDR1-AS–treated groups. No inhibitory effect of the oligonucleotides on normal bone marrow progenitors was observed. These findings suggest that MDR1-AS is useful to overcome multidrug resistance in the treatment of leukemia.

In vitro effect of r-verapamil on acute myelogenous leukemia blast cells: studies of cytokine secretion and cytokine-dependent blast proliferation.

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1 alpha, IL1 beta, TNF alpha, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.

Synergistic Reversal of Multidrug-Resistance Phenotype in Acute Myeloid Leukemia Cells by Cyclosporin A and Cremophor EL

AbstractCremophor (Crem) EL, the vehicle for intravenous delivery of cyclosporin A (CsA), has been reported to counteract multidrug resistance (MDR) in P-glycoprotein (Pgp)-over-expressing cell lines. Because of this, we sought to determine whether Crem functions independently as a modulator of MDR in blast cells obtained from acute myelogenous leukemia (AML) patients, and the nature of its interaction in combination with CsA in reversing an MDR phenotype. In the phenotypically classical MDR AML cell lines HL-60/Vine (overexpresses Pgp) or HL-60/AR (does not overex-press Pgp), the dose causing half-maximum enhancement (D50) of daunorubicin (DNR, 1 µg/mL, 3 hours) accumulation was achieved by the combination of CsA and Crem (CsA/Crem) at 1.2 µmol/L CsA. In contrast, the D50 for Crem alone was approached at an amount that would be needed to suspend 6.2 µmol/L CsA for HL-60/Vinc, and 81 µmol/L CsA for HL-60/AR. The D50 concentrations for CsA alone (dissolved in ethanol, which does not alter DNR accumulation) were also higher than those for CsA/Crem, being 6.5 µmol/L for HL-60/Vinc, and 3.1 µmol/L for HL-60/AR. The maximum absolute level of enhancement of DNR accumulation (Emax) in each cell line was approximately equivalent for CsA/Crem or CsA alone, and was equal to the 3 hr intracellular DNR accumulation observed in parental, drug sensitive HL-60/W cells. For Crem alone, HL-60/AR and HL-60/Vinc cells showed markedly different responses: HL-60/Vinc cells attained intracellular DNR content comparable to HL-60/W, whereas HL-60/AR cells achieved only approximately 35% of this level. Multiple-drug effects were analyzed by calculation of the Combination Index (Chou and Talalay, Adv Enzyme Regul 22:27, 1984), which indicated that CsA and Crem are synergistic in causing enhancement of DNR accumulation in these MDR HL-60 cell lines. In blasts from AML patients, 5 µmol/L CsA/Crem or an equivalent amount of Crem alone each caused significant (P < .001) enhancement of DNR accumulation (60 AML-patient marrow samples) or DNR retention (51 AML-patient marrows). Similarly, CsA/Crem or Crem alone caused significant (P < .01) enhancement of the cytotoxicity of DNR in 36 AML blast cell specimens. The degree of enhancement of accumulation/retention or cytotoxicity by CsA/Crem was approximately equivalent to that obtained with Crem alone. These studies indicate that Crem can reverse an MDR phenotype in patient AML blast cells. The equivalent degree of enhancement observed for Crem alone versus CsA/Crem also suggests that the Emax for DNR accumulation or retention was reached in AML cells by Crem alone (relative to 5 µmol/L CsA). Hence, synergism of CsA and Crem, indicated by the HL-60 studies, may permit the plasma concentrations obtained in recent phase I/II clinical trials (1 to 4 µmol/L CsA in Crem), to achieve maximal reversal of MDR in AML blast cells.

Synergistic reversal of multidrug-resistance phenotype in acute myeloid leukemia cells by cyclosporin A and cremophor EL

Cremophor (Crem) EL, the vehicle for intravenous delivery of cyclosporin A (CsA), has been reported to counteract multidrug resistance (MDR) in P-glycoprotein (Pgp)-over-expressing cell lines. Because of this, we sought to determine whether Crem functions independently as a modulator of MDR in blast cells obtained from acute myelogenous leukemia (AML) patients, and the nature of its interaction in combination with CsA in reversing an MDR phenotype. In the phenotypically classical MDR AML cell lines HL-60/Vinc (overexpresses Pgp) or HL-60/AR (does not overexpress Pgp), the dose causing half- maximum enhancement (D50) of daunorubicin (DNR, 1 micrograms/mL, 3 hours) accumulation was achieved by the combination of CsA and Crem (CsA/Crem) at 1.2 mumol/L CsA. In contrast, the D50 for Crem alone was approached at an amount that would be needed to suspend 6.2 mumol/L CsA for HL-60/Vinc, and 81 mumol/L CsA for HL-60/AR. The D50 concentrations for CsA alone (dissolved in ethanol, which does not alter DNR accumulation) were also higher than those for CsA/Crem, being 6.5 mumol/L for HL-60/Vinc, and 3.1 mumol/L for HL-60/AR. The maximum absolute level of enhancement of DNR accumulation (Emax) in each cell line was approximately equivalent for CsA/Crem or CsA alone, and was equal to the 3 hr intracellular DNR accumulation observed in parental, drug sensitive HL-60/W cells. For Crem alone, HL-60/AR and HL-60/Vinc cells showed markedly different responses: HL-60/Vinc cells attained intracellular DNR content comparable to HL-60/W, whereas HL-60/AR cells achieved only approximately 35% of this level. Multiple-drug effects were analyzed by calculation of the Combination Index (Chou and Talalay, Adv Enzyme Regul 22:27, 1984), which indicated that CsA and Crem are synergistic in causing enhancement of DNR accumulation in these MDR HL-60 cell lines. In blasts from AML patients, 5 mumol/L CsA/Crem or an equivalent amount of Crem alone each caused significant (P &lt; .001) enhancement of DNR accumulation (60 AML-patient marrow samples) or DNR retention (51 AML-patient marrows). Similarly, CsA/Crem or Crem alone caused significant (P &lt; .01) enhancement of the cytotoxicity of DNR in 36 AML blast cell specimens. The degree of enhancement of accumulation/retention or cytotoxicity by CsA/Crem was approximately equivalent to that obtained with Crem alone. These studies indicate that Crem can reverse an MDR phenotype in patient AML blast cells.

The Effects of Recombinant-DNA-Derived Interferons on the Growth of Myeloid Progenitor Cells

Interferons (IFNs) have been shown to have significant effects on hematopoietic cell growth. Previous studies defining these effects have utilized mouse and human alpha-, beta-, and gamma-IFN isolated from supernatants of stimulated cells. Despite purification, the possible presence of other lymphokines and soluble factors remains a concern. In this study, the effects of gene-cloned alpha- and gamma-IFN on colony-forming units of granulocyte/macrophage (CFU-GM) progenitors cultured from the peripheral blood of normal volunteers were examined. In addition, blast cell colonies from one patient with acute myelogenous leukemia (AML) were studied. The growth of normal CFU-GM and AML blast cell colonies was inhibited in a dose-dependent manner by gamma- and alpha-IFN. gamma-IFN was ten to 100 times more potent than alpha-IFN in that this species of IFN reduced colony formation by greater than 50% at concentrations of less than 15 antiviral U/mL. The effects of gamma-IFN were neutralized by a monoclonal antibody specific for gamma-IFN. These in vitro studies indicate that human gamma-IFN may be an important modulator of myelopoiesis. Although these data indicate a possible efficacy of gamma-IFN in the treatment of AML, the in vitro results should be considered for their in vivo significance.