Impact of Bone Marrow miR-21 Expression on Acute Myeloid Leukemia T Lymphocyte Fragility and Dysfunction

Background: Acute myeloid leukemia (AML) is a hematopoietic malignancy in which antitumor immunity is impaired. The therapeutic management of AML requires understanding the mechanisms involved in the fragility and immune dysfunction of AML T lymphocytes. Methods: In this study, T lymphocytes from healthy donors (HD) and AML patients were used. Extracellular vesicles (EVs) from leukemic cells were screened for their microRNA content and impact on T lymphocytes. Flow cytometry, transcriptomic as well as lentiviral transduction techniques were used to carry out the research. Results: We observed increased cell death of T lymphocytes from AML patients. EVs from leukemia myeloid cell lines harbored several miRNAs, including miR-21, and were able to induce T lymphocyte death. Compared to that in HD, miR-21 was overexpressed in both the bone marrow fluid and infiltrating T lymphocytes of AML patients. MiR-21 induces T lymphocyte cell death by upregulating proapoptotic gene expression. It also increases the immunosuppressive profile of T lymphocytes by upregulating the IL13, IL4, IL10, and FoxP3 genes. Conclusions: Our results demonstrate that miR-21 plays a significant role in AML T lymphocyte dysfunction and apoptosis. Targeting miR-21 may be a novel approach to restore the efficacy of the immune response against AML.

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Rationale for a Combination Therapy with the STAT5 Inhibitor AC-4-130 and the MCL1 Inhibitor S63845 in the Treatment of FLT3-Mutated or TET2-Mutated Acute Myeloid Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms22158092 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8092

Author(s):

Katja Seipel ◽

Carolyn Graber ◽

Laura Flückiger ◽

Ulrike Bacher ◽

Thomas Pabst

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Map Kinases ◽

Leukemic Cells ◽

Bone Marrow Stroma ◽

Flt3 Inhibitors ◽

Marrow Stroma ◽

Acute Myeloid

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor S63845. AML patient samples with a strong response to AC-4-130 and S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.

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High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy

Blood ◽

10.1182/blood.v81.11.3091.bloodjournal81113091 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3091-3096 ◽

Cited By ~ 8

Author(s):

L Campos ◽

JP Rouault ◽

O Sabido ◽

P Oriol ◽

N Roubi ◽

...

Keyword(s):

Cell Death ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mitochondrial Protein ◽

High Expression ◽

Leukemic Cells ◽

Stem Cell Marker ◽

Cell Counts ◽

Response To Chemotherapy ◽

Acute Myeloid

The BCL-2 proto-oncogene encodes a mitochondrial protein that blocks programmed cell death. High amounts of bcl-2 protein are found not only in lymphoid malignancies, but also in normal tissues characterized by apoptotic cell death, including bone marrow. Using a monoclonal antibody to bcl-2 protein, we analyzed 82 samples of newly diagnosed acute myeloid leukemia. The number of bcl-2+ cells in each sample was heterogeneous (range, 0% to 95%), with a mean of 23%. The percentage of bcl-2+ cells was higher in M4 and M5 types, according to French- American-British classification, and in cases with high white blood cell counts. bcl-2 expression was also correlated with that of the stem cell marker CD34. In vitro survival of leukemic cells maintained in liquid culture in the absence of growth factors was significantly longer in cases with a high percentage of bcl-2+ cells. High expression of bcl-2 was associated with a low complete remission rate after intensive chemotherapy (29% in cases with 20% or more positive cells v 85% in cases with less than 20% positive cells, P < 10(-5)) and with a significantly shorter survival. In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.

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Human acute myeloid leukemia cells bind to bone marrow stroma via a combination of beta-1 and beta-2 integrin mechanisms

Blood ◽

10.1182/blood.v82.10.3125.3125 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3125-3132 ◽

Cited By ~ 65

Author(s):

LJ Bendall ◽

K Kortlepel ◽

DJ Gottlieb

Keyword(s):

Bone Marrow ◽

Extracellular Matrix ◽

Acute Myeloid Leukemia ◽

Cell Adhesion ◽

Myeloid Leukemia ◽

Extracellular Matrix Proteins ◽

Matrix Proteins ◽

Leukemic Cells ◽

Beta 2 ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) cells respond to exogenous stimulation from myeloid growth factors that may be secreted by cells of the bone marrow (BM) stroma and retained by glycosaminoglycans in the extracellular matrix. We have analyzed the capacity of malignant cells from patients with AML to maintain close proximity to sites of growth factor production and retention by binding to BM stromal elements, including fibroblasts and extracellular matrix proteins. Leukemic cells from all cases of AML adhered to BM fibroblast (BMF) monolayers (mean +/- standard error [SE] percentage binding, 30.9% +/- 2.5%; n = 23) and to fibronectin and laminin (mean +/- SE percentage binding, 28.0% +/- 4.1% [n = 11] and 21.5% +/- 2.3% [n = 8], respectively). Binding to bovine and human collagen type 1, vitronectin, hyaluronic acid, and albumin was minimal. Analysis of binding mechanisms indicated that very late antigen-4 (VLA-4) and VLA-5 were responsible for AML cell binding to fibronectin. Binding to laminin could be inhibited by antibody to the alpha chain of VLA-6. In contrast, AML cell adhesion to BMF monolayers was not impaired by blocking antibodies to either beta 1 or beta 2 integrins used alone, although the combination of anti-CD11/CD18 and anti-VLA-4 inhibited binding in more than 50% of cases. When anti- VLA-5 was added in these cases, mean +/- SE inhibition of binding of 45.5% +/- 9.1% (P < .001) was observed. Binding of AML cells to extracellular matrix proteins fibronectin and laminin is predominantly beta 1-integrin-dependent, but AML cell adhesion to BMF relies on the simultaneous involvement of beta 1 and beta 2 integrins as well as other currently unrecognized ligands.

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Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a specific immunophenotype with CD2 expression

Blood ◽

10.1182/blood.v81.11.3043.3043 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3043-3051 ◽

Cited By ~ 84

Author(s):

HJ Adriaansen ◽

PA te Boekhorst ◽

AM Hagemeijer ◽

CE van der Schoot ◽

HR Delwel ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Low Frequency ◽

Leukemic Cell ◽

Leukemic Cells ◽

Terminal Deoxynucleotidyl Transferase ◽

Cell Populations ◽

Spontaneous Proliferation ◽

Acute Myeloid

Abstract Extensive immunologic marker analysis was performed to characterize the various leukemic cell populations in eight patients with inv(16)(p13q22) in association with acute myeloid leukemia with abnormal bone marrow eosinophilia (AML-M4Eo). The eight AML cases consisted of heterogeneous cell populations; mainly due to the presence of multiple subpopulations, which varied in size between the patients. However, the immunophenotype of these subpopulations was comparable, independent of their relative sizes. Virtually all AML-M4Eo cells were positive for the pan-myeloid marker CD13. In addition, the AML were partly positive for CD2, CD11b, CD11c, CD14, CD33, CD34, CD36, CDw65, terminal deoxynucleotidyl transferase (TdT), and HLA-DR. Double immunofluorescence stainings demonstrated coexpression of the CD2 antigen and myeloid markers and allowed the recognition of multiple AML subpopulations. The CD2 antigen was expressed by immature AML cells (CD34+, CD14-) and more mature monocytic AML cells (CD34-, CD14+), whereas TdT expression was exclusively found in the CD34+, CD14- cell population. The eight AML-M4Eo cases not only expressed the CD2 antigen, but also its ligand CD58 (leukocyte function antigen-3). Culturing of AML-M4Eo cell samples showed a high spontaneous proliferation in all three patients tested. Addition of a mixture of CD2 antibodies against the T11.1, T11.2, and T11.3 epitopes diminished cell proliferation in two patients with high CD2 expression, but no inhibitory effects were found in the third patient with low frequency and low density of CD2 expression. These results suggest that high expression of the CD2 molecule in AML-M4Eo stimulates proliferation of the leukemic cells, which might explain the high white blood cell count often found in this type of AML.

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High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy

Blood ◽

10.1182/blood.v81.11.3091.3091 ◽

1993 ◽

Vol 81 (11) ◽

pp. 3091-3096 ◽

Cited By ~ 451

Author(s):

L Campos ◽

JP Rouault ◽

O Sabido ◽

P Oriol ◽

N Roubi ◽

...

Keyword(s):

Cell Death ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Mitochondrial Protein ◽

High Expression ◽

Leukemic Cells ◽

Stem Cell Marker ◽

Cell Counts ◽

Response To Chemotherapy ◽

Acute Myeloid

Abstract The BCL-2 proto-oncogene encodes a mitochondrial protein that blocks programmed cell death. High amounts of bcl-2 protein are found not only in lymphoid malignancies, but also in normal tissues characterized by apoptotic cell death, including bone marrow. Using a monoclonal antibody to bcl-2 protein, we analyzed 82 samples of newly diagnosed acute myeloid leukemia. The number of bcl-2+ cells in each sample was heterogeneous (range, 0% to 95%), with a mean of 23%. The percentage of bcl-2+ cells was higher in M4 and M5 types, according to French- American-British classification, and in cases with high white blood cell counts. bcl-2 expression was also correlated with that of the stem cell marker CD34. In vitro survival of leukemic cells maintained in liquid culture in the absence of growth factors was significantly longer in cases with a high percentage of bcl-2+ cells. High expression of bcl-2 was associated with a low complete remission rate after intensive chemotherapy (29% in cases with 20% or more positive cells v 85% in cases with less than 20% positive cells, P < 10(-5)) and with a significantly shorter survival. In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.

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In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo

Blood ◽

10.1182/blood.v100.13.4622 ◽

2002 ◽

Vol 100 (13) ◽

pp. 4622-4628 ◽

Cited By ~ 100

Author(s):

Gunter Schuch ◽

Marcelle Machluf ◽

Georg Bartsch ◽

Masashi Nomi ◽

Henri Richard ◽

...

Keyword(s):

Bone Marrow ◽

Vascular Endothelial Growth Factor ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Vascular Endothelial ◽

Neuropilin 1 ◽

Endothelial Growth Factor ◽

Acute Myeloid

Recent findings implied that the progression of hematologic malignancies, like that of solid tumors, is dependent on neovascularization. Recent studies on patients with acute myeloid leukemia (AML) showed increased levels of leukocyte-associated vascular endothelial growth factor (VEGF) and neovascularization of the bone marrow. Murine (32D, M1) and human (HEL, U937, and UKE-1) leukemic cell lines and freshly isolated leukemic cells were analyzed for the expression of VEGF and VEGF receptor mRNA. The expression of VEGF and VEGF receptors KDR and neuropilin-1 (NRP-1) was detected in these cells. In a murine chloroma model, delivery of VEGF165using microencapsulation technology resulted in enhanced tumor growth and vascularization, whereas treatment with a VEGF antagonist soluble NRP-1 (sNRP-1) inhibited tumor angiogenesis and growth. In a systemic leukemia model, survival of mice injected with adenovirus (Ad) encoding for Fc-sNRP-1 (sNRP-1 dimer) was significantly prolonged as compared with mice injected with Ad-LacZ. Further analyses showed a reduction in circulating leukemic cells and infiltration of liver and spleen as well as bone marrow neovascularization and cellularity. Taken together, these results demonstrate that angiogenic factors such as VEGF promote AML progression in vivo. The use of VEGF antagonists as an antiangiogenesis approach offers a potential treatment for AML. Finally, our novel in vivo drug delivery model may be useful for testing the activities of other peptide antiangiogenic factors.

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Coagulopathy in HLA-DR Antigen-Negative Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v104.11.4457.4457 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4457-4457

Author(s):

Hideki Uchiumi ◽

Takafumi Matsushima ◽

Arito Yamane ◽

Hiroshi Handa ◽

Hiroyuki Irisawa ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Myeloid Cell ◽

Leukemic Cell ◽

Leukemic Cells ◽

Hla Dr ◽

Risk Of Mortality ◽

Acute Myeloid

Abstract Background: HLA-DR antigen is present on hematopoietic progenitors and granulocyte/monocyte, erythrocyte and megakaryocytic precursors but absent at the promyelocytic stage during myeloid cell maturation. In accordance with this, majority of promyelocytic leukemia (APL) cells were negative for HLA-DR. Meanwhile, some of non-APL acute myeloid leukemia (AML) cells is found to express HLA-DR. However, the clinical significance of HLA-DR antigen on AML cells is currently unclear. Purpose: We sought to determine the prevalence and clinical characteristics of negativity in HLA-DR expression by retrospectively analyzing 181 consecutive patients with de novo adult AML. Patients and Methods: AML patients examined in the current study (aged 15–86 years) had been diagnosed between August 1995 and July 2004, and categorized to M0 (8 patients), M1 (35), M2 (74), M3 (20), M4 (25), M5 (15), and M6 (4), based on the FAB classification. Median follow-up time was 19.3 months. Phenotypic analyses of leukemic cells were performed using CD45 gating methods. HLA-DR-negative AML was defined as HLA-DR expression less than 20% of cells in the CD45 leukemic cell gate. Results: Among 181 patients, HLA-DR antigens were not detected on AML cells from 46 patients; 20 with APL and 26 with non-APL (non-APL/DR(−)), the latter of which included M0 (2 patients), M1 (15), M2 (7), M4 (2). Leukemic cells from other non-APL patients were HLA-DR-positive (non-APL/DR(+)). None of non-APL/DR(−) patients had t(15;17) nor PML/RARa rearrangement on cytogenetic analysis. Twenty out of 26 patients with non-APL/DR(−) had normal chromosome, and 6 had abnormal karyotypes. In the non-APL/DR(−) group, various degrees of nuclear folding, convolution, or lobulation were observed in 9 patients. Although treatment response and overall survival rate were similar in the three groups (APL, non-APL/DR(−), and non-APL/DR(+)), both FDP levels at diagnosis (57.3 vs 13.2, p<0.05) and maximal FDP levels (232.6 vs 43.8, p<0.01) were significantly higher in non-APL/DR(−) compared with non-APL/DR(+). The maximal FDP levels in the non-APL/DR(−) patients were comparable to those in the APL patients. FDP levels greater than 40 mg/ml were significantly more prevalent in the non-APL/DR(−) than in the the non-APL/DR(−) group. Logistic regression analysis demonstrated that low HLA-DR expression was an independent risk factor for FDP > 40 mg/ml. Conclusion: Our study suggests that AML with negative HLA-DR antigen tend to be associated with abnormality in coagulation and fibrinolysis even if they are genetically non-APL. We propose that more attention should be paied for HLA-DR expression to avoid a devastating coagulopathy which carries a high risk of mortality unless specifically addressed.

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Detection of Nucleophosmin Gene Mutations in Plasma from Patients with Acute Myeloid Leukemia: Clinical Significance and Implications.

Blood ◽

10.1182/blood.v110.11.2855.2855 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2855-2855

Author(s):

Wanlong Ma ◽

Xi Zhang ◽

Iman Jilani ◽

Farhad Ravandi ◽

Elihu Estey ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Gene Mutations ◽

Leukemic Cells ◽

Striking Difference ◽

Plasma Dna ◽

Npm1 Mutation ◽

Acute Myeloid

Abstract Nucleotides insertion in the nucleophosphamin (NPM1) gene has been reported in about one third of patients with acute myeloid leukemia (AML). Multiple studies showed that the presence of NPM1 mutations associated with better outcome in patients with AML. Studies reported to date have analyzed leukemic cells obtained from bone marrow or peripheral blood. We tested for mutations in the NPM1 gene using peripheral blood plasma and compared results with clinical outcome from a single institution. Analyzing plasma from 98 newly diagnosed patient with AML showed NPM1 mutation in 24 (23%) of patient while only one (4%) of 28 previously untreated patients with myelodysplastic syndrome (MDS) showed NPM1 mutation. Compared with peripheral blood cells, 2 (8%) of the 24 positive patients were negative by cells; none were positive by cells and negative by plasma. Most of the mutations detected (45%) were in patients with FAB classification M2, M4 and M5. In addition to the reported 4 bp insertion, we also detected 4 bp deletion in one patient in cells and plasma. Patients with NPM1 mutation had a significantly higher white blood cell count (P = 0.0009) and a higher blast count in peripheral blood (P = 0.002) and in bone marrow (P = 0.002). Blasts in patients with NPM1 mutant expressed lower levels of HLA-DR (P = 0.005), CD13 (P = 0.02) and CD34 (P < 0.0001), but higher CD33 levels (P = 0.0004). Patients with NPM1 mutation appear to have better chance of responding to standard therapy (P = 0.06). Event free survival of patients with NPM1 mutation was longer (P = 0.056) than in patients with intermediate cytogenetic abnormalities. The most striking difference in survival was in patients who required >35 days to respond to therapy (Figure). Survival was significantly longer in patients with NPM1 mutation requiring >35 days to respond (P = 0.027). This data not only support that NPM1 plays a significant role in the biology and clinical behavior of AML, but also show that plasma DNA is enriched with leukemia-specific DNA and is a reliable source for testing. Figure Figure

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Clinical Activity, Pharmacokinetics, and Pharmacodynamics of Sorafenib In Pediatric Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v116.21.1073.1073 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1073-1073

Author(s):

Hiroto Inaba ◽

Jeffrey E Rubnitz ◽

Elaine Coustan-Smith ◽

Lie Li ◽

Brian D Furmanski ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

De Novo ◽

Leukemic Cells ◽

Clinical Activity ◽

Newly Diagnosed ◽

Pediatric Acute Myeloid Leukemia ◽

Rtk Signaling ◽

Acute Myeloid

Abstract Abstract 1073 Background: Aberrant receptor tyrosine kinase (RTK) signaling arising from genetic abnormalities, such as FLT3-internal tandem duplications (FLT3-ITD), is an important mechanism in the development and growth of acute myeloid leukemia (AML) and is often associated with a poor outcome. Hence, inhibition of RTK signaling is an attractive novel treatment option, particularly for disease that is resistant to conventional chemotherapy. We evaluated the clinical activity of the multikinase inhibitor sorafenib in children with de novo FLT3-ITD–positive AML or relapsed/refractory AML. Methods: Fourteen patients were treated. Six patients with newly diagnosed FLT3- ITD–positive AML (aged 9–16 years; median, 12 years) received 2 cycles of remission induction therapy and then started sorafenib (200 mg/m2 twice daily for 20 days) the day after completing induction II (low-dose cytarabine, daunorubicin, and etoposide). Nine patients (aged 6–17 years; median, 9 years) with relapsed AML (including one treated on the above regimen) received sorafenib alone (2 dose levels; 200 and 150 mg/m2) twice daily for the first week of therapy, concurrently with clofarabine and cytarabine on days 8–12, and then alone from days 13 to 28. Sorafenib pharmacokinetics were analyzed at steady-state on day 8 of sorafenib in patients with newly diagnosed AML and on day 7 in patients with relapsed AML. In patients with relapsed AML, the effect of sorafenib on signaling pathways in AML cells was assessed by flow cytometry. Results: All 6 newly diagnosed patients, including 2 whose AML was refractory to induction I, achieved a complete remission (CR) after induction II; 5 had negative minimal residual disease (MRD; <0.1% AML cells in bone marrow) after induction II. Both patients in this group who relapsed achieved second remissions, one with sorafenib alone and one on the relapse regimen described above. Of the 9 patients with relapsed AML, 6 (4 with FLT3-ITD) were treated with sorafenib 200 mg/m2. All 6 had a >50% decrease in blast percentage and/or bone marrow cellularity after 1 week of sorafenib. After concurrent sorafenib and chemotherapy, 5 of the 9 patients with relapsed AML achieved CR (2 had negative MRD) and 2 achieved a partial remission (PR; 5%-25% AML cells in bone marrow); all 4 patients with FLT3-ITD had a CR or PR. After sorafenib treatment, 6 patients underwent HSCT while 2 with FLT3-ITD who could not receive HSCT were treated with single-agent sorafenib and have maintained CR for up to 8 months. Hand-foot skin reaction (HFSR) or rash occurred in all patients and improved with cessation of sorafenib. Dose-limiting toxicity (DLT, grade 3 HFSR and/or rash) was observed in 3 of the 6 patients with relapsed AML treated with 200 mg/m2 of sorafenib; no DLT was observed at 150 mg/m2. The effect of sorafenib on downstream RTK signaling was tested in the leukemic cells of 4 patients: in most samples, phosphorylation of S6 ribosomal protein and 4E-BP1 was inhibited. The mean (± SD) steady-state concentration (Css) of sorafenib was 3.3 ± 1.2 mg/L in the newly diagnosed group and 6.5 ± 3.6 mg/L (200 mg/m2) and 7.3 ± 3.6 mg/L (150 mg/m2) in those with relapsed AML. In both groups, the mean conversion of sorafenib to sorafenib N-oxide was 27%-35% (approximately 3 times greater than previously reported), and mean sorafenib N-oxide Css was 1.0–3.2 mg/L (2.1-6.7 μM). In a 442-kinase screen, the inhibitory profiles of sorafenib N-oxide and sorafenib were similar, and FLT3-ITD phosphorylation was potently inhibited by both forms (sorafenib N-oxide Kd = 0.070 μM; sorafenib Kd = 0.094 μM). Sorafenib N-oxide inhibited the growth of an AML cell line with FLT3-ITD (IC50 = 0.026 μM) and 4 AML cell lines with wild-type FLT3 (IC50 = 3.9–13.3 μM) at approximately half the potency of sorafenib. Conclusion: In children with de novo FLT3-ITD and relapsed/refractory AML, sorafenib given alone or with chemotherapy induced dramatic responses and inhibited aberrant RTK signaling in leukemic cells. Sorafenib and its active metabolite (sorafenib N-oxide) likely contribute to both efficacy and toxicity. These results warrant the incorporation of sorafenib into future pediatric AML trials. Disclosures: Inaba: Bayer/Onyx: Research Funding. Off Label Use: Sorafenib and clofarabine: both used for treatment of pediatric acute myeloid leukemia.

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P2X7 Receptor Activation By ATP As Target of Novel Therapies in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v126.23.3684.3684 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3684-3684

Author(s):

Valentina Salvestrini ◽

Stefania Orecchioni ◽

Francesca Reggiani ◽

Giovanna Talarico ◽

Elisa Orioli ◽

...

Keyword(s):

Cell Death ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Pore Formation ◽

Cytotoxic Effect ◽

Leukemic Cells ◽

Cell Compartment ◽

Hematopoietic Stem ◽

Direct Cytotoxicity ◽

Acute Myeloid

Abstract ATP is the key energy molecule as well as an ubiquitous extracellular messenger. Depending on its dose and the engaged purinergic P2 receptor (P2R) subtype, ATP can trigger many different cell responses, including proliferation and cell death. Recent studies have shown that high ATP level exhibits direct cytotoxicity on many tumor cell types. Among the receptors engaged by ATP, P2X7 is the most consistently expressed by tumor cells and its overexpression is related to tumor growth and progression. The P2X7 is an ATP-gated ion channel that, upon sustained stimulation with millimolar ATP concentrations, drives the opening of a non-selective large conductance pore, triggering cell-death signal. We previously demonstrated that ATP is a potent stimulator of normal hematopoietic stem cell compartment while inhibiting acute myeloid leukemia (AML) cells. Based on this observation, we studied AML samples (n=20) collected from the bone marrow or the peripheral blood of leukemic patients at diagnosis before treatment (percentage of circulating blasts >90%). In addition, normal hematopoietic stem cells (HSC) were isolated from leukapheresis products of 5 healthy donors receiving G-CSF. Our data demonstrate that AML cells express high level of P2X7 and that its activation with high dose of ATP reduces blast cell viability while is not effective on normal CD34+ cells. The cytotoxic effect is due to the induction of apoptosis, associated with reduction of mithocondrial membrane potential and activation of caspase cascade. Interestingly, P2X7 is also expressed by leukemic stem/progenitor cells (LSC) and ATP treatment exerts a direct cytotoxicity on different subsets of stem/progenitor cell compartment i.e. CD34- CD38-, CD34+ CD38-, CD34+ CD38+ and CD34- CD38+. Of note, this cytotoxic effect was not observed on HSC subpopulations. Furthermore, we transplanted 1x106 human AML cells into NSG immunodeficient mice followed by intraperitoneal administration of ATP every other day for thirty days post-transplantation. Our results show a 40% inhibition of AML engraftment in ATP-treated mice vs controls. Different P2X7 splice variants have been identified among which only two are functional: P2X7A, which shows both pro-apoptotic and trophic activity and P2X7B, which retains only the growth promoting phenotype. In order to explain ATP different effects on LSCs and normal HSCs, we assumed a different P2X7 isoforms expression on normal and leukemic cells. Preliminary results showed a reduced expression of both P2X7A and P2X7B on normal CD34+ compared to leukemic cells. In particular normal CD34+ express very low level of P2X7A, which is responsible for pore formation after ATP stimulation. Moreover, since P2X7 pore formation facilitates the passage of hydrophilic chemotherapeutic agents, we hypothesized that ATP may potentiate the cytotoxic effect of antineoplastic drugs. Our results showed that ATP potentiates the cytotoxic effect of ARA-C, by significantly reducing cell proliferation and increasing apoptosis of leukemia cell lines. In conclusion, overall survival of adult AML remains poor due to the lack of novel and effective therapies. Novel compounds that have the potential to improve the treatment efficacy with low toxicity are highly warranted. Overall, our results may provide the biological rationale to use P2X7 as a target for novel therapeutical approaches against AML. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

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