Differential cytotoxic activity of pharmacological inhibitors of IGF1R-related pathways in JAK2V617F driven cells.

Myeloproliferative neoplasms (MPN) belong to a group of clonal diseases of hematopoietic stem cells characterized by aberrant proliferation of mature myeloid lineages. The constitutive activation of the JAK2/STAT signaling pathway is now well established to play a central role in MPN pathogenesis; however, accumulating evidence now indicates that the IGF1R-mediated signaling pathway contributes to the maintenance of the malignant phenotype. Studies using inhibitors of IGF1-mediated signaling have reported cytotoxic effects in cellular and murine models of MPN, but no consensus has been reached regarding the potency and efficacy of inhibitors of the IGF1R-related pathway in this context. In the present study, we compared the potency and efficacy of three inhibitors of IGF1R-related pathways in a JAK2V617F-driven cellular model. These inhibitors (NT157, OSI-906, and NVP-AEW54) present antineoplastic activity with similar efficacy in Ba/F3 JAK2V617F cells, with NT157 showing the greatest potency. Both the induction of apoptosis and reduction in cell proliferation were associated with the observed reduction in cell viability. Downregulation of JAK2/STAT signaling was an advantageous off-target effect of all three inhibitors. These preclinical studies reinforce the potential of the IGF1R-related pathway as a therapeutic target in MPN.

Phenformin increases early hematopoietic progenitors in the Jak2V617F murine model.

BACKGROUND: Myeloproliferative neoplasms (MPN) are disorders characterized by an alteration at the hematopoietic stem cell (HSC) level, where the JAK2 mutation is the most common genetic alteration found in classic MPN (polycythemia vera, essential thrombocythemia, and primary myelofibrosis). We and others previously demonstrated that metformin reduced splenomegaly and platelets counts in peripheral blood in JAK2V617F pre-clinical MPN models, which highlighted the antineoplastic potential of biguanides for MPN treatment. Phenformin is a biguanide that has been used to treat diabetes, but was withdrawn due to its potential to cause lactic acidosis in patients. AIMS: We herein aimed to investigate the effects of phenformin in MPN disease burden and stem cell function in Jak2V617F-knockin MPN mice. RESULTS: In vitro phenformin treatment reduced cell viability and increased apoptosis in SET2 JAK2V67F cells. Long-term treatment with 40 mg/kg phenformin in Jak2V617F knockin mice increased the frequency of LSK, myeloid progenitors (MP), and multipotent progenitors (MPP) in the bone marrow. Phenformin treatment did not affect peripheral blood counts, spleen weight, megakaryocyte count, erythroid precursors frequency, or ex vivo clonogenic capacity. Ex vivo treatment of bone marrow cells from Jak2V617F knockin mice with phenformin did not affect hematologic parameters or engraftment in recipient mice. CONCLUSIONS: Phenformin increased the percentages of LSK, MP, and MPP populations, but did not reduce disease burden in Jak2V617F-knockin mice. Additional studies are necessary to further understand the effects of phenformin on early hematopoietic progenitors.

STMN1 is highly expressed and contributes to clonogenicity in acute promyelocytic leukemia cells.

Stathmin 1 (STMN1) is a microtubule-destabilizing protein highly expressed in hematological malignancies and involved in proliferation and differentiation. Although a previous study found that the PML-RARα fusion protein, which contributes to the pathophysiology of acute promyelocytic leukemia (APL), positively regulates STMN1 at the transcription and protein activity levels, little is known about the role of STMN1 in APL. In this study, we aimed to investigate the STMN1 expression levels and their associations with laboratory, clinical, and genomic data in APL patients. We also assessed the dynamics of STMN1 expression during myeloid cell differentiation and cell cycle progression, and the cellular effects of STMN1 silencing and pharmacological effects of microtubule-stabilizing drugs on APL cells. We found that STMN1 transcripts were significantly increased in samples from APL patients compared with those of healthy donors (all p < 0.05). However, this had no effect on clinical outcomes. STMN1 expression was associated with proliferation- and metabolism-related gene signatures in APL. Our data confirmed that STMN1 was highly expressed in early hematopoietic progenitors and reduced during cell differentiation, including the ATRA-induced granulocytic differentiation model. STMN1 phosphorylation was predominant in a pool of mitosis-enriched APL cells. In NB4 and NB4-R2 cells, STMN1 knockdown decreased autonomous cell growth (all p < 0.05) but did not impact ATRA-induced apoptosis and differentiation. Finally, treatment with paclitaxel (as a single agent or combined with ATRA) induced microtubule stabilization, resulting in mitotic catastrophe with repercussions for cell viability, even in ATRA-resistant APL cells. This study provides new insights into the STMN1 functions and microtubule dynamics in APL.

NT157, an IGF1R-IRS1/2 inhibitor, exhibits antineoplastic effects in pre-clinical models of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

Interleukin-8 is not a predictive biomarker for the development of the acute promyelocytic leukemia differentiation syndrome.

BACKGROUND: Differentiation syndrome (DS) is the main life-threatening adverse event that occurs in acute promyelocytic leukemia (APL) patients treated with all-trans retinoic acid (ATRA). Cytokine imbalances have been reported to play role during the developing of acute promyelocytic leukemia differentiation syndrome (APL-DS). However, the relationship between the plasma cytokine levels and their prognostic value for the prediction of DS developing in patients with APL during the treatment with ATRA and anthracyclines has not been previously reported. METHODS: In this study, we followed an APL cohort (n = 17) over 7 days of ATRA therapy in DS (n = 6) and non-DS groups (n = 11). Interleukin (IL)-1ß, IL-6, IL-8, IL-10, IL-12p70 and TNF-α were measured in the peripheral blood plasma from 17 patients with APL and 11 healthy adult controls by using the cytometric bead array method. RESULTS: In non-DS patients, IL-8 plasma levels were significantly reduced in the seventh day of ATRA treatment (34.16; 6.99 to 147.11 pg mL- 1 in D0 vs. 10.9; 0 to 26.81 pg mL- 1 in D7; p = 0.02) whereas their levels did not discriminate between DS and non-DS development during the entire induction period (all p > 0.05 in D0, D3, and D7). No significant differences were found in IL-6 levels between groups (p > 0.05 in D0-D7). Other cytokines tested were all undetectable in patients with APL or healthy controls. CONCLUSIONS: We demonstrated that the modulation of IL-8 following ATRA treatment may occur regardless of the development of DS and, therefore, does not appear to be a predictive biomarker to monitor the APL-DS.

NTAL is associated with treatment outcome, cell proliferation and differentiation in acute promyelocytic leukemia.

Non-T cell activation linker (NTAL) is a lipid raft-membrane protein expressed by normal and leukemic cells and involved in cell signaling. In acute promyelocytic leukemia (APL), NTAL depletion from lipid rafts decreases cell viability through regulation of the Akt/PI3K pathway. The role of NTAL in APL cell processes, and its association with clinical outcome, has not, however, been established. Here, we show that reduced levels of NTAL were associated with increased all-trans retinoic acid (ATRA)-induced differentiation, generation of reactive oxygen species, and mitochondrial dysfunction. Additionally, NTAL-knockdown (NTAL-KD) in APL cell lines led to activation of Ras, inhibition of Akt/mTOR pathways, and increased expression of autophagy markers, leading to an increased apoptosis rate following arsenic trioxide treatment. Furthermore, NTAL-KD in NB4 cells decreased the tumor burden in (NOD scid gamma) NSG mice, suggesting its implication in tumor growth. A retrospective analysis of NTAL expression in a cohort of patients treated with ATRA and anthracyclines, revealed that NTAL overexpression was associated with a high leukocyte count (P = 0.007) and was independently associated with shorter overall survival (Hazard Ratio: 3.6; 95% Confidence Interval: 1.17-11.28; P = 0.026). Taken together, our data highlights the importance of NTAL in APL cell survival and response to treatment.

NT157 has antineoplastic effects and inhibits IRS1/2 and STAT3/5 in JAK2V617F-positive myeloproliferative neoplasm cells.

Recent data indicate that IGF1R/IRS signaling is a potential therapeutic target in BCR-ABL1-negative myeloproliferative neoplasms (MPN); in this pathway, IRS2 is involved in the malignant transformation induced by JAK2V617F, and upregulation of IGF1R signaling induces the MPN phenotype. NT157, a synthetic compound designed as an IGF1R-IRS1/2 inhibitor, has been shown to induce antineoplastic effects in solid tumors. Herein, we aimed to characterize the molecular and cellular effects of NT157 in JAK2V617F-positive MPN cell lines (HEL and SET2) and primary patient hematopoietic cells. In JAK2V617F cell lines, NT157 decreased cell viability, clonogenicity, and cell proliferation, resulting in increases in apoptosis and cell cycle arrest in the G2/M phase (p < 0.05). NT157 treatment inhibited IRS1/2, JAK2/STAT, and NFκB signaling, and it activated the AP-1 complex, downregulated four oncogenes (CCND1, MYB, WT1, and NFKB1), and upregulated three apoptotic-related genes (CDKN1A, FOS, and JUN) (p < 0.05). NT157 induced genotoxic stress in a JAK2/STAT-independent manner. NT157 inhibited erythropoietin-independent colony formation in cells from polycythemia vera patients (p < 0.05). These findings further elucidate the mechanism of NT157 action in a MPN context and suggest that targeting IRS1/2 proteins may represent a promising therapeutic strategy for MPN.

Autophagy inhibition potentiates ruxolitinib-induced apoptosis in JAK2V617F cells.

JAK2V617F can mimic growth factor signaling, leading to PI3K/AKT/mTOR activation and inhibition of autophagy. We hypothesized that selective inhibition of JAK1/2 by ruxolitinib could induce autophagy and limit drug efficacy in myeloproliferative neoplasms (MPN). Therefore, we investigated the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, to determine the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify the frequency and clinical impact of autophagy-related gene mutations in patients with MPNs. In SET2 JAK2V617F cells, ruxolitinib treatment induced autophagy and modulated 26 out of 79 autophagy-related genes. Ruxolitinib treatment reduced the expressions of important autophagy regulators, including mTOR/p70S6K/4EBP1 and the STAT/BCL2 axis, in a dose- and time-dependent manner. Pharmacological inhibition of autophagy was able to significantly suppress ruxolitinib-induced autophagy and increased ruxolitinib-induced apoptosis. Mutations in autophagy-related genes were found in 15.5% of MPN patients and were associated with increased age and a trend towards worse survival. In conclusion, ruxolitinib induces autophagy in JAK2V617F cells, potentially by modulation of mTOR-, STAT- and BCL2-mediated signaling. This may lead to inhibition of apoptosis. Our results suggest that the combination of ruxolitinib with pharmacological inhibitors of autophagy, such as chloroquine, may be a promising strategy to treat patients with JAK2V617F-mutated MPNs.

IGF1R/IRS1 targeting has cytotoxic activity and inhibits PI3K/AKT/mTOR and MAPK signaling in acute lymphoblastic leukemia cells.

The IGF1R/IRS1 signaling is activated in acute lymphoblastic leukemia (ALL) and can be targeted by the pharmacological inhibitors NT157 (IGF1R-IRS1/2 inhibitor) and OSI-906 (IGF1R/IR inhibitor). Here we investigate the cellular and molecular effects of NT157 and OSI-906 in ALL cells. NT157 and OSI-906 treatment reduced viability, proliferation and cell cycle progression in ALL cell lines. Similarly, in primary samples of patients with ALL, both OSI-906 and NT157 reduced viability, but only NT157 induced apoptosis. NT157 and OSI-906 did not show cytotoxicity in primary samples from healthy donor. NT157 and OSI-906 significantly decreased Jurkat cell migration, but did not modulate Namalwa migration. Consistent with the more potent effect of NT157 on cells, NT157 significantly modulated expression of 25 genes related to the MAPK signaling pathway in Jurkat cells, including oncogenes and tumor suppressor genes. Both compounds inhibited mTOR and p70S6K activity, but only NT157 inhibited AKT and 4-EBP1 activation. In summary, in ALL cells, NT157 has cytotoxic activity, whereas OSI-906 is cytostatic. NT157 has a stronger effect on ALL cells, and thus the direct inhibition of IRS1 may be a potential therapeutic target in ALL.

The lipid raft protein NTAL participates in AKT signaling in mantle cell lymphoma.

Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold (n = 8) and decreased tumor weight by 2.7-fold (n = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells.

INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Metformin exerts multitarget antileukemia activity in JAK2V617F-positive myeloproliferative neoplasms.

The recurrent gain-of-function JAK2V617F mutation confers growth factor-independent proliferation for hematopoietic cells and is a major contributor to the pathogenesis of myeloproliferative neoplasms (MPN). The lack of complete response in most patients treated with the JAK1/2 inhibitor ruxolitinib indicates the need for identifying novel therapeutic strategies. Metformin is a biguanide that exerts selective antineoplastic activity in hematological malignancies. In the present study, we investigate and compare effects of metformin and ruxolitinib alone and in combination on cell signaling and cellular functions in JAK2V617F-positive cells. In JAK2V617F-expressing cell lines, metformin treatment significantly reduced cell viability, cell proliferation, clonogenicity, and cellular oxygen consumption and delayed cell cycle progression. Metformin reduced cyclin D1 expression and RB, STAT3, STAT5, ERK1/2 and p70S6K phosphorylation. Metformin plus ruxolitinib demonstrated more intense reduction of cell viability and induction of apoptosis compared to monotherapy. Notably, metformin reduced Ba/F3 JAK2V617F tumor burden and splenomegaly in Jak2V617F knock-in-induced MPN mice and spontaneous erythroid colony formation in primary cells from polycythemia vera patients. In conclusion, metformin exerts multitarget antileukemia activity in MPN: downregulation of JAK2/STAT signaling and mitochondrial activity. Our exploratory study establishes novel molecular mechanisms of metformin and ruxolitinib action and provides insights for development of alternative/complementary therapeutic strategies for MPN.

Paclitaxel induces Stathmin 1 phosphorylation, microtubule stability and apoptosis in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by abnormal proliferation and accumulation of lymphoblasts in the hematopoietic system. Stathmin 1 is a proliferation marker for normal lymphocytes, which has been described as highly expressed in ALL patients and functionally important for leukemia phenotype. In the present study, we expand our previous observations and aim to investigate Stathmin 1 expression and its impact on laboratory features and clinical outcomes in an independent cohort of ALL patients, and to verify the effects of paclitaxel treatment on Stathmin 1 phosphorylation and cell viability in ALL cell lines. In ALL patients, Stathmin 1 expression was significantly increased, associated with lower age onset and positively correlated with white blood cell counts, but did not impact on clinical outcomes. Functional assays revealed that paclitaxel induces Stathmin 1 phosphorylation at serine 16 (an inhibitory site), microtubule stability and apoptosis in Jurkat and Namalwa cell lines. Paclitaxel treatment did not modulate cell viability of normal peripheral blood leukocytes. In conclusion, our data confirm increased levels of Stathmin 1 in ALL patients and that therapeutic doses of paclitaxel inhibits Stathmin 1 function and promote microtubule stability and apoptosis in ALL cells.

Comparison of microRNA expression in high-count monoclonal B-cell lymphocytosis and Binet A chronic lymphocytic leukemia

Abstract Background Evidence suggests that monoclonal B-cell lymphocytosis precedes all chronic lymphocytic leukemia cases, although the molecular mechanisms responsible for disease progression are not understood. Aberrant miRNA expression may contribute to the pathogenesis of chronic lymphocytic leukemia. The objective of this study was to compare miRNA expression profiles of patients with Binet A chronic lymphocytic leukemia with those of subjects with high-count monoclonal B-cell lymphocytosis and healthy volunteers (controls). Methods Twenty-one chronic lymphocytic leukemia patients, 12 subjects with monoclonal B-cell lymphocytosis and ten healthy volunteers were enrolled in this study. Flow cytometry CD19+CD5+-based cell sorting was performed for the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups and CD19+ cells were sorted to analyze the control group. The expressions of miRNAs (miR-15a, miR-16-1, miR-29b, miR-34a, miR-181a, miR-181b and miR-155) were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Results Significant differences between the expressions in the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups were restricted to the expression of miR-155, which was higher in the former group. A comparison between healthy controls and monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia patients revealed higher miR-155 and miR-34a levels and lower miR-15a, miR-16-1, miR-181a and miR-181b in the latter group. Conclusions Our results show a progressive increase of miR-155 expression from controls to monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. The role of miR-155 in the development of overt chronic lymphocytic leukemia in individuals with monoclonal B-cell lymphocytosis must be further analyzed.

Comparison of microRNA expression in high-count monoclonal B-cell lymphocytosis and Binet A chronic lymphocytic leukemia.

BACKGROUND: Evidence suggests that monoclonal B-cell lymphocytosis precedes all chronic lymphocytic leukemia cases, although the molecular mechanisms responsible for disease progression are not understood. Aberrant miRNA expression may contribute to the pathogenesis of chronic lymphocytic leukemia. The objective of this study was to compare miRNA expression profiles of patients with Binet A chronic lymphocytic leukemia with those of subjects with high-count monoclonal B-cell lymphocytosis and healthy volunteers (controls). METHODS: Twenty-one chronic lymphocytic leukemia patients, 12 subjects with monoclonal B-cell lymphocytosis and ten healthy volunteers were enrolled in this study. Flow cytometry CD19+CD5+-based cell sorting was performed for the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups and CD19+ cells were sorted to analyze the control group. The expressions of miRNAs (miR-15a, miR-16-1, miR-29b, miR-34a, miR-181a, miR-181b and miR-155) were determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Significant differences between the expressions in the chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis groups were restricted to the expression of miR-155, which was higher in the former group. A comparison between healthy controls and monoclonal B-cell lymphocytosis/chronic lymphocytic leukemia patients revealed higher miR-155 and miR-34a levels and lower miR-15a, miR-16-1, miR-181a and miR-181b in the latter group. CONCLUSIONS: Our results show a progressive increase of miR-155 expression from controls to monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia. The role of miR-155 in the development of overt chronic lymphocytic leukemia in individuals with monoclonal B-cell lymphocytosis must be further analyzed.

Reversine triggers mitotic catastrophe and apoptosis in K562 cells.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative neoplasm of the hematopoietic stem cell characterized by presence of the oncoprotein BCR-ABL1, which have constitutive tyrosine kinase activity. BCR-ABL1 activation induces aurora kinase A (AURKA) and aurora kinase B (AURKB) expression, which are serine-threonine kinases that play an important function in chromosome alignment, segregation and cytokinesis during mitosis. Acquisition of resistance to tyrosine kinase inhibitors has emerged as a problem for CML patients and the identification of novel targets with an important contribution for CML phenotype is of interest. In the present study, we explored the cellular effects of reversine, an AURKA and AURKB inhibitor, in the BCR-ABL1+ K562 cells. Our results indicate that reversine reduces AURKA and AURKB expression, leads to reduction of cell viability and increased apoptosis in a dose- and time-dependent manner, as well as, induces mitotic catastrophe in K562 cells. Our preclinical study establishes that reversine presents an effective antileukemia activity against K562 cells and provide new insights on anticancer opportunities for CML.

Apoptosis induction by (+)alpha-tocopheryl succinate in the absence or presence of all-trans retinoic acid and arsenic trioxide in NB4, NB4-R2 and primary APL cells.

We analyzed the effect of (+)alpha-tocopheryl succinate (alpha-TOS) alone or associated with arsenic trioxide (ATO) or all-trans retinoid acid (ATRA) in acute promyelocytic leukemia (APL). alpha-TOS-induced apoptosis in APL clinical samples and in ATRA-sensitive (NB4) and ATRA-resistant (NB4-R2) APL cell lines. The effective dose 50% (ED-50) was calculated to be 71 and 58muM, for NB4 and NB4-R2, respectively. alpha-TOS neither induced nor modified ATRA-induced differentiation of APL cells, and did not affect the proliferation and differentiation of normal CD34(+) hematopoietic progenitors in methylcellulose assays. alpha-TOS exerted a moderate antagonistic effect to ATO-induced apoptosis when treatment was done simultaneously but when alpha-TOS was added 24h after ATO, an additive effect was observed. Our results support the concept of alpha-TOS as an anti-leukemic compound which spares normal hematopoiesis.