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We characterize the metabolic background in distinct Acute Myeloid Leukemias (AMLs), by comparing the metabolism of primary AML blasts isolated at diagnosis with that of normal hematopoietic maturing progenitors, using the Seahorse XF Agilent. Leukemic cells feature lower spare respiratory (SRC) and glycolytic capacities as compared to hematopoietic precursors (i.e. day 7, promyelocytes). According with Proton Leak (PL) values, AML blasts can be grouped in two well defined populations. The AML group with blasts presenting high PL or high basal OXPHOS plus high SRC levels had shorter overall survival time and significantly overexpressed myeloid cell leukemia 1 (MCL1) protein. We demonstrate that MCL1 directly binds to Hexokinase 2 (HK2) on the outer mitochondrial membrane (OMM). Overall, these results suggest that high PL and high SRC plus high basal OXPHOS levels at disease onset, arguably with the concourse of MCL1/HK2 action, are significantly linked with shorter overall survival time in AML. Our data describe a new function for MCL1 protein in AMLs’ cells: by forming a complex with HK2, MCL1 co-localizes to VDAC on the OMM, thus inducing glycolysis and OXPHOS, ultimately conferring metabolic plasticity and promoting resistance to therapy.

Acute myeloid leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) stress and oxidative stress that could otherwise be overcome. Here, we show that the triple combination of the differentiating agent retinoic acid (RA), the ER stress–inducing drug tunicamycin (Tm), and arsenic trioxide (ATO), able to generate oxidative stress, leads to the death of AML cell lines expressing fusion proteins involving the gene MLL and the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, the combination of RA, Tm, and ATO decreased the colony-forming capacity of primary leukemic blasts bearing the FLT-ITD mutation without affecting healthy hematopoietic progenitor cells. We demonstrate in cell lines that combination of these drugs generates ER and oxidative stresses and impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stresses combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation.

Acute myeloid leukemia (AML) is often characterized by the expression of fusion or mutant proteins that cause impaired differentiation and enhanced proliferation and survival. The presence of mutant proteins prone to misfolding can render the cells sensitive to endoplasmic reticulum (ER) stress and oxidative stress that could otherwise be overcome. Here, we show that the triple combination of the differentiating agent retinoic acid (RA), the ER stress–inducing drug tunicamycin (Tm), and arsenic trioxide (ATO), able to generate oxidative stress, leads to the death of AML cell lines expressing fusion proteins involving the gene MLLand the internal tandem duplication (ITD) in the FLT3 tyrosine kinase receptor. Importantly, the combination of RA, Tm, and ATO decreased the colony-forming capacity of primary leukemic blasts bearing the FLT-ITD mutation without affecting healthy hematopoietic progenitor cells. We demonstrate in cell lines that combination of these drugs generates ER and oxidative stresses and impairs maturation and causes accumulation of FLT3 protein in the ER. Our data provide a proof of concept that low amounts of drugs that generate ER and oxidative stresses combined with RA could be an effective targeted therapy to hit AML cells characterized by MLL fusion proteins and FLT3-ITD mutation.

Somatic mutations of the spliceosome machinery have been recently identified by whole genome analysis in hematologic diseases, such as myelodysplastic syndrome, chronic lymphocytic leukemia, myeloproliferative neoplasms, acute myeloid leukemia, and advanced forms of mastocytosis, and also in nonhematologic conditions. SRSF2is a member of the serine/arginine–rich family pre-mRNA splicing factors that plays a role in mRNA export from the nucleus and translation. We describe a high-resolution melting (HRM) curve analysis to screen for SRSF2hotspot mutations in a fast, sensitive, and reliable way. Fifty bone marrow samples from patients with myelodysplastic syndrome were analyzed by the HRM assay and by direct sequencing. HRM screening identified four melting patterns corresponding to a negative (wild-type) group and three different mutated groups. Each mutated group was identified according to the positive control used: P95H, P95L, and P95R, respectively. An HRM mutated pattern was identified in seven patients. Positive and negative results from HRM were compared with direct sequencing results with a sensitivity and specificity of 100% (95% CI, 0.56–1, and 95% CI, 0.89–1, respectively). Analytical sensitivity analysis revealed a detection threshold of up to 1:9 (mutated/wild type) dilution. This rapid screening method may provide useful information for clinical decision making and be helpful to optimize laboratory resources and reduce turnaround time.

Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-A promoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-A promoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-A transcriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.

Epigenetic modifications regulate developmental genes involved in stem cell identity and lineage choice. NFI-A is a posttranscriptional microRNA-223 (miR-223) target directing human hematopoietic progenitor lineage decision: NFI-A induction or silencing boosts erythropoiesis or granulopoiesis, respectively. Here we show that NFI-Apromoter silencing, which allows granulopoiesis, is guaranteed by epigenetic events, including the resolution of opposing chromatin “bivalent domains,” hypermethylation, recruitment of polycomb (PcG)–RNAi complexes, and miR-223 promoter targeting activity. During granulopoiesis, miR-223 localizes inside the nucleus and targets the NFI-Apromoter region containing PcGs binding sites and miR-223 complementary DNA sequences, evolutionarily conserved in mammalians. Remarkably, both the integrity of the PcGs-RNAi complex and DNA sequences matching the seed region of miR-223 are required to induce NFI-Atranscriptional silencing. Moreover, ectopic miR-223 expression in human myeloid progenitors causes heterochromatic repression of NFI-A gene and channels granulopoiesis, whereas its stable knockdown produces the opposite effects. Our findings indicate that, besides the regulation of translation of mRNA targets, endogenous miRs can affect gene expression at the transcriptional level, functioning in a critical interface between chromatin remodeling complexes and the genome to direct fate lineage determination of hematopoietic progenitors.

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD184352 reduces the level of ΔNp73 and blunts the arsenic-mediated up-regulation of ΔNp73, thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1(p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73 gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD184352 reduces the level of ΔNp73 and blunts the arsenic-mediated up-regulation of ΔNp73, thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1 (p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.

We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, strikingly enhances ATO-mediated apoptosis in NB4, K562 cell lines (Blood104: 519–525, 2004), and in primary acute myelogenous leukemia (AML) cases. The aim of this study was to investigate whether the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. P73 exists as multiple TA (transactivation competent, pro-apoptotic and anti-proliferative) p73 COOH-terminal splicing isoforms (α,β,γ,δ,ε,ζ) of which the two major forms are p73α and p73β. In addition, dominant negative (ΔN) variants are expressed from a second promoter, that possess anti-apoptotic and pro-proliferative potential. In order to evaluate whether the combined treatment modulates p73 isoforms in AML blasts, the protein expression of TA-p73α, TA-p73β and ΔN-p73α were evaluated before and after the treatment with PD and/or ATO (1–2 μM) in 13 cases of AML and the TA/ΔN-p73 ratio was calculated. The basal expression of TA-p73α and TA-p73β were clearly evident in 11/13 and 12/13 cases respectively. In addition ΔN-p73α expression was detectable in 11/13 cases. Interestingly, in the PD+ATO responsive cases (11/13) we found that MEK1 inhibitor reduced the levels of ΔNp73 proteins and promoted the accumulation of endogenous TA-p73α and/or TA-p73β elevating the TA/ΔN ratio. ATO alone promoted the increase of both TA and ΔN-p73 proteins expression failing, or resulting less efficient than PD or PD+ATO in elevating TA/ΔN ratio. The lack of p73 proteins expression or the inability of PD in elevating TA/ΔN-p73 ratio resulted in loss of PD+ATO efficacy treatment. We then evaluated whether the changes in TA/ΔN-p73 ratio observed in PD+ATO vs ATO treated cells resulted in an increased expression of pro-apoptotic p73/p53 target genes, Bax, PUMA (p53 Up-regulated Modulator of Apoptosis), and P53AIP1 (p53-regulated Apoptosis-Inducing Protein 1) a primary effector gene of wild type p53 and TAp73-induced apoptosis whose overexpression induces massive mithocondrial apoptotic cell death. Interestingly, after a 48 hours treatment we found that Bax and PUMA proteins accumulated to a greater extent when cells were challenged with ATO treatment than with PD+ATO in 6/9 AML whereas p53AIP1 expression was greatly enhanced after PD+ATO treatment compared to ATO alone (2 or more fold increase). Conversely no differences in p53AIP1, Bax and PUMA expression between ATO and PD+ATO treatment were observed in no responsive cases. Interestingly, both p73 and p53 proteins, accumulated in AML blasts treated with 2μM of ATO, whereas lower concentration of ATO (1μM) induced only p73. In the responsive cases the combined treatment also led to an increased poly (ADP-ribose) polymerase (PARP) fragmentation that reflected the increased apoptosis. Finally, the selective downregulation of ΔNp73 by using specific siRNA, sensitized both K562 and NB4 leukemic cell lines to ATO-induced apoptosis suggesting that it may contribute to the ATO-resistance in leukemia cells. Our results support ATO plus PD combination as potential anti-leukemic treatment.

We recently reported that PD184352 (PD) (Pfizer), a highly selective inhibitor of MEK1 phosphorylation and activation, strikingly enhances ATO-mediated apoptosis in NB4, K562 cell lines (Blood 104: 519–525, 2004), and in primary acute myelogenous leukemia (AML) cases. The aim of this study was to investigate whether the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. P73 exists as multiple TA (transactivation competent, pro-apoptotic and anti-proliferative) p73 COOH-terminal splicing isoforms (α,β,γ,δ,ε,ζ) of which the two major forms are p73α and p73β. In addition, dominant negative (ΔN) variants are expressed from a second promoter, that possess anti-apoptotic and pro-proliferative potential. In order to evaluate whether the combined treatment modulates p73 isoforms in AML blasts, the protein expression of TA-p73α, TA-p73β and ΔN-p73α were evaluated before and after the treatment with PD and/or ATO (1–2 μM) in 13 cases of AML and the TA/ΔN-p73 ratio was calculated. The basal expression of TA-p73α and TA-p73β were clearly evident in 11/13 and 12/13 cases respectively. In addition ΔN-p73α expression was detectable in 11/13 cases. Interestingly, in the PD+ATO responsive cases (11/13) we found that MEK1 inhibitor reduced the levels of ΔNp73 proteins and promoted the accumulation of endogenous TA-p73α and/or TA-p73β elevating the TA/ΔN ratio. ATO alone promoted the increase of both TA and ΔN-p73 proteins expression failing, or resulting less efficient than PD or PD+ATO in elevating TA/ΔN ratio. The lack of p73 proteins expression or the inability of PD in elevating TA/ΔN-p73 ratio resulted in loss of PD+ATO efficacy treatment. We then evaluated whether the changes in TA/ΔN-p73 ratio observed in PD+ATO vs ATO treated cells resulted in an increased expression of pro-apoptotic p73/p53 target genes, Bax, PUMA (p53 Up-regulated Modulator of Apoptosis), and P53AIP1 (p53-regulated Apoptosis-Inducing Protein 1) a primary effector gene of wild type p53 and TAp73-induced apoptosis whose overexpression induces massive mithocondrial apoptotic cell death. Interestingly, after a 48 hours treatment we found that Bax and PUMA proteins accumulated to a greater extent when cells were challenged with ATO treatment than with PD+ATO in 6/9 AML whereas p53AIP1 expression was greatly enhanced after PD+ATO treatment compared to ATO alone (2 or more fold increase). Conversely no differences in p53AIP1, Bax and PUMA expression between ATO and PD+ATO treatment were observed in no responsive cases. Interestingly, both p73 and p53 proteins, accumulated in AML blasts treated with 2μM of ATO, whereas lower concentration of ATO (1μM) induced only p73. In the responsive cases the combined treatment also led to an increased poly (ADP-ribose) polymerase (PARP) fragmentation that reflected the increased apoptosis. Finally, the selective downregulation of ΔNp73 by using specific siRNA, sensitized both K562 and NB4 leukemic cell lines to ATO-induced apoptosis suggesting that it may contribute to the ATO-resistance in leukemia cells. Our results support ATO plus PD combination as potential anti-leukemic treatment.

We recently demonstrated that the combined treatment with PD184352, a highly selective inhibitor of MEK1 phosphorylation and activation (kindly provided to us by Dr J. S. Sebolt-Leopold, Cancer Molecular Sciences, Pfizer Global Research & Development, Ann Arbor, MI) and arsenic trioxide (ATO) strikingly potentiates apoptotic cell death induced by ATO in K562 and NB4 leukemic cells through the up-regulation of the proapoptotic p53AIP1 protein (p53-regulated Apoptosis-Inducing Protein 1), via p73 activation (Blood104: 519–525, 2004). Moreover, we showed that MEK1 inhibition sensitizes both parental and arsenic resistant NB4 cell lines to ATO-induced apoptosis through the MEK1 inhibition-mediated dephosphorylation of Bad, increasing its capacity to heterodimerize with Bcl-xL and Bcl-2 thereby blocking theirs antiapoptotic functions (ASH 2003, Abstract 61). p53AIP1 interacts with Bcl-2, affecting p53AIP1-mediated apoptosis through regulation of the mitochondrial transmembrane potential, thus supporting a model in which both pathways (p73-p53AIP1 and Bad phosphorylation) contribute to ATO induced apoptosis and its enhancement by MEK1 inhibitor co-treatment. Collectively, our results prompted us to investigate whether the combination of PD184352 and ATO was effective in inducing apoptosis of primary acute myeloid leukemia (AML) blasts. Twenty primary AML samples with different FAB classification were analyzed at diagnosis. Immunological and genetic characteristics were defined. Primary AML samples were cultured in presence of escalating doses of PD184352 (0.1–20 μM), ATO (0.125–10 μM) or combinations of the 2 agents at a 1:1 ratio (0.25/0.25, 0.5/0.5, 1/1, 1.5/1.5, 2/2). After 48 h of treatment the cells were harvested for mitochondrial transmembrane potential, annexin V and sub-G1 DNA content detection. Combination Index (CI) plots were then generated using the Chou-Talalay method and Calcusyn software (Biosoft, Ferguson, MO). CI less than 1.0 indicates synergism; CI = 1.0 indicates additive effect; CI more than 1.0 indicates antagonistic effect. Interestingly, MEK1 inhibition strikingly increased the apoptosis induced by ATO in sixteen of twenty primary AML samples analyzed. Isobologram analysis confirmed the additive or synergistic nature of this interaction. Five responder patients showed additive effect (one M0, two M2, two M4), eleven synergism (one M1, three M2, three M3, two M4, two M5). In the responsive AML samples the combined treatment with PD184352 and ATO strikingly potentiated loss of mitochondrial transmembrane potential induced by ATO. Treatment with MEK1 inhibitor down-modulated the steady level of ERK and Bad phosphorylation and prevented theirs phosphorylation in rensponse to ATO treatment. Moreover, p53AIP1 expression was enhanced after PD184352 plus ATO treatment compared to ATO alone. In conclusion these results demonstrated a cooperation between these two agents in this setting, and could provide an experimental basis for combined or sequential MEK1 inhibition plus ATO therapy in AML.

We recently demonstrated that the combined treatment with PD184352, a highly selective inhibitor of MEK1 phosphorylation and activation (kindly provided to us by Dr J. S. Sebolt-Leopold, Cancer Molecular Sciences, Pfizer Global Research & Development, Ann Arbor, MI) and arsenic trioxide (ATO) strikingly potentiates apoptotic cell death induced by ATO in K562 and NB4 leukemic cells through the up-regulation of the proapoptotic p53AIP1 protein (p53-regulated Apoptosis-Inducing Protein 1), via p73 activation (Blood 104: 519–525, 2004). Moreover, we showed that MEK1 inhibition sensitizes both parental and arsenic resistant NB4 cell lines to ATO-induced apoptosis through the MEK1 inhibition-mediated dephosphorylation of Bad, increasing its capacity to heterodimerize with Bcl-xL and Bcl-2 thereby blocking theirs antiapoptotic functions (ASH 2003, Abstract 61). p53AIP1 interacts with Bcl-2, affecting p53AIP1-mediated apoptosis through regulation of the mitochondrial transmembrane potential, thus supporting a model in which both pathways (p73-p53AIP1 and Bad phosphorylation) contribute to ATO induced apoptosis and its enhancement by MEK1 inhibitor co-treatment. Collectively, our results prompted us to investigate whether the combination of PD184352 and ATO was effective in inducing apoptosis of primary acute myeloid leukemia (AML) blasts. Twenty primary AML samples with different FAB classification were analyzed at diagnosis. Immunological and genetic characteristics were defined. Primary AML samples were cultured in presence of escalating doses of PD184352 (0.1–20 μM), ATO (0.125–10 μM) or combinations of the 2 agents at a 1:1 ratio (0.25/0.25, 0.5/0.5, 1/1, 1.5/1.5, 2/2). After 48 h of treatment the cells were harvested for mitochondrial transmembrane potential, annexin V and sub-G1 DNA content detection. Combination Index (CI) plots were then generated using the Chou-Talalay method and Calcusyn software (Biosoft, Ferguson, MO). CI less than 1.0 indicates synergism; CI = 1.0 indicates additive effect; CI more than 1.0 indicates antagonistic effect. Interestingly, MEK1 inhibition strikingly increased the apoptosis induced by ATO in sixteen of twenty primary AML samples analyzed. Isobologram analysis confirmed the additive or synergistic nature of this interaction. Five responder patients showed additive effect (one M0, two M2, two M4), eleven synergism (one M1, three M2, three M3, two M4, two M5). In the responsive AML samples the combined treatment with PD184352 and ATO strikingly potentiated loss of mitochondrial transmembrane potential induced by ATO. Treatment with MEK1 inhibitor down-modulated the steady level of ERK and Bad phosphorylation and prevented theirs phosphorylation in rensponse to ATO treatment. Moreover, p53AIP1 expression was enhanced after PD184352 plus ATO treatment compared to ATO alone. In conclusion these results demonstrated a cooperation between these two agents in this setting, and could provide an experimental basis for combined or sequential MEK1 inhibition plus ATO therapy in AML.