Clinical significance of mitochondrial DNA content in acute promyelocytic leukaemia.

Although a growing body of evidence demonstrates that altered mtDNA content (mtDNAc) has clinical implications in several types of solid tumours, its prognostic relevance in acute promyelocytic leukaemia (APL) patients remains largely unknown. Here, we show that patients with higher-than-normal mtDNAc had better outcomes regardless of tumour burden. These results were more evident in patients with low-risk of relapse. The multivariate Cox proportional hazard model demonstrated that high mtDNAc was independently associated with a decreased cumulative incidence of relapse. Altogether, our data highlights the possible role of mitochondrial metabolism in APL patients treated with ATRA.

PML/RARA inhibits expression of HSP90 and its target AKT.

Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.

Cytotoxic effects of high concentrations of sodium ascorbate on human myeloid cell lines.

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7 mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50 % lethal concentration (LC50) of 3 mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

Polycombs and microRNA-223 regulate human granulopoiesis by transcriptional control of target gene expression.

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.

Rapid detection of IDH2 (R140Q and R172K) mutations in acute myeloid leukemia.

NADP-dependent enzyme isocitrate dehydrogenase (IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10⁻³ dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup.

Benznidazole modulates cell proliferation in acute leukemia cells.

CONTEXT: We have previously reported that benznidazole (BZL), known for its trypanocidal action, has anti-proliferative activity against different cell lines like HeLa and Raw 264.7 among others. At the moment, it has not been reported if the anti-proliferative effect of BZL is similar for non-adherent hematopoietic cells like was reported for adherent cancer cell lines. OBJECTIVE: We aimed to investigate the efficacy of BZL on the growth of the leukemic cell lines THP-1 and OCI/AML3. MATERIALS AND METHODS: We evaluated cell proliferation by [³H]-thymidine incorporation and MTT reduction as well as cell death by lactate dehydrogenase (LDH) activity. We assessed apoptosis by flow cytometry for detection of annexin V-positive and propidium iodide-negative cells, along with nuclear morphology by diamidino-2-phenolindole (DAPI) staining. Western blot studies were performed to evaluate changes in cell cycle proteins in BZL-treated cells. RESULTS: BZL significantly reduced proliferation of both cell lines without inducing cell death. Likewise it produced no significant differences in apoptosis between treated cells and controls. In addition, flow cytometry analysis indicated that BZL caused a larger number of THP-1 cells in G0/G1 phase and a smaller number of cells in S phase than controls. This was accompanied with an increase in the expression of the CDK inhibitor p27 and of cyclin D1, with no significant differences in the protein levels of CDK1, CDK2, CDK4, cyclins E, A and B as compared to controls. CONCLUSION: BZL inhibits the proliferation of leukemic non-adherent cells by controlling cell cycle at G0/G1 cell phase through up-regulation of p27.

Effects of Triheptanoin on Mitochondrial Respiration and Glycolysis in Cultured Fibroblasts from Neutral Lipid Storage Disease Type M (NLSD-M) Patients.

Neutral lipid storage disease type M (NLSD-M) is an ultra-rare, autosomal recessive disorder that causes severe skeletal and cardiac muscle damage and lipid accumulation in all body tissues. In this hereditary pathology, the defective action of the adipose triglyceride lipase (ATGL) enzyme induces the enlargement of cytoplasmic lipid droplets and reduction in the detachment of mono- (MG) and diglycerides (DG). Although the pathogenesis of muscle fiber necrosis is unknown, some studies have shown alterations in cellular energy production, probably because MG and DG, the substrates of Krebs cycle, are less available. No tests have been tried with medium-chain fatty acid molecules to evaluate the anaplerotic effect in NLSD cells. In this study, we evaluated the in vitro effect of triheptanoin (Dojolvi®), a highly purified chemical triglyceride with seven carbon atoms, in fibroblasts obtained from five NLSD-M patients. Glycolytic and mitochondrial functions were determined by Seahorse XF Agylent Technology, and cellular viability and triglyceride content were measured through colorimetric assays. After the addition of triheptanoin, we observed an increase in glycolysis and mitochondrial respiration in all patients compared with healthy controls. These preliminary results show that triheptanoin is able to induce an anaplerotic effect in NLSD-M fibroblasts, paving the way towards new therapeutic strategies.

YY1 Knockdown Relieves the Differentiation Block and Restores Apoptosis in AML Cells.

In this study we analyzed the expression of Yin and Yang 1 protein (YY1), a member of the noncanonical PcG complexes, in AML patient samples and AML cell lines and the effect of YY1 downregulation on the AML differentiation block. Our results show that YY1 is significantly overexpressed in AML patient samples and AML cell lines and that YY1 knockdown relieves the differentiation block. YY1 downregulation in two AML cell lines (HL-60 and OCI-AML3) and one AML patient sample restored the expression of members of the CEBP protein family, increased the expression of extrinsic growth factors/receptors and surface antigenic markers, induced morphological cell characteristics typical of myeloid differentiation, and sensitized cells to retinoic acid treatment and to apoptosis. Overall, our data show that YY1 is not a secondary regulator of myeloid differentiation but that, if overexpressed, it can play a predominant role in myeloid differentiation block.

MCL1 regulates AML cells metabolism via direct interaction with HK2. Metabolic signature at onset predicts overall survival in AMLs' patients.

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.

Analysis of mutational status, SNP rs16754, and expression levels of Wilms tumor 1 (WT1) gene in acute promyelocytic leukemia.

Overexpression, polymorphisms, and mutations of the WT1 gene have been reported in several human tumors including acute myeloid leukemia (AML) and variably correlated with prognosis. Acute promyelocytic leukemia (APL) represents the AML subset disclosing higher WT1 expression levels; however, no WT1 studies specifically focused on APL have been conducted. We screened for the presence of mutations, SNP rs16754, and expression levels of WT1 gene in 103 adult patients with newly diagnosed APL. Fms-like tyrosine kinase (FLT3) mutations were analyzed as well. WT1 mutations were identified in four (4 %) patients. At least one copy of the minor SNP rs16754 allele (WT1(AG) or WT1(GG)) was detected in 30 (29 %) patients. Six patients (6 %) were homozygous for the minor allele (WT1(GG)) and this genotype was associated with higher WT1 mRNA copies (p = 0.018). FLT3 mutations were found in 37 % of patients and correlated with high WT1 mRNA expression (p = 0.004). Patients heterozygous or homozygous for the minor allele and patients homozygous for major (WT1(AA)) allele did not differ in terms of presenting features. In adult APL, WT1 gene mutational and polymorphic profile shows similarities with pediatric AML rather than with adult AML.

Ascorbate Plus Buformin in AML: A Metabolic Targeted Treatment.

In the present study, we characterized the metabolic background of different Acute Myeloid Leukemias' (AMLs) cells and described a heterogeneous and highly flexible energetic metabolism. Using the Seahorse XF Agilent, we compared the metabolism of normal hematopoietic progenitors with that of primary AML blasts and five different AML cell lines. We assessed the efficacy and mechanism of action of the association of high doses of ascorbate, a powerful oxidant, with the metabolic inhibitor buformin, which inhibits mitochondrial complex I and completely shuts down mitochondrial contributions in ATP production. Primary blasts from seventeen AML patients, assayed for annexin V and live/dead exclusion by flow cytometry, showed an increase in the apoptotic effect using the drug combination, as compared with ascorbate alone. We show that ascorbate inhibits glycolysis through interfering with HK1/2 and GLUT1 functions in hematopoietic cells. Ascorbate combined with buformin decreases mitochondrial respiration and ATP production and downregulates glycolysis, enhancing the apoptotic effect of ascorbate in primary blasts from AMLs and sparing normal CD34+ bone marrow progenitors. In conclusion, our data have therapeutic implications especially in fragile patients since both agents have an excellent safety profile, and the data also support the clinical evaluation of ascorbate-buformin in association with different mechanism drugs for the treatment of refractory/relapsing AML patients with no other therapeutic options.

The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells.

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34+ samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34+ samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients.

Hb S-San Martin: a new sickling hemoglobin with two amino acid substitutions [ß6(A3)Glu→Val;ß105(G7)Leu→Pro] in an Argentinean family.

A new sickling hemoglobin (Hb) detected in an Argentinean family from San Martín, Buenos Aires, Argentina, is hereby described. Two mutations were identified on the same ß-globin gene resulting in a new variant named Hb San Martin. One mutation was found on exon 1, corresponding to Hb S [ß6Glu→Val, GAG>GTG] and the second one on exon 3 at ß105(G7)Leu→Pro, CTC>CCC. The replacement of leucine by proline will likely impair the structure breaking helix G and causing instability of the molecule and the clinical manifestations typical of unstable Hbs. The mutation at ß105 seemed to be a de novo one in our patients, arising on a previously mutated gene, due to the fact that Hb S is the most frequent structural variant.