Systemic Metabolomic Profiling of Acute Myeloid Leukemia Patients before and During Disease-Stabilizing Treatment Based on All-Trans Retinoic Acid, Valproic Acid, and Low-Dose Chemotherapy

Acute myeloid leukemia (AML) is an aggressive malignancy, and many elderly/unfit patients cannot receive intensive and potentially curative therapy. These patients receive low-toxicity disease-stabilizing treatment. The combination of all-trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid can stabilize the disease for a subset of such patients. We performed untargeted serum metabolomic profiling for 44 AML patients receiving treatment based on ATRA and valproic acid combined with low-dose cytotoxic drugs (cytarabine, hydroxyurea, 6-mercaptopurin) which identified 886 metabolites. When comparing pretreatment samples from responders and non-responders, metabolites mainly belonging to amino acid and lipid (i.e., fatty acid) pathways were altered. Furthermore, patients with rapidly progressive disease showed an extensively altered lipid metabolism. Both ATRA and valproic acid monotherapy also altered the amino acid and lipid metabolite profiles; however, these changes were only highly significant for valproic acid treatment. Twenty-three metabolites were significantly altered by seven-day valproic acid treatment (p < 0.05, q < 0.05), where the majority of altered metabolites belonged to lipid (especially fatty acid metabolism) and amino acid pathways, including several carnitines. These metabolomic effects, and especially the effects on lipid metabolism, may be important for the antileukemic and epigenetic effects of this treatment.publishedVersio

Histone deacetylase inhibition in the treatment of acute myeloid leukemia: the effects of valproic acid on leukemic cells, and the clinical and experimental evidence for combining valproic acid with other antileukemic agents

Several new therapeutic strategies are now considered for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy, including modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs). These enzymes alter the acetylation of several proteins, including histones and transcription factors, as well as several other proteins directly involved in the regulation of cell proliferation, differentiation and apoptosis. Valproic acid (VPA) is a HDAC inhibitor that has been investigated in several clinical AML studies, usually in combination with all-trans retinoic acid (ATRA) for treatment of patients unfit for intensive chemotherapy, for example older patients, and many of these patients have relapsed or primary resistant leukemia. The toxicity of VPA in these patients is low and complete hematological remission lasting for several months has been reported for a few patients (<5% of included patients), but increased peripheral blood platelet counts are seen for 30 to 40% of patients and may last for up to 1 to 2 years. We review the biological effects of VPA on human AML cells, the results from clinical studies of VPA in the treatment of AML and the evidence for combining VPA with new targeted therapy. However, it should be emphasized that VPA has not been investigated in randomized clinical studies. Despite this lack of randomized studies, we conclude that disease-stabilizing treatment including VPA should be considered especially in unfit patients, because the possibility of improving normal blood values has been documented in several studies and the risk of clinically relevant toxicity is minimal

The combination of valproic acid, all-trans retinoic acid and low-dose cytarabine as disease-stabilizing treatment in acute myeloid leukemia

Background: A large proportion of patients with acute myeloid leukemia (AML) are not fit for intensive and potentially curative therapy due to advanced age or comorbidity. Previous studies have demonstrated that a subset of these patients can benefit from disease-stabilizing therapy based on all-trans retinoic acid (ATRA) and valproic acid. Even though complete hematological remission is only achieved for exceptional patients, a relatively large subset of patients respond to this treatment with stabilization of normal peripheral blood cell counts. Methods: In this clinical study we investigated the efficiency and safety of combining (i) continuous administration of valproic acid with (ii) intermittent oral ATRA treatment (21.5 mg/m2 twice daily) for 14 days and low-dose cytarabine (10 mg/m2 daily) for 10 days administered subcutaneously. If cytarabine could not control hyperleukocytosis it was replaced by hydroxyurea or 6-mercaptopurin to keep the peripheral blood blast count below 50 × 109/L. Results: The study included 36 AML patients (median age 77 years, range 48 to 90 years) unfit for conventional intensive chemotherapy; 11 patients responded to the treatment according to the myelodysplastic syndrome (MDS) response criteria and two of these responders achieved complete hematological remission. The most common response to treatment was increased and stabilized platelet counts. The responder patients had a median survival of 171 days (range 102 to > 574 days) and they could spend most of this time outside hospital, whereas the nonresponders had a median survival of 33 days (range 8 to 149 days). The valproic acid serum levels did not differ between responder and nonresponder patients and the treatment was associated with a decrease in the level of circulating regulatory T cells. Conclusion: Treatment with continuous valproic acid and intermittent ATRA plus low-dose cytarabine has a low frequency of side effects and complete hematological remission is seen for a small minority of patients. However, disease stabilization is seen for a subset of AML patients unfit for conventional intensive chemotherapy

Systemic Metabolomic Profiling of Acute Myeloid Leukemia Patients before and During Disease-Stabilizing Treatment Based on All-Trans Retinoic Acid, Valproic Acid, and Low-Dose Chemotherapy

Acute myeloid leukemia (AML) is an aggressive malignancy, and many elderly/unfit patients cannot receive intensive and potentially curative therapy. These patients receive low-toxicity disease-stabilizing treatment. The combination of all-trans retinoic acid (ATRA) and the histone deacetylase inhibitor valproic acid can stabilize the disease for a subset of such patients. We performed untargeted serum metabolomic profiling for 44 AML patients receiving treatment based on ATRA and valproic acid combined with low-dose cytotoxic drugs (cytarabine, hydroxyurea, 6-mercaptopurin) which identified 886 metabolites. When comparing pretreatment samples from responders and non-responders, metabolites mainly belonging to amino acid and lipid (i.e., fatty acid) pathways were altered. Furthermore, patients with rapidly progressive disease showed an extensively altered lipid metabolism. Both ATRA and valproic acid monotherapy also altered the amino acid and lipid metabolite profiles; however, these changes were only highly significant for valproic acid treatment. Twenty-three metabolites were significantly altered by seven-day valproic acid treatment (p < 0.05, q < 0.05), where the majority of altered metabolites belonged to lipid (especially fatty acid metabolism) and amino acid pathways, including several carnitines. These metabolomic effects, and especially the effects on lipid metabolism, may be important for the antileukemic and epigenetic effects of this treatment

Disease-stabilizing treatment based on all-trans retinoic acid and valproic acid in acute myeloid leukemia – identification of responders by gene expression profiling of pretreatment leukemic cells

Abstract Background Acute myeloid leukemia (AML) is an aggressive malignancy only cured by intensive therapy. However, many elderly and unfit patients cannot receive such treatment due to an unacceptable risk of treatment-related morbidity and mortality. Disease-stabilizing therapy is then the only possible strategy, one alternative being treatment based on all-trans retinoic acid (ATRA) combined with the histone deacetylase inhibitor valproic acid and possibly low-toxicity conventional chemotherapy. Methods Primary AML cells were derived from 43 patients included in two clinical studies of treatment based on ATRA, valproic acid and theophyllamine; low toxicity chemotherapy (low-dose cytarabine, hydroxyurea, 6-mercaptopurin) was also allowed. Pretreatment leukemic cells were analyzed by mutation profiling of 54 genes frequently mutated in myeloid malignancies and by global gene expression profiling before and during in vivo treatment. Results Patients were classified as responders and non-responders to the treatment, however response to treatment showed no significant associations with karyotype or mutational profiles. Significance analysis of microarray (SAM) showed that responders and non-responders significantly differed with regard to the expression of 179 different genes. The differentially expressed genes encoding proteins with a known function were further classified based on the PANTHER (protein annotation through evolutionary relationship) classification system. The identified genes encoded proteins that are involved in several important biological functions, but a main subset of the genes were important for transcriptional regulation. These pretherapy differences in gene expression were largely maintained during treatment. Our analyses of primary AML cells during in vivo treatment suggest that ATRA modulates HOX activity (i.e. decreased expression of HOXA3, HOXA4 and HOXA5 and their regulator PBX3), but altered function of DNA methyl transferase 3A (DNMT3A) and G-protein coupled receptor signaling may also contribute to the effect of the overall treatment. Conclusions Responders and non-responders to AML stabilizing treatment based on ATRA and valproic acid differ in the pretreatment transcriptional regulation of their leukemic cells, and these differences may be important for the clinical effect of this treatment. Trial registrations ClinicalTrials.gov no. NCT00175812 ; EudraCT no. 2004–001663-22, registered September 9, 2005 and ClinicalTrials.gov no. NCT00995332 ; EudraCT no. 2007–2007–001995-36, registered October 14, 2009

Molecular mechanisms of nutlin-3 involve acetylation of p53, histones and heat shock proteins in acute myeloid leukemia

Background The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. Results Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intracellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. Conclusions Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML

The Possible Diagnostic and Prognostic Use of Systemic Chemokine Profiles in Clinical Medicine&amp;#8212;The Experience in Acute Myeloid Leukemia from Disease Development and Diagnosis via Conventional Chemotherapy to Allogeneic Stem Cell Transplantation

Chemokines are important regulators of many different biological processes, including (i) inflammation with activation and local recruitment of immunocompetent cells; (ii) angiogenesis as a part of inflammation or carcinogenesis; and (iii) as a bridge between the coagulation system and inflammation/immune activation. The systemic levels of various chemokines may therefore reflect local disease processes, and such variations may thereby be used in the routine clinical handling of patients. The experience from patients with myeloproliferative diseases, and especially patients with acute myeloid leukemia (AML), suggests that systemic plasma/serum cytokine profiles can be useful, both as a diagnostic tool and for prognostication of patients. However, cytokines/chemokines are released by a wide range of cells and are involved in a wide range of biological processes; the altered levels may therefore mainly reflect the strength and nature of the biological processes, and the optimal clinical use of chemokine/cytokine analyses may therefore require combination with organ-specific biomarkers. Chemokine levels are also altered by clinical procedures, therapeutic interventions and the general status of the patients. A careful standardization of sample collection is therefore important, and the interpretation of the observations will require that the overall clinical context is considered. Despite these limitations, we conclude that analysis of systemic chemokine/cytokine profiles can reflect important clinical characteristics and, therefore, is an important scientific tool that can be used as a part of future clinical studies to identify clinically relevant biomarkers

Expression of the potential therapeutic target CXXC5 in primary acute myeloid leukemia cells -high expression is associated with adverse prognosis as well as altered intracellular signaling and transcriptional regulation

International audienceThe CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication. INTRODUCTION CXXC5 is a retinoid-responsive gene localized to the 5q31.3 chromosomal region [1] and encoding a retinoid-inducible nuclear factor (RINF) [2] that is a protein containing a CXXC-type zinc-finger domain and acting as a transcription regulator [3]. Expression studies as well as gene silencing experiments suggest that CXXC5 is important in normal myelopoiesis [2] and for differentiation of endothelial cells [3]. Furthermore, we recently described that CXXC5 is expressed in primary acute myeloid leukemia (AML) cells; this expression shows a wide variation between patients and high levels are associated with an adverse prognosis and resistance to chemotherapy-induced apoptosis [4]. Another study recently confirmed our observations and CXXC5 expression was then of independent prognostic significance in multivariate analyses after adjustmen

Additional file 1: Table S1. of Disease-stabilizing treatment based on all-trans retinoic acid and valproic acid in acute myeloid leukemia – identification of responders by gene expression profiling of pretreatment leukemic cells

The characteristics of the 60 patients included in the two clinical studies. Table S2. Clinical and biological characteristics of the patients included in the study. Table S3. AML-stabilizing treatment based on ATRA plus valproic acid; a summary and comparison of the two treatment regimen. Table S4. Analysis of 54 submikrocopic mutations in primary human AML cells. Table S5. Differences in global gene expression profiles by primary human AML cells derived from responders and non-responders to AML-stabilizing treatment. Table S6. Differences in global gene expression profiles by primary human AML cells derived during AML-stabilizing treatment. Table S7. Differentially expressed genes identified from comparison of primary AML cells before and after treatment. (DOCX 55 kb