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.

BackgroundA 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.MethodsIn 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.ResultsThe 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.ConclusionTreatment 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.

Heat shock protein (HSP) 70 and HSP90 are released by primary human acute myeloid leukemia (AML) cells during stress-induced spontaneous in vitro apoptosis. The AML cells also show constitutive release of several cytokines and the systemic serum levels of several soluble mediators are altered in patients with untreated AML. In the present study, we have investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving AML-stabilizing palliative treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Patients with untreated AML showed increased HSP90 levels and a distinct serum cytokine profile when compared with healthy controls, and low pre-therapy HSP90 levels were associated with a prolonged survival during treatment with ATRA 1 valproic acid 1 theophyllin. Hierarchical cluster analysis showed a close association between HSP70, HSP90, IL-1 receptor antagonist (IL-1ra), and hepatocyte growth factor (HGF) levels. Furthermore, disease-stabilizing therapy altered the serum-cytokine profile, but the correlations between HSP70/HSP90/IL-1ra/HGF were maintained only when ATRA 1 valproic acid were combined with theophyllin but not when combined with cytarabine. We conclude that both HSP levels and serum cytokine profiles are altered and may represent possible therapeutic targets or prognostic markers in human AML. Am. J. Hematol. 87:368-376, 2012. V

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ABSTRACT:The retinoid-responsive gene CXXC5 localizes to the 5q31.2 chromosomal region and encodes a retinoid-inducible nuclear factor (RINF) that seems important during normal myelopoiesis. We investigated CXXC5/RINF expression in primary human acute myeloid leukemia (AML) cells derived from 594 patients, and a wide variation in CXXC5/RINF mRNA levels was observed both in the immature leukemic myeloblasts and in immature acute lymphoblastic leukemia cells. Furthermore, patients with lowrisk cytogenetic abnormalities showed significantly lower levels compared to patients with high-risk abnormalities, and high RINF/CXXC5/ mRNA levels were associated with decreased overall survival for patients receiving intensive chemotherapy for newly diagnosed AML. This association with prognosis was seen both when investigating (i) an unselected patient population as well as for patients with (ii) normal cytogenetic and (iii) core-binding factor AML. CXXC5/RINF knockdown in AML cell lines caused increased susceptibility to chemotherapy-induced apoptosis, and regulation of apoptosis also seemed to differ between primary human AML cells with high and low RINF expression. The association with adverse prognosis together with the antiapoptotic effect of CXXC5/RINF suggests that targeting of CXXC5/RINF should be considered as a possible therapeutic strategy, especially in high-risk patients who show increased expression in AML cells compared with normal hematopoietic cells.

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.

Abstract. several previous studies have demonstrated that both conventional cytotoxic drugs as well as targeted therapeutics can induce apoptosis in primary human acute myelogenous leukemia (AMl) cells. However, the apoptotic phenotype of dying AMl cells has been less extensively characterized. even though specific antileukemic immune reactivity is important in AMl, especially for allotransplanted patients, it has not been investigated whether dying primary human AMl cells show phenotypic characteristics consistent with immunogenic apoptosis [calreticulin exposure, heat shock protein (Hsp) release]. We therefore investigated whether in vitro cultured primary human acute myeloid leukemia (AMl) cells show calreticulin exposure and Hsp70/Hsp90 release during spontaneous (stress-induced) apoptosis when cultured in medium alone and when cultured in the presence of antileukemic drugs. Both surface exposure of calreticulin and release of Hsp70 and Hsp90 was detected but showed a wide variation between patients. this variation was also maintained when the AMl cells were cultured in the presence of cytotoxic drugs (cytarabine, daunorubicin, mitomycin), all-trans retinoic acid (AtrA) and valproic acid. Finally, AMl cells collected during in vivo AtrA therapy showed increased calreticulin exposure during spontaneous in vitro apoptosis, suggesting that in vivo pharmacotherapy can modulate the apoptotic phenotype. to conclude, apoptotic AMl cells can show phenotypic characteristics consistent with immunogenic apoptosis, but there is a wide variation between patients and the level of calreticulin exposure/Hsp release seems to depend on individual patient characteristics rather than the apoptosisinducing agent.

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.