A dual role for Hdac1: oncosuppressor in tumorigenesis, oncogene in tumor maintenance.

Aberrant recruitment of histone deacetylases (HDACs) by the oncogenic fusion protein PML-RAR is involved in the pathogenesis of acute promyelocytic leukemia (APL). PML-RAR, however, is not sufficient to induce disease in mice but requires additional oncogenic lesions during the preleukemic phase. Here, we show that knock-down of Hdac1 and Hdac2 dramatically accelerates leukemogenesis in transgenic preleukemic mice. These events are not restricted to APL because lymphomagenesis driven by deletion of p53 or, to a lesser extent, by c-myc overexpression, was also accelerated by Hdac1 knock-down. In the preleukemic phase of APL, Hdac1 counteracts the activity of PML-RAR in (1) blocking differentiation; (2) impairing genomic stability; and (3) increasing self-renewal in hematopoietic progenitors, as all of these events are affected by the reduction in Hdac1 levels. This led to an expansion of a subpopulation of PML-RAR-expressing cells that is the major source of leukemic stem cells in the full leukemic stage. Remarkably, short-term treatment of preleukemic mice with an HDAC inhibitor accelerated leukemogenesis. In contrast, knock-down of Hdac1 in APL mice led to enhanced survival duration of the leukemic animals. Thus, Hdac1 has a dual role in tumorigenesis: oncosuppressive in the early stages, and oncogenic in established tumor cells.

Outcome of children treated for relapsed acute myeloid leukemia in Central America.

BACKGROUND: Relapsed childhood acute myeloid leukemia (AML) outcomes have not been documented in resource-limited settings. We examined survival after relapse for children with AML (non-APML) and acute promyelocytic leukemia (APML) in Central America. PROCEDURE: We retrospectively evaluated outcomes of children with first relapse of AML (non-APML) and APML in Guatemala, Honduras, or El Salvador diagnosed between 1997 and 2011. Predictors of subsequent event-free survival (EFS) and overall survival (OS) were examined. RESULTS: We identified 140 children with relapsed AML (non-APML), and 24 with relapsed APML. Two-year subsequent EFS and OS (±SE) were 7.0 ± 2.5% and 9.1 ± 2.8%, respectively. Worse outcomes were associated with Hispanic or Indigenous heritage, white blood cell count at diagnosis ≥50 × 10(9) /L, and time to relapse <18 months. For those with relapsed APML, subsequent 2-year EFS and OS were 36.7 ± 10.8% and 43.4 ± 12.1%, although few patients survived beyond 3 years. 15.2% of all patients were managed solely with palliative intent following first relapse. CONCLUSIONS: Children with relapsed AML in Central America rarely survive, so palliative strategies should be considered following relapse in this population. However, children with late relapse or with APML may have a prolonged period of remission with second treatment, and consideration of re-treatment may be appropriate.

Who benefits from maintenance therapy in acute promyelocytic leukemia?

Acute promyelocytic leukemia (APL) is remarkable for its upfront mortality rate from disseminated intravascular coagulation and its high cure rate with therapy. Although induction and consolidation regimens continue to evolve, newer approaches combine an anthracycline with or without cytarabine and the highly effective differentiating drugs all-trans retinoic acid and arsenic trioxide. Early trials showed a benefit of maintenance therapy on overall survival, although this benefit has been less clear in subsequent trials. This review assesses the differences in these trials and outlines a rational approach to maintenance therapy in APL, generally advising against maintenance in patients who underwent adequate consolidation therapy, particularly if they presented with low-risk disease (WBC < 10,000) and experienced molecular complete remission after completion of consolidation.

Oral arsenic trioxide for relapsed acute promyelocytic leukemia in pediatric patients.

Four patients (age 3-11 years at diagnosis) with relapsed acute promyelocytic leukemia (APL), 12-38 months from diagnosis, were treated with oral arsenic trioxide (As(2) O(3) ). One patient was treated with oral As(2) O(3) monotherapy and chemotherapy. Three patients failed initial oral or intravenous As(2) O(3) monotherapy were treated with oral As(2) O(3) plus ATRA followed by long-term oral maintenance (cumulative As(2) O(3) dose 280-2,100 mg). All patients achieved molecular remission, at a median follow up of 122 (10-132) months with no adverse effects. Oral As(2) O(3) , particularly in prolonged maintenance with oral ATRA may obviate the need of stem cell transplantation in relapsed pediatric APL.

PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia.

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.

Long-term remission after first-line single-agent treatment with arsenic trioxide of relapsed acute promyelocytic leukemia in an 8-year-old boy.

Arsenic trioxide (ATO) has been proven to be highly effective in adults with newly diagnosed or relapsed acute promyelocytic leukemia (APL). Only very limited data are published on the use of ATO as a single agent for first-line therapy of relapsed APL. The authors present a case of a 8-year-old boy with a bone marrow relapse of APL 7 years after first diagnosis, who achieved durable molecular remission with ATO as single agent: induction therapy for 12 weeks, consolidation for 4 weeks, then 6 cycles of 10 days over a period of 6 months. In total, 140 doses of ATO (0.15 mg/kg/day) were given (21 mg/kg). Consecutive promyelocytic leukemia-retinoic acid receptor α (PML-RARα) RT-PCR analyses were negative with a follow-up of 48 months. Acute or late side effects of arsenic were not observed. At present, the boy is in complete remission 4 years after the diagnosis of the relapse.

[Recurrences of acute promyelocytic leukemia in children: experience with arsenic trioxide therapy and autologous hematopoietic cell transplantation].

AIM: To analyze the specific features of recurrences of acute promyelocytic leukemia (APL) in children after standard therapy with daunorubicin, cytosine arabinoside (Ara-C), all-trans retinoic acid (ATRA) and to develop further programmed treatment policy. SUBJECTS AND METHODS: The study included 9 patients with recurrent APL. The recurrences developed significantly more frequently in a very high-risk group (patients with minimal residual disease being preserved after the intensive therapy phase). Induction used arsenic trioxide (ATO) and/or standard chemotherapy + ATRA; ATO monotherapy was in consolidation. CD34+ cells were mobilized until molecular remission was achieved with high-dose Ara-C and granulocyte colony-stimulating factor. Pretransplantation conditioning involved melfalan as a basic drug in combination with high-dose AraC (5 pts), treosulfan (1 pt) or bisulfan (1 pt). Six patients received gemtusumab ozogamicin, 3-9 mg/m2, at different stages of therapy. RESULTS: Before therapy one patient died; 8 patients achieved the second molecular remission; CD34+ cell mobilization and sampling were effective in 7 cases. Five patients were in long-term molecular remission after autologous hemopoietic stem cell transplantation (autoHSCT). Follow-up was 23-40 months. One patient is being prepared for transplantation. Following autoHSCT, another patient with a developed repeat recurrence died from complications due to related partially compatible transplantation. Visceral, including cardiological, toxicity of therapy was insignificant. In the APL-2003 protocol, overall and event-free survival rates were 93 +/- 3 and 76 +/- 6%, respectively. CONCLUSION; The application of ATO and autoHSCT in recurrent APL makes it possible to achieve and preserve the second molecular remission in case of insignificant extrahematological toxicity. Russian clinics should have access to ATO.

Sustained remission achieved with ATRA and chemotherapy in second relapse of acute promyelocytic leukemia in Down syndrome.

Children with Down syndrome (DS) are at an increased risk of developing acute leukemia. Acute myeloid leukemia predominates among DS children below 4 years of age but acute promyelocytic leukemia (APL) has rarely been reported in DS. Acute myeloid leukemia in DS is extremely sensitive to treatment but the optimum treatment of de novo or relapsed APL in DS is not known. We describe a child with DS and APL, who despite having a multiply relapsing course, achieved a third remission with ATRA and chemotherapy, which is sustained with maintenance therapy. A brief review of literature is also presented.

Inhibition of promyelocytic leukemia (PML)/retinoic acid receptor-alpha and PML expression in acute promyelocytic leukemia cells by anti-PML peptide nucleic acid.

The fusion protein promyelocytic leukemia (PML)/retinoic acid receptor (RAR)alpha is tightly linked to the pathogenesis of acute promyelocytic leukemia (APL); hence, it represents a tumor-associated, transformation-related molecule. In this study, three anti-PML adamantyl-conjugated peptide nucleic acid (PNA) oligomers previously described as in vitro inhibitors of PML/RARalpha translation were combined and used to block PML/RARalpha synthesis in NB4 cells. Cationic liposomes were used to achieve sufficient delivery of PNAs into the cells. Upon treatment of cells with the liposome/PNA mixture, enhanced cellular uptake of PNA (approximately 5-fold compared with control) was obtained. Concomitantly, a substantial reduction (>90%) of the expression of PML/RARalpha was observed when all of the three PNAs were used together. This resulted in a dramatic effect on the number and viability of NB4 cells in culture after 48 h of treatment. This phenomenon was preceded by induction of apoptosis that could be observed 24 h after treatment. No sign of granulocytic differentiation was observed after treatment. These effects were also noted on other leukemic cell lines that express PML but not the fusion transcript. These results show that it is possible to deliver PNA into hematopoietic cells and obtain specific gene inhibition, and they suggest that a growth inhibitory effect on acute promyelocytic leukemia cells can be obtained through the block of PML/RARalpha and PML expression.

Granulocytic differentiation of human NB4 promyelocytic leukemia cells induced by all-trans retinoic acid metabolites.

The metabolism of all-trans retinoic acid (ATRA) has been reported to be partly responsible for the in vivo resistance to ATRA seen in the treatment of human acute promyelocytic leukemia (APL). However, ATRA metabolism appears to be involved in the growth inhibition of several cancer cell lines in vitro. The purpose of this study was to evaluate the in vitro activity of the principal metabolites of ATRA [4-hydroxy-retinoic acid (4-OH-RA), 18-hydroxy-retinoic acid (18-OH-RA), 4-oxo-retinoic acid (4-oxo-RA), and 5,6-epoxy-retinoic acid (5,6-epoxy-RA)] in NB4, a human promyelocytic leukemia cell line that exhibits the APL diagnostic t(15;17) chromosomal translocation and expresses the PML-RAR alpha fusion protein. We established that the four ATRA metabolites were indeed formed by the NB4 cells in vitro. NB4 cell growth was inhibited (69-78% at 120 h) and cell cycle progression in the G1 phase (82-85% at 120 h) was blocked by ATRA and all of the metabolites at 1 microM concentration. ATRA and its metabolites could induce NB4 cells differentiation with similar activity, as evaluated by cell morphology, by the nitroblue tetrazolium reduction test (82-88% at 120 h) or by the expression of the maturation specific cell surface marker CD11c. In addition, nuclear body reorganization to macropunctated structures, as well as the degradation of PML-RAR alpha, was found to be similar for ATRA and all of its metabolites. Comparison of the relative potency of the retinoids using the nitroblue tetrazolium reduction test showed effective concentrations required to differentiate 50% of cells in 72 h as follows: ATRA, 15.8 +/- 1.7 nM; 4-oxo-RA, 38.3 +/- 1.3 nM; 18-OH-RA, 55.5 +/- 1.8 nM; 4-OH-RA, 79.8 +/- 1.8 nM; and 5,6-epoxy-RA, 99.5 +/- 1.5 nM. The ATRA metabolites were found to exert their differentiation effects via the RAR alpha nuclear receptors, because the RAR alpha-specific antagonist BMS614 blocked metabolite-induced CD11c expression in NB4 cells. These data demonstrate that the principal ATRA Phase 1 metabolites can elicit leukemia cell growth inhibition and differentiation in vitro through the RAR alpha signaling pathway, and they suggest that these metabolites may play a role in ATRA antileukemic activity in vivo.

Potentiation of Acute Promyelocytic Leukemia Cell Differentiation and Prevention of Leukemia Development in Mice by Oleanolic Acid.

Although differentiation therapy with all-trans retinoic acid (ATRA) induces complete remission in most acute promyelocytic leukemia (APL) patients, it is associated with organ toxicity. The present study focused on investigating the effects of the natural compounds oleanolic acid (OA) and ursolic acid (UA) on proliferation and differentiation of human APL HL-60 cells in vitro and murine APL WEHI-3 cells in vivo. Results demonstrated that OA and UA significantly inhibited cellular proliferation of HL-60 in a concentration- and time-dependent manner. Non-cytotoxic concentration of OA exhibited a marked differentiation-inducing effect on HL-60 and enhanced ATRA-induced HL-60 differentiation. In contrast, UA showed only a moderate effect. Activation of MAPK/NF-κB signaling pathway was likely found to be involved in the mechanism. Moreover, OA increased survival duration of WEHI-3 transplanted BALB/c mice, and decreased leukemia cells infiltration in the liver and spleen. Thus, these results may provide new insight for developing alternative therapy in APL patients.

Conformationally defined retinoic acid analogues. 4. Potential new agents for acute promyelocytic and juvenile myelomonocytic leukemias.

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.

Hematopoiesis and retinoids: development and disease.

Retinoids function as activating ligands for a class of nuclear receptors that control gene expression programs for a wide range of tissues and organs during embryogenesis and throughout life. Over the years, three sets of observations have spurred interest in the function of retinoids with respect to development and disease of hematopoietic cells. Since the 1920s, epidemiological studies indicated altered hematopoiesis in vitamin A-deficient (VAD) human populations. More recently, the ability of retinoids to affect various aspects of hematopoietic development has been demonstrated in vitro. Finally, it was discovered that the gene encoding a retinoid receptor is a key target for chromosomal translocations that cause acute promyelocytic leukemia (APL). More recent investigations using targeted gene disruptions, VAD animal models, and mouse models of leukemia have continued to shed light on the function of the retinoid pathway in blood cells. It is now clear that retinoids are required for normal hematopoiesis during both yolk sac and fetal liver stages of hematopoiesis, while the pathway has at least modulatory functions for bone marrow derived progenitors. Studies of normal development and APL have provided complementary insight into the molecular control of blood cell differentiation. Here we review the evidence for retinoid requirements in hematopoiesis and also summarize current ideas regarding how this pathway is subverted in leukemia.

Cancer chemoprevention by hydroxytyrosol isolated from virgin olive oil through G1 cell cycle arrest and apoptosis.

Recent epidemiological evidence and animal studies suggest a relationship between the intake of olive oil and a reduced risk of several malignancies. The present study assesses the effect of hydroxytyrosol, a major antioxidant compound of virgin olive oil, on proliferation, apoptosis and cell cycle of tumour cells. Hydroxytyrosol inhibited proliferation of both human promyelocytic leukaemia cells HL60 and colon adenocarcinoma cells HT29 and HT29 clone 19A. The con-centrations of hydroxytyrosol which inhibited 50% of cell proliferation were approximately 50 and approximately 750 micromol/l for HL60 and both HT29 and HT29 clone 19A cells, respectively. At concentrations ranging from 50 to 100 micromol/l, hydroxytyrosol induced an appreciable apoptosis in HL60 cells after 24 h of incubation as evidenced by flow cytometry, fluorescence microscopy and internucleosomal DNA fragmentation. Interestingly, no effect on apoptosis was observed after similar treatment of freshly isolated human lymphocytes and polymorphonuclear cells. The DNA cell cycle analysis, quantified by flow cytometry, showed that the treatment of HL60 cells with hydroxytyrosol 50-100 micromol/l arrested the cells in the G0/G1 phase with a concomitant decrease in the cell percentage in the S and G2/M phases. These results support the hypothesis that hydroxytyrosol may exert a protective activity against cancer by arresting the cell cycle and inducing apoptosis in tumour cells, and suggest that hydroxytyrosol, an important component of virgin olive oil, may be responsible for its anticancer activity.

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells.

Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60). The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Cellular cytosols and intact cell suspensions were assayed. The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays. This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.

[Prevention and treatment of solid tumors with retinoids].

Retinoids regulate terminal differentiation of various cells and morphogenesis of various organs. Disorders of such regulations induce cellular and structural anomalies, and lead directly to carcinogenesis. According to these physiological roles of retinoids, many experimental studies have been conducted to test, and successfully demonstrate, the effect of retinoids for the treatment of cancers or the prevention of carcinogenesis. In clinical studies, however, retinoid therapy has not been established as a first choice treatment for solid tumors, although the effect of all-trans retinoic acid for acute promyelocytic leukemia is excellent. Primary cancer chemoprevention in general populations using beta-carotene has failed in all but one of four recently reported trials. In contrast, secondary chemoprevention by retinoids is promising for cancers such as head and neck cancer, uterine cervical cancer and hepatoma.

Ponatinib suppresses the development of myeloid and lymphoid malignancies associated with FGFR1 abnormalities.

Myeloid and lymphoid malignancies associated with fibroblast growth factor receptor-1 (FGFR1) abnormalities are characterized by constitutively activated FGFR1 kinase and rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. Molecular targeted therapies have not been widely used for stem cell leukemia/lymphoma (SCLL). Ponatinib (AP24534), which potently inhibits native and mutant BCR-ABL, also targets the FGFR family. Using murine BaF3 cells, stably transformed with six different FGFR1 fusion genes, as well as human KG1 cells expressing activated chimeric FGFR1 and five newly established murine SCLL cell lines, we show that ponatinib (<50 nM) can effectively inhibit phosphoactivation of the fusion kinases and their downstream effectors, such as PLCγ, Stat5 and Src. Ponatinib also significantly extended survival of mice transplanted with different SCLL cell lines. Ponatinib administered at 30 mg/kg daily also significantly delayed, or even prevented, tumorigenesis of KG1 cells in xenotransplanted mice. Furthermore, we demonstrate that ponatinib specifically inhibits cell growth and clonogenicity of normal human CD34+ progenitor cells transformed by chimeric FGFR1 fusion kinases. Overall, our data provide convincing evidence to suggest that pharmacologic inhibition of FGFR1 fusion kinases with ponatinib is likely to be beneficial for patients with SCLL and perhaps for other human disorders associated with dysregulated FGFR1 activity.