Camilla Stapnes

Histone Deacetylase Inhibitors in Cancer Treatment: A Review of the Clinical Toxicity and the Modulation of Gene Expression in Cancer Cells

Characterization of epigenetic events in carcinogenesis has led to the discovery of a new class of oncogenes and thereby a new class of therapeutic targets. Among the new therapeutic approaches are modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs). HDACs deacetylate histones as well as transcription factors and can modulate gene expression through both these mechanisms in normal and malignant cells. Furthermore, acetylation is an important posttranslational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis in normal as well as cancer cells. Even though several HDAC inhibitors have been characterized in vitro, only a limited number of these agents are in clinical trials. Various HDAC inhibitors differ in their toxicity profile when comparing the side effects described in the available clinical studies of HDAC inhibition in the treatment of cancer. These drugs may also affect normal hematopoiesis; hematologic toxicity is common to many drugs but stimulation of hematopoiesis seems to occur for others. HDAC inhibitors usually affect < 10% of the genes in cancer cells. Divergent effects of HDAC inhibition on the global gene expression profiles have been described when testing various cancer cells, and this is further complicated by altered HDAC expression induced by HDAC inhibitors. However, increased p21 expression seems to be a common characteristic for most studies, suggesting an important role of this molecule during HDAC inhibitory treatment. Even though the initial studies are encouraging, additional in vitro and in vivo pharmacological characterization is definitely needed.

The proteasome inhibitors bortezomib and PR-171 have antiproliferative and proapoptotic effects on primary human acute myeloid leukaemia cells.

Proteasome inhibitors represent a new class of antineoplastic drugs that are considered in the treatment of haematological malignancies. We compared the effects of the reversible proteasome inhibitor bortezomib (Velcade®) and the epoxomicin derivative PR‐171, an irreversible inhibitor, on primary human acute myeloid leukaemia (AML) cells. Both drugs inhibited autocrine‐ and cytokine‐dependent proliferation of primary AML blasts when tested at nanomolar levels (0·1–100 nmol/l). The antiproliferative effect was independent of basal chymotrypsin‐like proteasome activity (showing a 20‐fold variation between patients), genetic abnormalities, morphological differentiation and CD34 expression when testing a large group of consecutive patients (n = 54). The effect was retained in cocultures with bone marrow stromal cells. In addition, both drugs enhanced apoptosis. The effect of PR‐171 could be detected at lower concentrations than for bortezomib, especially when testing the influence on clonogenic AML cell proliferation. Both drugs had divergent effects on AML cells’ constitutive cytokine release. Furthermore, both drugs caused a decrease in proliferation and viability when tested in combination with idarubicin or cytarabine. An antiproliferative effect on primary human acute lymphoblastic leukaemia cells was also detected. We conclude that nanomolar levels of the proteasome inhibitors tested had dose‐dependent antiproliferative and proapoptotic effects on primary AML cells in vitro.

A Novel, Extraneuronal Role for Cyclin-dependent Protein Kinase 5 (CDK5) MODULATION OF cAMP-INDUCED APOPTOSIS IN RAT LEUKEMIA CELLS

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.

Protein lysine acetylation in normal and leukaemic haematopoiesis: HDACs as possible therapeutic targets in adult AML.

Several new therapeutic strategies are now considered for acute myelogenous leukaemia (AML), including modulation of protein lysine acetylation through inhibition of histone deacetylases (HDACs): a large group of enzymes that alters the acetylation and, thereby, the function of a wide range of nuclear and cytoplasmic proteins. Firstly, HDACs can deacetylate histones as well as transcription factors, and can modulate gene expression through both these mechanisms. Secondly, acetylation is an important post-translational modulation of several proteins involved in the regulation of cell proliferation, differentiation and apoptosis (e.g., p53, tubulin, heat-shock protein 90). The only HDAC inhibitors that have been investigated in clinical studies of AML are butyrate derivatives, valproic acid and depsipeptide. In the first studies, the drugs have usually been used as continuous therapy for several weeks or months, and in most studies the drugs were used alone or in combination with all-trans retinoic acid for treatment of patients with relapsed or primary resistant AML. Neurological toxicity and gastrointestinal side effects seem to be common for all three drugs. Complete haematological remission lasting for several months has been reported for a few patients (< 5% of included patients), whereas increased peripheral blood platelet counts seem more common and have been described both for patients with AML and myelodysplastic syndromes. Taken together, these studies suggest that HDAC inhibition can mediate antileukaemic effects in AML, but for most patients the clinical benefit seems limited and further studies of combination therapy are required.

A subset of patients with high-risk acute myelogenous leukemia shows improved peripheral blood cell counts when treated with the combination of valproic acid, theophylline and all-trans retinoic acid.

Acute myelogenous leukemia (AML) patients (24 consecutive patients, median age 71 years, 17 high-risk disease) were treated with all-trans retinoic acid, theophylline and valproic acid. Among 22 evaluable patients 9 responded with increased normal peripheral blood cell counts. The responses could be classified as hematological improvement according to response criteria for patients with myelodysplastic syndromes (MDS) for four patients only. The nine patients with increased normal cell counts had a median survival from start of therapy of 147 days compared with 48 days for the other patients. Four patients fulfilling the MDS criteria had a survival ranging from 112 to 644 days. The treatment was associated with decreased in vitro cytokine-dependent AML cell proliferation and increased blood levels of Endocan and angiopoietin-2 both for responders and non-responders. We conclude that the therapy causes disease stabilization for a subset of AML patients.

Targeted therapy in acute myeloid leukaemia: current status and future directions.

Background: The limit of acceptable toxicity for standard chemotherapeutic drugs used in acute myeloid leukaemia (AML) therapy has been reached. New therapeutic strategies are therefore needed. Objective: This review summarizes development in new strategies, and gives an overview of the clinical status on new drugs for non-promyelocytic AML in adults. Methods: Information was principally gathered from the databases ClinicalTrials.gov and PubMed.gov. Results/conclusion: The major improvements in AML treatment during the last two decades has not been the introduction of new therapeutic agents, but rather the more optimal use of well-known drugs (e.g., high-dose cytarabine therapy, the use of ATRA in maintenance therapy of acute promyelocytic leukaemia) and improvement in the diagnosis and treatment of potentially life-threatening complications in patients treated with allogeneic stem cell transplantation. However, further investigations based on specific targeted therapy and stratification of patients according to knowledge of the individual disease and health status will probably be necessary in future studies of new targeted therapy.

In vivo biological effects of ATRA in the treatment of AML

Background: All-trans retinoic acid (ATRA) is mandatory in the treatment of acute promyelocytic leukaemia (APL). Experimental studies suggest that ATRA can induce differentiation and apoptosis in leukaemia cells also for other acute myelogenous leukaemia (AML) subtypes, but the clinical observations are conflicting. Design and methods: Twenty-two AML patients with non-APL disease received oral ATRA alone (22.5 mg/m2 twice daily) for two days, the patients thereafter continued ATRA together with valproic acid and theophylline. We investigated the biological effects of the initial 2 days treatment with ATRA alone. Serum/plasma samples were collected before and after 2 days of ATRA, peripheral blood AML cells were collected from all 12 patients with circulating leukaemia cells (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22). Results: AML cells collected during therapy had altered flow cytometric forward and right angle light scatters but no morphological signs of differentiation. ATRA increased the percentage of circulating AML cells in G0/G1 phase for 9 out of 12 patients (p = 0.043). Circulating leukaemia cells derived during therapy had increased intracellular levels of P21 (mean increase in mean fluorescence intensity (MFI) being 18.2%, p = 0.017), and decreased levels of Gata-2 (mean decrease in MFI 19%, p = 0.026), NF-κB p65 (mean decrease in MFI 15.4%, p = 0.033) and Bcl-2 (mean decrease in MFI 7.2%, p = 0.005). In addition, increased systemic levels of the endothelial marker endocan (plasma) and the angioregulatory mediator angiopoietin-2 (serum) were observed. Conclusions: In vivo ATRA treatment in AML affects leukaemic cell morphology, regulation of cell cycle progression and apoptosis, and possibly also microvascular endothelial cell functions.

Acute myelogenous leukemia in a patient with Li–Fraumeni syndrome treated with valproic acid, theophyllamine and all-trans retinoic acid: a case report

Acute myelogenous leukemia in a patient with Li–Fraumeni syndrome treated with valproic acid, theophyllamine and all-trans retinoic acid: a case report

Treatment with valproic acid, all-trans retinoic acid (ATRA) and theophyllamine for 9 days caused a persistent increase in peripheral blood platelet counts for a patient with acute myelogenous leukemia.

New therapeutic strategies are now considered in acute myelogenous leukemia (AML), including the use of histone deacetylase (HDAC) inhibitors, alltrans retinoic acid (ATRA) and the cyclic AMP increasing agent theophyllamine [1 /5]. Firstly, the presently used and relatively unspecific HDAC inhibitors would be expected to alter acetylation of many intracellular proteins and not only histones [6], and modulation of gene expression through decreased histone acetylation is probably one of several pharmacological effects. Valproic acid is a short chain fatty acid that is used as an anticonvulsant but can also function as an HDAC inhibitor and induce differentiation and apoptosis of AML cells in vitro [1,2]. Secondly, ATRA is regarded as mandatory in the treatment of acute promyelocytic leukemia (APL), but its use in nonAPL disease has not improved long-term AML-free survival even though in vivo effects on AML blasts have been detected for a relatively large fraction of treated patients [7]. Finally, increasing the intracellular levels of the second messenger cyclic AMP (e.g. by theophyllamine) is another therapeutic possibility [8]. For our present AML patient we combined these three therapeutic strategies. Increased peripheral blood platelet counts have been described after treatment with several HDAC inhibitors (i.e. valproic acid, butyrate derivatives, depsipeptide) for a subset of AML and myelodysplastic syndrome (MDS) patients [1 /5]. Most of these studies investigated continuous treatment with HDAC inhibitor plus ATRA. Even though hematological remission was very rare and was not observed in most studies, increased platelet counts may be an important effect for these patients who often experience severe thrombocytopenia. In the present report we present evidence that increased thrombopoiesis can occur early during treatment with valproic acid /ATRA /theophyllamine, and the platelet levels may continue to increase and persist at high levels for several weeks following short-term treatment.