Current Drug Targets - Volume 8, Issue 6, 2007

Pediatric acute lymphoblastic leukemia, the most common childhood malignancy, represents a profound cancer therapy success story of the 20th Century. Well-designed clinical trials, coupled with improvements in supportive care, have increased the likelihood of cure from almost nil prior to the 1960s to approaching 80% at the present day. Nevertheless, our rejoicing that the most common pediatric malignancy is also one of the most curable should be tempered by the realization that a significant number of children still relapse and die from the disease, that survivors suffer significant longterm effects of cytotoxic chemotherapy, and that the cure rates for other less common acute leukemia subtypes remain at less than 50%. The identification of novel targets for drug development in pediatric acute leukemia provides the vision to protect our most valuable resource, by saving those children who have relapsed, by developing drugs that more specifically target leukemia cells (and thereby spare the normal cells of the body), and by improving the likelihood of survival from the leukemias currently considered for the most part incurable. A substantial number of biomedical researchers remain committed to understanding the molecular basis for pediatric acute leukemogenesis, its progression, and in too many cases its resistance to conventional treatment. The current issue highlights exciting developments in this field of research. It has long been established that the microenvironment influences the development and progression of acute leukemia. The chapter by Konopleva and Andreef summarizes our current understanding, and explores the possibility of targeting microenvironment/leukemia interactions in the development of novel therapies. Furthermore, it has become increasingly evident that dysregulation of tyrosine kinases is intimately involved in leukemogenesis. Stubbs and Armstrong describe the role of mutations in the FLT3 receptor tyrosine kinase in leukemia, and summarize current clinical development of FLT3 inhibitors. Inappropriate activation of the RAS signaling pathway is commonly observed in juvenile myelomonocytic leukemia (JMML), a rapidly fatal myeloproliferative disorder of early childhood. The chapter by Flotho and colleagues summarizes current understanding of JMML, and offers insight into how the RAS signaling pathway might be exploited in the development of novel therapeutics to improve the outcome for this devastating disease. Transcription factors are dysregulated in many childhood acute leukemias by chromosomal translocations that result in their repressed activity or inappropriate activation. Berman and Look review the current state of knowledge in the field, and focus on recent attempts to target transcription factors in leukemia, and in particular acute promyelocytic leukemia and T-lineage acute lymphoblastic leukemia, as well as predictions for future drug targeting. Tubulin-binding agents, in particular the Vinca alkaloids vincristine and vinblastine, have become central components of combination chemotherapy regimens used in the treatment of a wide range of malignancies, including pediatric acute leukemia. Liaw and colleagues review the mechanism action of tubulin-binding agents, how drug resistance can emerge, and provide insight into current and future clinical development of novel tubulin-binding drugs. While tubulin-binding drugs exert their anti-leukemic effects via induction of apoptosis (programmed cell death), the chapter by Grant and Dent explores the rational combination of drugs that simultaneously target signal transduction and cell cycle regulatory pathways to specifically induce apoptosis in acute leukemia cells. Den Boer and Pieters then review the use of microarray analysis of gene expression to identify new drug targets in acute leukemia subtypes.........

Normal hematopoiesis is maintained by dynamic interactions between hematopoietic cells and the bone marrow microenvironment. In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow microenvironment such as stroma, osteoblasts and endothelium. In this review, we will discuss the influence of the microenvironment on the evolution of the leukemic phenotype. We propose that specific niches within the bone marrow microenvironment may provide a sanctuary for subpopulations of leukemic cells to evade chemotherapyinduced death and allow acquisition of a drug-resistant phenotype. We will also discuss recent studies that suggest novel therapeutic interventions targeting the microenvironment/leukemia interaction. Focus on this stroma-leukemia crosstalk may result in the development of strategies that alleviate the acquisition of a chemoresistant phenotype and enhance the efficacy of therapies in hematological malignancies.

During the past few years, a major focus of leukemia research has centered on tyrosine kinases as potential therapeutic targets. This is due in large part to the success of imatinib mesylate (STI571, Gleevec), which has proven effective as a therapy for chronic myeloid leukemias bearing the t(9;22) encoding the BCR-ABL kinase. It has become increasingly evident that mutations producing constitutively active tyrosine kinases play a role in leukemogenesis. Another kinase that has drawn significant attention is the FMS-like tyrosine kinase 3 (FLT3). FLT3 is expressed in most childhood acute leukemias. Select genetic subgroups possess particularly high-level expression, with a significant percentage therein harboring activating mutations. In this review we will discuss FLT3 as a potential therapeutic target in childhood acute leukemias. We will highlight the role of FLT3 in hematopoiesis, and how when activated, it may play a role in the development of acute myeloid or acute lymphoblastic leukemia. We will examine the successes in elucidating FLT3 function in acute leukemias, highlight current FLT3 targeted therapeutics, and discuss how FLT3 inhibitors might be used in combination therapies in the future.

The RAS proteins function as fundamental signaling switches that control normal cell growth and differentiation. Deregulated activation of RAS-dependent signaling pathways constitutes a potent mechanism of malignant cell transformation. Juvenile myelomonocytic leukemia (JMML) is a rapidly fatal myeloproliferative disorder of early childhood for which no effective treatment other than hematopoietic stem cell transplantation is currently available. Many aspects of JMML pathobiology are linked to deregulated RAS signaling. Hence, targeting RAS or its interactors on a molecular level is a promising strategy in the development of novel rational therapies for this menacing disease. Here we give an overview of current concepts on the pathogenesis of JMML, present important aspects of cellular RAS biology that can be exploited for pharmacologic manipulation, and discuss mouse models that have greatly advanced our understanding of the role RAS plays in JMML. In addition, we review recent approaches to develop agents that interfere with the RAS network at the level of the granulocyte-macrophage colony-stimulating factor receptor, posttranslational RAS processing (prenylation and endoprotease cleavage), RAF serine/threonine kinase, MEK mitogen-activated protein kinase, and target of rapamycin activity. Preclinical and clinical data of these pharmaceuticals in JMML and other myeloid malignancies is discussed.

Transcription factors play essential roles in controlling normal blood development and their alteration leads to abnormalities in cell proliferation, differentiation and survival. In many childhood acute leukemias, transcription factors are altered through chromosomal translocations that change their functional properties resulting in repressed activity or inappropriate activation. The development of therapies that specifically target these molecular abnormalities holds promise for improving the outcome in diseases that remain challenging to treat, such as childhood T-cell acute lymphoblastic leukemia and acute myeloid leukemia, with improved toxicity profiles. All trans-retinoic acid and arsenic trioxide have already demonstrated efficacy in acute promyelocytic leukemia in both adults and children. Newer agents, such as histone deacetylase inhibitors, drugs targeting the NOTCH pathway, and short interfering RNAs have shown encouraging results in pre-clinical studies and are likely to enter the clinical arena in the near future. Through an improved understanding of the pathways and mechanisms underlying the malignant transformation induced by altered transcription factors, new targeted therapies will be designed that should greatly enhance current available treatments.

Antimitotic agents that interfere with the tubulin/microtubule system are important in the treatment of a range of cancers. Natural product tubulin-binding agents such as the Vinca alkaloids have proven highly effective in the treatment of leukemia. Improved understanding of the mechanisms of action of these and related drugs has led to the identification of distinct binding sites on tubulin that cause inhibition of spindle microtubule dynamics, mitotic arrest and cell death. Despite the efficacy of these agents, treatment failure caused by the emergence of drug resistant leukemic cells is a significant clinical problem. Alterations in the cellular target of tubulin-binding agents have been strongly implicated in resistance to these agents. This review will focus on the microtubule cytoskeleton and its role in drug resistance in leukemia. The identification of novel protein pathways involved in drug response and the development of new drugs targeted against microtubules, offers opportunities to treat resistant disease, improve outcome and potentially reduce toxicity for leukemia patients.

The last decade has witnessed the introduction of a large number of novel, molecularly targeted agents into the therapeutic armamentarium against diverse forms of cancer, including leukemia. Such agents include signal transduction, cell cycle, histone deacetylase, Hsp90, proteasome, and Bcl-2 family member inhibitors, among others. While most of these agents have been or are currently being evaluated in adult patients with acute leukemia, experience in childhood leukemia is very limited. Although the use of such targeted agents as potentiators of conventional cytotoxic agent activity represents a logical approach, an emerging body of evidence suggests that neoplastic cells in general, and leukemic cells in particular, are highly susceptible to a therapeutic strategy in which survival signaling and cell cycle regulatory pathways are simultaneously disrupted. In in vitro studies, highly synergistic antileukemic interactions have been reported between CDK and HDAC inhibitors; HDAC and proteasome inhibitors; Bcl-2 antagonists and CDK inhibitors; MEK/ERK and Chk1 inhibitors, and proteasome and CDK inhibitors, among other combinations. Some of these strategies, including combinations of HDAC and CDK inhibitors, and CDK and proteasome inhibitors, have now entered the clinical arena in patients with leukemia and other hematologic malignancies. Based upon preclinical results to date, there is reason to suspect that such strategies might prove to be active against several types of childhood leukemia. Thus, over the next decade, the introduction of molecularly targeted agents, alone and in combination, into the therapeutic armamentarium against childhood leukemia may have significant implications for children with this disease.

The efficacy of current treatment protocols for childhood cancer is mainly based on empirical studies by adding drugs, changing drug dosages and changing drug combinations. In pediatric acute lymphoblastic leukemia (ALL), this approach has resulted into ∼80% 5-year disease-free survival whereas less favorable results have yet been obtained for acute myeloid leukemia (AML), i.e. ∼50%, and other types of tumors, e.g. ∼60% for medulloblastoma. A further optimization of therapy results requires more insights into the molecular biology of tumor cells, including genetic defects and aberrant expression of genes. This knowledge is needed to rationally develop more specific therapies in which relapse-risk and side-effects of therapy are reduced using targeted drugs. Genome-wide analysis of gene expression levels (mRNA) has revealed many new insights into the biology of leukemic cells. In this review we will discuss the recent progress that has been made in the use of microarrays for identifying new markers and targets for treatment of acute leukemia in children.

The mainstay of clinical anti-neoplastic chemotherapy is multi-agent combinations, most of which were developed empirically. To speed research and decrease costs, there is increasing interest in moving new drugs into clinical trials in potentially active combinations based upon pre-clinical testing data. Because testing drug combinations in animals is expensive and complex, defining drug combinations initially in cell culture assays is essential. For in vitro testing we employ a panel of well-characterized cell lines and DIMSCAN, a semi-automatic fluorescence-based digital image microscopy system that quantifies relative total (using a DNA stain) or viable [using fluorescein diacetate (FDA)] cell numbers in tissue culture multi-well-plates ranging from 6 to 384 wells per plate. DIMSCAN is a rapid and efficient tool for conducting in vitro cytotoxicity assays across a 4 log dynamic range. The specificity of detecting viable cells with FDA is achieved by use of digital image processing and chemical quenching of fluorescence in non-viable cells with eosin Y. Cytotoxicity measured by DIMSCAN was found to be comparable to manual trypan blue dye exclusion counts or colony formation in soft agar, but with a significantly wider dynamic range, that enables drug combination studies used to detect synergistic or antagonistic interactions in cell lines from both solid tumors and leukemias. While different mathematical models have been proposed for evaluating drug interactions, which can be classified as synergistic (combinations demonstrating greater than the additive activity expected from each agent alone), additive, or antagonistic (drugs showing less activity in combination than expected from the sum of each agent alone), we generally find the Combination Index method (as developed by Chou, et al.) to be the most suitable for evaluating of drug interactions in cell culture assays.

The number of new anti-cancer drugs emerging for clinical trials in humans far exceeds the availability of pediatric acute leukemia patients to be entered into clinical trials. Therefore, preclinical testing of new agents for the treatment of childhood acute leukemia is essential to ensure that the most promising drugs are prioritized to enter clinical trials. Historically, the murine system has been central to modeling human leukemia in vivo. A greater knowledge of the molecular lesions underlying particular subtypes of leukemia has led to the generation of genetically engineered murine models, generally involving the knockin or knockout of certain genes and fusion genes at their normal genetic locus. However, the most predominant in vivo models for preclinical drug testing have been human leukemia xenografts. Successful engraftment of all subtypes of acute lymphoblastic leukemia, most subtypes of acute myeloid leukemia as well as juvenile myelomonocytic leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia have been described in various immune- deficient murine hosts. Preclinical testing of novel therapeutics in vivo will likely identify the most promising new agents to enter clinical trials, and will allow their future use to be optimized in combination with other novel and conventional chemotherapeutics.