Leukemia is a disease of the blood or bone marrow, which is characterized by increased numbers of abnormal white blood cells. The abnormality of leukemic cells lies in their inhibited differentiation and increased proliferation rate. Leukemia is divided into acute and chronic, and further subdivided into lymphocytic and myeloid [1]. Within these groups further divisions are often necessary. The most heterogeneous group being a group of acute myeloid leukemias (AML) [2]. In leukemias differentiation block occurs in early hematopoietic progenitors, and resulting malignant cells are named blast cells. Acute leukemias are diagnosed either on the basis of presence of over 20% of blasts in the blood or in bone marrow or on the basis of presence of specific cytogenetic or molecular abnormalities [1]. There are more than 200 known chromosome translocations and mutations in leukemic cells of patients diagnosed with AML [3]. Which of these 200 are initial mutations responsible for clonal expansion of abnormal cells, and which are accumulated during the progress of disease, is largely unknown. AML is rare in children, it constitutes 80–85% of acute leukemia in adults, and its incidence increases with age [4]. The progress in understanding molecular background of the disease has led to significant changes in the classification of AML subtypes. Before 2001, the French-American-British (FAB) classification was used, in which AML was divided into eight subtypes (M0—M7), based on the type of cell from which the leukemia developed and on its degree of maturity [5]. In 2001 WHO introduced a more accurate division that incorporates cytogenetic abnormalities and prognostic significance. WHO classification was further revised in 2008 and has classified AML into four main groups: AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes, therapy-related myeloid neoplasms, and AML not otherwise specified [6]. It should be also remembered that the disease is not common. In our country, Poland, with a population of about 38 million people, there are about 1000 cases of AML diagnosed per year [7]. Taking together the numbers and the variety of genetic alterations, it becomes clear that finding of a specific treatment for AML is not an easy task.

The idea of targeted therapies is not new. In his speech at the ceremonial opening of the Georg-Speyer-Haus in September 1906, Paul Ehrlich proposed that drugs should work as “magic bullets” that kill pathogens, and leave normal tissue unaffected [8]. Ehrlich's “magic bullets” were directed towards microorganisms, but later on the idea was adopted for anticancer treatment. Of course, every drug has its target, and most of the currently used anticancer chemotherapeutic drugs target, in a more or less direct manner, synthesis of DNA and cell division. Since cancer cells are not the only ones that need to proliferate in human body, chemotherapeutic drugs are often toxic and cannot be considered as “magic bullets” or in other words, targeted drugs. Therefore targeted anticancer therapies should be based on compounds that interfere with cellular components that are altered or present only in cancer cells [9].

Paradigmatic targeted therapy is embodied by treatment of patients with chronic myeloid leukemia (CML) using Imatinib [10]. Over 90% of CML patients carry chromosomal abnormality called the Philadelphia (Ph) chromosome [11,12]. Therefore the drug that interferes selectively with the tyrosine kinase activity of the fusion protein Bcr-Abl, should be toxic only to CML malignant cells [13]. The introduction of Imatinib (and similar specific inhibitors of Bcr-Abl) has revolutionized treatment of CML patients and increased rates of complete hematological response to 97% and complete cytogenetic response to 85% [14].

The most important difficulty in finding appropriate regimens of targeted therapy for AML patients originates from the above mentioned heterogeneity of the disease. Among 200 known chromosome translocations and mutations in AML, some are more common than others. One of the most common mutations seen in AML is internal tandem duplication in fms-like tyrosine kinase 3 (FLT3-ITD). This mutation is apparent in about 25% of all AML patients and confers unfavorable prognosis [2,15]. There is also another mutation in FLT3 receptor, point mutation in the tyrosine kinase domain (FLT3-TDK), seen in approximately 7–8% of AML patients, with less defined prognostic significance [2,16]. FLT3 receptor tyrosine kinase is normally expressed in immature precursors of myeloid and B-lymphoid lineages [17,18]. FLT3 ligand (FLT3-L) together with other colony stimulating factors and interleukins can stimulate proliferation of hematopoietic cells [19,20]. The two above mentioned mutations result in a ligand-independent activation of the receptor and give survival advantage to blast cells over their normal counterparts [21]. Aberrant FLT3 receptor seemed to be an attractive therapeutic target in AML, so several small molecules FLT3 tyrosine kinase inhibitors (TKI) have been developed and examined in vitro and in vivo. Eight of them, after successful preclinical screening, have been introduced into clinical trials [22]. The data available at the moment show that TKIs, used as single agents did not fulfill expectations and that the quality of clinical response was unsatisfactory [22].

Leukemic cells are inhibited in their hematopoietic differentiation by either genetic abnormalities or by gene expression abnormalities [2]. These cells proliferate rapidly, but often do not express proteins important for function of their normal counterparts. Even though white blood cells counts are high in these patients, the immune functions are lacking. Therefore, finding a method of forced differentiation of leukemic cells always seemed attractive to researchers and clinicians. Differentiation therapy seems to be a particularly attractive solution for AML patients. These patients are often elderly and rapidly progressing disease causes poor tolerability of intensive cytotoxic protocols [1]. Forced differentiation of myloid precursors should, in principle, improve the immune status of patients without massive lysis of blast cells seen in some cytotoxic regimens [23].

It should be remembered that inhibited differentiation may result not only from presence of mutated proteins, but also from epigenetic changes like DNA hypermetylation or aberrant acetylation of histones [9]. On the contrary to the loss of gene function caused by mutations, epigenetic changes can be reversed via pharmacologic inhibition of DNA methyltransferases (DnmT) and histone deacetylases (HDAC). In normal cells, histone acetylation and DNA methylation are maintained in equilibrium, allowing temporal expression of the genes. In leukemic cells, this balance is disturbed, hypermethylation occurs, HDACs are overexpressed, what leads to the transcriptional repression [24]. Therefore, several HDAC and DnmT inhibitors have been under development for the treatment of patients with hematological malignancies, such as AML or MDS, either as monotherapies or in combination with other agents [25-28]. Treatment of leukemia cells with such inhibitors, results in chromatin remodeling that unblocks a set of genes whose transcriptional activation induces cellular differentiation, cell cycle arrest, apoptosis or autophagy [26,29,30]. miRs, small non-coding RNAs 19–25 nucleotides long, provide an additional level of control between proliferation and differentiation [31,32]. They regulate gene expression post-transcriptionally via degradation of target mRNAs or/and via inhibition of protein translation [33,34]. A single miR can control levels of hundreds different target genes. Many miRs have been linked to the specification of hematopoietic cell lineages, and have been found altered by chromosomal translocations associated with leukemia and therefore constitute potential therapeutic targets [35-38].

Acute Promyelocytic Leukemia (APL) is the first hematological malignancy in which therapeutic approach specifically targeting the underlying molecular lesion has been successfully introduced into clinical practice. APL is a subset of AML characterized by uncontrolled expansion of leukemic blast cells, blocked at promyelocytic stage of hematopoiesis, in the bone marrow [39]. Morphologically, it is classified as a subtype M3 of AML [5], cytogenetically is characterized by a reciprocal translocation between the long arms of chromosomes 15 and 17 [t(15;17)] [40-43]. These aberrations lead to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, and to the formation of the resultant chimeric oncoprotein PML-RARA [42,44]. The fusion transcript of PML-RARA is detectable in more than 95% of APL patients with t(15;17), and becomes a major player disturbing proper promyelocytic differentiation, as well as a molecular marker for this disease [41,45]. In the remaining minority of patients, alternative fusions of RARA may occur [46-49]. These findings enabled the introduction of all- trans-retinoic acid (ATRA) as a differentiation agent for APL treatment.

The major role of 1,25D in human body is maintenance of calcium/phosphate homeostasis, but many other so called non-classical actions of 1,25D are known. One of these non-classical actions is, the above mentioned, monocytic differentiation of AML cells [96,97]. In order to separate calcemic properties of 1,25D from other activities of the compound, many low-calcemic vitamin D analogs (VDAs) have been synthesized for various clinical purposes [106-108]. Some VDAs have shown very promising anti-leukemic activities in vitro and in vivo [109-113]. Encouraged by early observations of 1,25D-induced differentiation of AML cells, few clinical trials were conducted to test the ability of 1,25D to treat myelodysplastic syndrome (MDS) and AML [114,115]. Since hypercalcemia is a limiting factor in clinical use of 1,25D in cancer patients, a low-calcemic analog, paricalcitol was also used to treat MDS in a small clinical trial [116]. Results of these trials were disappointing, therefore at present the use of VDAs in combination therapy is postulated. Several different combinations of drugs for use together with VDAs have been proposed and in most of them the goal is to potentiate differentiating effects of 1,25D or VDAs. This way calcemic effects could be avoided by lowering doses of 1,25D or VDAs necessary to obtain differentiation of leukemic cells. A series of papers has shown that antioxidants, such as carnosic acid, silibinin and curcumin effectively potentiate 1,25D-induced cell differentiation in vitro [117-119] and extend the life span of mice inoculated with murine leukemia [110,120]. Similar effect of enhanced differentiation could be obtained using everolismus, immunosuppressant used in transplantation medicine and in oncology, which inhibits mammalian target of rapamycin (mTOR) [121]. Interestingly inhibitors of p38 kinases α and β [122], as well as inhibitors of phospholipase A₂ [123] and non-specific inhibitors of cyclooxygenase (COX) [124] do the same. All compounds mentioned above share the ability to interfere with 1,25D-induced cell signaling in leukemic cells and, what is important, most of them are accepted drugs. The second strategy is to combine 1,25D or VDAs with agents that trigger cell death, for example with nutlin-3, which enhanced pro-apoptotic and downregulated anti-apoptotic proteins [125]. Many studies have shown that 1,25D potentiates the anti-tumor activities of chemotherapeutics agents [126], predominantly in solid cancers. However, there are also examples of clinical usefulness of 1,25D and cytarabine combination, which prolonged remission in elderly patients with acute AML and MDS [127,128].

Another approach is to select from a great variety of AMLs, only these which are susceptible to 1,25D. A good analogy exists in ATRA treatment, which is very effective in APL patients, but not in other subtypes of AML. It has been shown that individual responses of ex vivo cultured blast cells from patients with AML to differentiation-inducing effect of 1,25D are variable [118]. The analysis performed in order to look for correlations between mutations that are often diagnosed in blast cells of AML patients, with the susceptibility of the blasts towards VDA-induced differentiation, has shown that the most susceptible cells are these that carry monosomy 7 or partial loss of 7q [129]. Monosomy 7 or losses in the long arm of this chromosome occur in about 18% of the AML cases [130] and are often connected with prior MDS or with earlier therapy with alkylating agents [131]. Adult patients with this mutation have a very aggressive disease, great susceptibility to infections and poor prognosis [131]. If the correlation found in ex vivo cultured blasts could be translated into therapeutic use of VDAs in patients with monosomy 7 or partial loss of 7q, the differentiation-inducing activity of analogs together with their immunostimulating potential [132] might have beneficial effects in this group of patients.

Significant clinical improvement of patients with APL treated with ATRA raised reasonable hope for other agents, such as 1,25D and VDAs, to be effective differentiation-inducing drugs. Further improvements of APL therapy with ASO and detailed understanding of molecular mechanisms of both drugs have shown that therapies may be tailored for specific abnormalities present in neoplastic cells. Novel insights into the etiology of leukemia are of major importance for clinical utility in future drugs [144-146] and may help to select susceptible targets for differentiation therapy using 1,25D and VDAs. It is obvious that the new therapeutic approach should be directed towards leukemic cells, without general cytotoxicity to the organism.