CHAPTER II

LITERATURE REVIEW

"Acute myeloid leukemia (AML) goes by many names, including acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute non-lymphocytic leukemia"

(Detailed Guide: Leukemia).

The word "acute" in acute myeloid leukemia refers to the fact the leukemia may progress at a rapid rate, and if/when the disease is not treated, within a few months, it would likely prove to be fatal. AML begins to develop in cells that traditionally develop into various types of blood cells. Most AML cases develop from cells that would transform into white blood cells (other than lymphocytes), however, a number AML cases develop in other kinds of blood-forming cells. AML starts to develop in the bone marrow, albeit, in the majority of cases it rapidly moves into the blood. At times, it may spread to other body parts, "including the lymph nodes, liver, spleen, central nervous system (brain and spinal cord), and testes" (Detailed Guide: Leukemia). Some other cancer types that may start in these organs and/or elsewhere, and then spread to the bone marrow are not leukemia.

Information presented in this literature review chapter, the researcher notes, "names" or reports a number of relevant points regarding AML. The three clinical studies presented in this study's introduction relate particularly relevant data regarding treatments of/for AML. The following three studies the researcher examines during this thesis include:

1. Study I: "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias" (Shiozawa, et al.).

2. Study II: "Nuclear Factor- B Modulation in Patients Undergoing Induction Chemotherapy for Acute Myelogenous Leukemia" (Strair, et al.).

3. Study III: "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias" (Shiozaw, et al.).

Treatment of AML

As researchers continue to study the suspected causes, diagnosis, and treatment of AML at various medical centers, university hospitals, and other institutions, they have begun to make progress in understanding ways changes in an individual's DNA may contribute to normal bone marrow cells developing into leukemia. With an increased understanding of the genes involved in particular translocations or other chromosomal changes frequently occurring in AML, more insight into why these cells become abnormal is gained, along with the potential for the development of newer targeted therapies against AML.

The following list depicts a number of these newer target therapies, however, the newer therapies are not limited to these:

Gene Expression Profiling: This lab technique to help identify and classify different cancers utilizes microarrays to simultaneously examines the patterns of various genes in the cancer cells, rather than examining single genes.

Detection of Minimal Residual Disease: "The polymerase chain reaction (PCR) test can identify AML cells based on their gene translocations or rearrangements. This test can find one leukemia cell among a million normal cells. A PCR test can be useful in determining how completely the treatment has destroyed the AML cells" (Detailed Guide: Leukemia).

Improving Chemotherapy

Stem Cell Transplants

Targeted Therapies

New targeted drugs that specifically attack some of the genetic changes seen in AML are now being developed.

About 1 person out of 3 with AML has a mutation in the FLT3 gene. Several new drugs, called FLT3 inhibitors, target this gene. They have shown activity against AML in early studies, especially when combined with chemotherapy. So far, they are only available in clinical trials. Other gene mutations, such as changes in the c-KIT gene, also appear to be important in some cases of AML, and may become important targets for new therapies. Drugs that target this gene, such as imatinib (Gleevec) and dasatinib (Sprycel) are already used against other types of leukemia, and are now being studied against AML.

Gemtuzumab ozogamicin (Mylotarg) is a monoclonal antibody that is often used in older patients if AML doesn't respond to chemotherapy or comes back after treatment. Doctors are now studying whether this drug might be useful if given with chemotherapy earlier in the course of the disease (Detailed Guide: Leukemia).

Study I

During Study I, "Epigenetic Modification of CCAAT/Enhancer Binding Protein Expression in Acute Myeloid Leukemia," Hackanson, et al., as noted earlier, confirm C/EBP mRNA as a target for miRNA-124a. Bjorn, et al. signify that C/EBP epigenetic alterations denote a frequent event in AML and epigenetic treatment may result in down-regulation of a key hematopoietic transcription facto. Hackanson, et al. point out that the "functional loss of CCAAT/enhancer binding protein (C/EBP), a master regulatory transcription factor in the hematopoietic system, can result in a differentiation...

Here, we show the effect of epigenetic aberrations in regulating C/EBP expression in acute myeloid leukemia (AML)" (Abstract section, para. 1). Results from the study by Hackanson, et al. suggest that epigenetic alterations of C/EBP frequently occur in AML. In addition, epigenetic treatment result in down-regulation of a key hematopoietic transcription factor (Ibid.1).
Acute myeloid leukemia (AML) has been extensively studied at the cytogenetic, molecular, and transcriptional level. This knowledge has contributed to the subclassification of AML and translated into significant improvement of therapies (1 -- 3). Leukemic transformation to AML is a multistep process requiring the alterations of genes involved in proliferation/survival and hematopoietic differentiation (4). One such gene, CCAAT/enhancer binding protein (C/EBP), is a key transcription factor involved in the regulation of cell proliferation and differentiation in a variety of cell types, particularly in the hematopoietic system (5, 6). Whereas under physiologic conditions C/EBP is a master regulator for myeloid differentiation and granulocytic maturation, its absence results in a block of granulopoiesis, as shown in several studies (6, 7). C/EBP has gained interest in the AML field, because it has been shown that down-regulation of C/EBP protein through mutations, posttranslational modifications, and protein-protein interactions with fusion proteins AML1/ETO or CBFB-SMMHC plays a key role in leukemic transformation (6, 8 -- 11) (Hackanson, et al. Introduction section, para. 1).

Besides genetic aberrations, epigenetic modifications, such as DNA methylation and histone-tail modifications, have been shown to initiate or augment malignant transformation (12 -- 14). Global promoter studies, as well as gene-specific approaches, have revealed that aberrant promoter methylation is a common event in AML (13, 15). Because of the pharmacologic reversibility of epigenetic changes by drugs, such as the DNA-demethylating agent 5-aza-2'deoxycytidine (DAC) or the histone deacetylase (HDAC) inhibitor valproic acid, epigenetic therapy seems prominently among novel leukemia treatment strategies (16 -- 19). Whereas it is commonly seen that epigenetic treatment leads primarily to up-regulation of genes, several groups have recently shown that DNA demethylation and HDAC inhibition can also result in down-regulation of gene expression (20, 21). The molecular mechanisms underlying these findings are largely unknown, but potential mechanisms include alterations in gene expression profiles as a result of drug treatment triggering additional changes in gene expression that are independent of promoter demethylation (20) (Hackanson, et al. Introduction section, para. 2).

A third epigenetic mechanism has recently gained attention: gene expression regulation through microRNAs (miRNA). These short noncoding RNAs have been shown to down-regulate gene expression by targeting the 3' untranslated region (UTR) of their target genes. Depending on whether the specific miRNA is entirely or partially complementary to its 3' UTR binding site, down-regulation is accomplished by either mRNA degradation or translational repression, respectively (22). Besides their role in cell proliferation, differentiation, and apoptosis, recent studies have provided evidence that miRNAs are also involved in leukemogenesis (23, 24) (Hackanson, et al. Introduction section, para. 3).

Until recently, little has been known about the regulation of miRNAs, but seminal studies have now shown that hematopoietic transcription factors C/EBP and PU.1 are capable of steering miRNA-223 (miR-223) expression, which is a crucial factor in granulocytic differentiation (25, 26). Moreover, it is becoming evident that miRNAs are not only effectors of the epigenetic machinery, but they themselves can be regulated by DNA methylation (27 -- 29) (Hackanson, et al. Introduction section, para..4).

In a recent study, DNA methylation of the C/EBP core promoter was found in a small subset of AML patients and biologically linked to T-cell lineage infidelity (30). Furthermore, we have shown in two studies in lung cancer and head and neck squamous cell carcinoma that C/EBP expression is down-regulated by epigenetic mechanisms, including DNA methylation of the C/EBP upstream promoter region (31, 32). Encouraged by these results, we sought to comprehensively investigate the role of epigenetic regulation of C/EBP in AML (Hackanson, et al. Introduction section, para. 5).

Patient samples and cell lines. One hundred forty-six bone marrow samples from AML patients were obtained from the Cancer and Leukemia Group B. tissue bank and the University of Freiburg tissue bank. Bone marrow samples from seven healthy donors were collected after obtaining informed consent under a protocol approved by Ohio State University Institutional Review Board. Cell lines U937, THPI, HL60, K562, and Kasumi1 were obtained from the American Type Culture Collection, and NB4 from the German Collection of Cell Cultures. U937, THPI, and NB4 were cultured in RPMI…