Genetic abnormalities and challenges in the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematopoietic disorder in which there are too many immature blood-forming cells accumulating in the bone marrow and interfering with the production of normal blood cells. It has long been recognized that AML is a clinically heterogeneous disease characterized by a multitude of chromosomal abnormalities and gene mutations, which translate to marked differences in responses and survival following chemotherapy. The cytogenetic and molecular genetic aberrations associated with AML are not mutually exclusive and often coexist in the leukemic cells. AML is a disease of the elderly, with a mean age of diagnosis of 70 years. Adverse cytogenetic abnormalities increase with age, and within each cytogenetic group, prognosis with standard treatment worsens with age. In the past 20 years, there has been little improvement in chemotherapeutic regimens and hence the overall survival for patients with AML. A huge unmet need exists for efficacious targeted therapies for elderly patients that are less toxic than available chemotherapy regimens. The multitude of chromosomal and genetic abnormalities makes the treatment of AML a challenging prospect. A detailed understanding of the molecular changes associated with the chromosomal and genetic abnormalities in AML is likely to provide a rationale for therapy design and biomarker development. This review summarizes the variety of cytogenetic and genetic changes observed in AML and gives an overview of the clinical status of new drugs in development.

Activation of AKT kinases in cancer: implications for therapeutic targeting.

The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention.

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP)-based couple d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z' of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC trade mark and validated with the protein kinase C inhibitor staurosporine. The IC(50) value generated was comparable to the value obtained by the radioactive (33)P-gamma-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

Adenoviral-mediated expression of a kinase-dead mutant of Akt induces apoptosis selectively in tumor cells and suppresses tumor growth in mice.

Akt/protein kinase B is a serine/threonine kinase that plays a critical role in cell survival signaling, and its activation has been linked to tumorigenesis in several human cancers. Up-regulation of Akt, as well as its upstream regulator phosphatidylinositol 3-kinase, has been found in many tumors, and the negative regulator of this pathway, mutated in multiple advanced cancers suppressor (MMAC; also known as phosphatase and tensin homologue deleted on chromosome 10), is a tumor suppressor gene. We have investigated the effects of inhibiting Akt signaling in tumor cells by expression of an Akt kinase-dead mutant in which the two regulatory phosphorylation sites have been mutated to alanines. This mutant, which functions in a dominant negative manner (Akt-DN), was introduced into tumor cells using a replication-defective adenovirus expression system. As controls we used adenoviruses expressing p53, MMAC, beta-galactosidase, and empty virus. We show that in vitro proliferation of human and mouse tumor cells expressing high levels of activated/phosphorylated Akt was inhibited by both Akt-DN and p53, in comparison with control viruses expressing beta-galactosidase. Similarly, Akt-DN mutant expression led to selective induction of apoptosis in tumor cells expressing activated Akt. On the other hand, Akt-DN expression had minimal effect in normal and tumor cells expressing low levels of activated Akt. Expression of MMAC induced selective apoptosis in tumor cell lines in which MMAC is inactivated but not in tumor cells expressing wild-type levels of MMAC. In addition, the growth of tumor cells in a mouse model was also significantly inhibited by intratumoral injection of Akt-DN virus. These studies validate the usefulness of targeting Akt for new drug discovery efforts and suggest that inhibition of Akt may have a selective antitumor effect.

Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis.

The integrin receptor alphavbeta3 has been shown to play a critical role in several distinct processes, such as angiogenesis, osteoclast-mediated bone resorption and tumor metastasis. Its expression is upregulated in newly synthesized blood vessels produced in response to a variety of tumors and purified angiogenic factors. Studies show that alphavbeta3 is a critical target downstream from perhaps all angiogcnic factors. Proof-of-principle that alphavbeta3 antagonists such as monoclonal antibodies and small molecules block angiogenesis and tumor growth has been obtained in several animal models. Many endogenous inhibitors of angiogenesis such as angiostatin, endostatin and tumstatin seem to work through the alphavbeta3 receptor further emphasizing the critical role of this receptor in angiogenesis. In addition, the alphavbeta3 receptor has been clearly implicated in several pathological processes such as rheumatoid arthritis, osteoporosis, and metastasis of prostate cancer to bone. Thus alphavbeta3 may prove to be an important target for pharmacological intervention in more than one clinical setting.