Hong-Gang Wang - US grants

DESCRIPTION: (adapted verbatim from the investigator's abstract) Overexpression of Bcl-2 contributes not only to the origins of cancer but also to difficulties in treating it, because Bcl-2 can block or markedly impair the induction of apoptosis by essentially all chemotherapeutic drugs and radiation. Tumor cells can also become resistant to therapy by reducing their expression of pro-apoptosis proteins such as Bax, as opposed to increasing their levels of Bcl-2. The precise biochemical mechanisms by which Bcl-2 family proteins exert their influence on cell life and death however remain far from clear. The investigator and other researchers have shown that the kinases Raf- and Akt can phosphorylate BAD, a pro-apoptotic member of Bcl-2 family, and abrogate its apoptosis-inducing effects in cells. In addition, their preliminary investigations demonstrated that the Ca2+-activated protein phosphatase calcineurin induces dephosphorylation of BAD, promoting its association with anti-apoptotic Bcl-2 family proteins and nullifying their cell survival activity. These findings suggest that the activity of BAD can be modulated by specific signal transduction molecules and suggest a need of better understanding the mechanisms that regulate the post-translational modifications of this cell death promoter. The goals of this proposal are to define the mechanisms that account for the post-translational modifications of BAD and to explore the physiological significance of this potentially important signaling pathway for controlling cell death in human cancer cells. The role of calcineurin-mediated dephosphorylation of BAD with regards to tumor cell responses to chemotherapeutic drugs and radiation will also be determined. The results obtained might provide insights as to how to render human cancer cells more sensitive to chemotherapeutic drugs and radiotherapy.

DESCRIPTION (PROVIDED BY APPLICANT):MECHANISMS OF RAD9-MEDIATED CHECKPOINTS AND APOPTOSIS: Cell-cycle checkpoints play a critical role in the maintenance of genomic integrity by inhibiting progression through the cell cycle or initiating programmed cell death in the presence of damaged DNA or incomplete DNA replication. Studies in fission yeast implicate that members of the Rad family of checkpoint proteins including Radi, Rad3, Rad9, Radl7, Rad26, and Hus1 play important roles in the activation of DNA damage and replication checkpoints. We have reported that Rad9 can interact with Bcl-2 and Bcl-xL through a BH3-like region located in the amino terminus of the Rad9 protein, and can promote apoptosis in mammalian cells. DNA damage enhances Rad9 phosphorylation and induces Rad9 to move to the nuclear envelope, where it colocalizes with Bcl-2. In addition, our preliminary data indicate that c-Abl-mediated phosphorylation of Rad9 on Y28 induces increased association of Rad9 with BcI-xL and enhances the effect of Rad9 on apoptosis induction. Interestingly, the Rad9 protein is hyperphosphorylated and some form appears to be cell cycle-dependent. Moreover, we have recently found that Hus1 forms a protein complex with PCNA in human skin Flow2000 fibroblasts when DNA is damaged or replication is inhibited. Exposure of Flow2000 cells to 8 Gy ionizing radiation (that induces G2/M-arrest but not apoptosis) or hydroxyurea triggered translocation of Husi from the cytosol to the nucleus, where it colocalized with PCNA and Rad9. This nuclear translocation and the complex formation of Husl with PCNA correlate closely with changes in cell cycle distribution in response to radiation exposure. The goal of this proposal is to test the hypothesis that Rad9 is an important modulator of the DNA integrity checkpoint pathway determining whether a cell should transiently delay cell-cycle progression or die after DNA damage and that damage induced complex formation with a discrete set of cellular proteins plays a critical role in Rad9 function.

The Program's broad long-term objective is to develop new targeted therapeutics for acute myelogenous leukemia (AML). The overarching hypothesis of the Program Project is that sphingolipid metabolism is altered in AML and can be used to direct therapeutic regimens. A corollary of this hypothesis suggests that novel therapeutics that target dysfunctional sphingolipid metabolism may be highly efficacious in AML. The integration of the Program follows the metabolism of ceramide. Project 1 targets ceramide metabolism utilizing ceramide-based nanotherapeutics; Projects 2 and 3 target acid ceramidase and sphingosine kinase, which coordinately generate the pro-mitogenic and anti-apoptotic ceramide metabolite, sphingosine- 1-phosphate, and Project 4 targets P-glycoprotein-mediated glycosylation of ceramide. All Projects have validated therapeutic modalities in both in vitro and in vivo models of AML. The Program is supported by five integral Cores. These include the: Synthesis and Nanoformulation Core, which provides synthesized compounds not available commercially for biologic studies; Targeted Sphingomics Core, which is essential for quantification of sphingolipid metabolism; Animal Modeling and Clinical Resources Core, which provides state-of-the-art molecularly defined AML samples with annotated clinical outcomes and murine leukemia stem cells models; Biostatistics Core, which provides critical research design and analysis; Administrative Core, which provides oversight and coordination of all scientific, administrative, and fiscal activities. Development of targeted therapeutics for AML will be pursued in the following overall Specific Aims of the Program: 1. Engineer, characterize and optimize novel lipomimetic- or small molecule-based therapeutics for AML. 2. Validate the efficacy and toxicology of sphingolipid-targeted therapeutics in vivo using murine leukemia stem cells models. 3. Define the role of altered sphingolipid metabolism in cell survival, apoptosis, autophagy, and drug resistance in AML. To accomplish these Aims, we have assembled a transdisciplinary team of clinical and basic scientists, organic chemists, and material scientists. We are fortunate that NCI NanoCharacterization Laboratory has accelerated pre-clinical development of the Penn State ceramide liposomal nanoplatform. The clinical significance of the Program rests on the urgent and unmet needs for development of new therapeutics in AML. In the revised application, we have specifically responded to all of the reviewer's critiques, in particular, addressing the major issues associated with AML heterogeneity and humanized AML murine models. Importantly, we have documented engraftment in NSG mice of AML subsets defined by integrated genetic profiling.