The genotoxic effect of conventional anticancer therapy involving many chemotherapy agents and gamma irradiation results in the induction of apoptosis in cancer cells. The ability to inhibit apoptosis appears to be a principal mechanism by which resistant cancer cells are protected from chemotherapy and radiation. Cellular mechanisms that protect cancer cells against apoptosis include lack of a functional response mechanism to apoptotic stimuli (e.g. mutated or deleted p53 tumor suppressor gene), presence of an inhibitor to apoptosis (e.g. Bcl-2, IAP) and the expression of the multidrug resistance gene (MDR). Recently, we found that the inducible activation of the nuclear transcription factor NF-kB inhibits the apoptotic response to chemotherapy and irradiation. NF-kB, a key transcription factor involved with immune and inflammatory responses as well as cell growth, is regulated primarily through interactions with an inhibitor protein known as IkB. Stimuli which activate NF-kB typically induce the recently identified IkB kinase (IKK) to phosphorylate IkB on N-terminal serines which leads to ubiquitination and subsequent degradation of the inhibitor by the proteasome. Following IkB degradation, NF-kB translocates to the nucleus where it regulates genes involved in a variety of processes, including the suppression of apoptosis.

We recently described a gene therapy approach that uses a recombinant adenovirus to transfer a modified form of IkBa. In these experiments, transfer of super-repressor IkBa resulted in significant augmentation of chemosensitivity and enhanced induction of apoptosis in a xenograft tumor model in response to chemotherapy treatment. These findings suggested that NF-kB may represent an important molecular target for the purpose of enhancing the sensitivity of certain cancer cells to apoptotic stimuli. The use of gene therapy to deliver NF-kB inhibitors is relevant to certain cancers but is limited when considering widely-disseminated metastases. An alternative strategy for the inhibition of NF-kB activation is facilitated by inhibition of proteasome function. The inhibition of proteolytic function effectively blocks degradation of cellular proteins that have undergone ubiquitination, such as IkB. In fact, proteasome inhibition is a well-established mechanism to block NF-kB in response to a variety of stimuli. A clear advantage to this therapeutic approach is the clinical availability of a systemically administered small molecule (PS-341, a potent, boronic acid dipeptide that is highly-selective for proteasome inhibition) that can potentially inhibit chemotherapy induced activation of NF-kB and enhance the apoptotic response to chemotherapy. To evaluate this therapeutic approach, we determined whether inactivation of proteasome function would inhibit inducible NF-kB activation, and result in increased levels of apoptosis in response to chemotherapy, and enhanced antitumor effects in vivo. To test our hypothesis we utilized PS-341 to evaluate the effects of proteasome inhibition on 1) chemotherapy-induced NF-kB activation in colorectal cancer cells, 2) the levels of apoptosis following treatment with chemotherapy; and 3) tumor growth in a xenograft model treated with chemotherapy. Phase I clinical trials in which we explore the ability of this approach to augment chemosensitivity in patients with refractory malignancy are nearing completion. Phase II studies will be initiated in the Fall of 2002. Correlative studies will include pharmacokinetic and pharmacogenomic analysis of patient samples. Future laboratory studies will explore the role of newly developed small molecular compounds to selectively inhibit chemotherapy-induced NF-kB activation by targeting the downstream mediators of this response mechanism.

Role of NF-*B in Inhibition of Chemotherapy-Induced Apoptosis
Principal Investigator: James C. Cusack, Jr, MD
Group Members: Rong Liu, MD; John Flannery, MD; Lijun Xia, MD