Understanding the molecular mechanisms by which myeloma cells evade signals leading to chemotherapy-induced apoptosis is the long term goal of this project. Glucocorticoids such as dexamethasone (Dex], all potent inducers of apoptosis are frequently used to treat multiple myeloma [MM], a clonal B cell-malignancy. Using standard doses of these agents, complete remissions are rare [5% of patients]. Drug resistance increases with prolonged treatment, suggesting primary resistance of tumor cells as a major obstacle to cure. This resistance maybe due to MM cell expression of moderate to high levels of the anti-apoptotic protein bcl-2. There is compelling evidence that bcl-2 imparts its anti-apoptotic signal by ducking and modulating the expression of genes critical for apoptosis, including transcription factors such as NF-kappaB. We hypothesize that chemoresistance in MM by bcl-2 requires activation of NF-kappaB. To investigate this hypothesis we will address the following specific aims: 1. Test whether persistent NF-kappaB activation observed in MM cells confers resistance in chemotherapy-induced apoptosis in MM cell lines. P50:p65 NF-kappaB subunits will be over-expressed in drug-sensitive MM cell lines to inactivated NF-kB. Antisense phosphorothiorate oligonucleotides for p50 and p65 will also be used to inhibit NF-kappaB. 2. Determine whether bcl-2 contributes to maintaining NF-kappaB activity through modulation of drug-mediated post-translational regulation of IkappaB proteins and/or p65 transactivation. Different modes of IkBalpha, Ikbeta and Ikepsilon protein regulation will be investigated: 1] basal and signal-induced IkappaB phosphorylation and degradation 2) IKK-alpha and -beta functions and 3) inhibitory properties of IkappaB proteins. For p65 transactivation: 1] a Gal4-BASED hybrid system will be used to test whether clinically relevant drugs and enforced bcl-2 interfere with the transactivation potential of p65 and 2] a bcl-2 deletion mutant that lacks the transmembrane domain will be expressed in MM cell lines to determine whether membrane-anchored bcl-2 augments p65 transactivation. 3. Investigate whether the inhibitory effects of Dex on NF-kappaB activity predict response to chemotherapy. Longitudinal studies will be conducted to determine whether patient responsiveness to glucocorticoid based therapy can predicted from their in vitro response to Dex in terms of NF-kappaB DNA binding, p65 immunostaining. There studies will hopefully provide deeper insight into the molecular basis of drug resistance in MM and should result in the identification of potential targets for therapeutic intervention.
