Dynamic Effect of Bortezomib on Nuclear Factor-B Activity and Gene Expression in Tumor Cells

ABSTRACT Nuclear factor-B (NF-B) influences the initiation, progression, and maintenance of diverse cancer types. Despite current therapeutic efforts to block hyperactive NF-B in cancer cells, the in vivo effects of a drug upon this complex pathway are unclear. We monitored NF-B activity and a fast-expressing reporter level simultaneously in head and neck squamous carcinoma cells by quantitative live microscopy. The real-time single cell assay revealed the tumor necrosis factor-␣-induced oscillation of NF-B was echoed by equally dynamic reporter expression rate. Bortezomib is a proteasome inhibitor whose anticancer action is partly mediated through inhibition of NF-B. When administered to preactivated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway "blockade," the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways. Recent trends in clinical investigations clearly tend toward molecularly targeted approaches that are based on mechanisms underlying the pathophysiology of the disease. Often, the goal is to block the activity of a specific pathway that has been implicated in the disease process, by targeting a key component with a small molecule or an antibody, for example. It is seldom known whether the desired "blockade" is achieved in the relevant tissue and why paradoxical outcomes occur in certain cases. Here, we show that NF-B activity in tumor cells is altered by the action of a proteasome inhibitor in a complex way that cannot sufficiently be explained by the simplistic notion of pathway blockade. NF-B/Rel is a master regulator of inflammatory processes and has a growing list of cancers and other common diseases that require its aberrant activit

Dynamic Effect of Bortezomib on NF-κB Activity and Gene Expression in Tumor Cells Running Title: Inhibition dynamics of Bortezomib on NF-κB in living cells

Abstract 523 words in Introduction 710 words in Discussion Abbreviations: NF-κB, nuclear factor kappa B; TNF-α, tumor necrosis factor alpha; IκB, inhibitor of kappa B; IKK, IκB kinase; HNSCC, head and neck squamous cell carcinoma; EGFP, enhanced green fluorescent protein. MOL #49114 3 Abstract NF-κB influences the initiation, progression, and maintenance of diverse cancer types. Despite current therapeutic efforts to block hyperactive NF-κB in cancer cells, the in vivo effects of a drug upon this complex pathway are unclear. We monitored NF-κB activity and a fast-expressing reporter level simultaneously in head and neck squamous carcinoma cells by quantitative live microscopy. The real time single cell assay revealed the TNF-α induced oscillation of NF-κB was echoed by equally dynamic reporter expression rate. Bortezomib is a proteasome inhibitor whose anti-cancer action is partly mediated through inhibition of NF-κB. When administered to pre-activated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway 'blockade', the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways. MOL #49114

Phase 1 and pharmacokinetic study of bolus-infusion flavopiridol followed by cytosine arabinoside and mitoxantrone for acute leukemias

Flavopiridol is a protein bound, cytotoxic, cyclin-dependent kinase inhibitor. Flavopiridol given by 1-hour bolus at 50 mg/m2 daily 3 times followed by cytosine arabinoside and mitoxantrone (FLAM) is active in adults with poor-risk acute leukemias. A pharmacologically derived “hybrid” schedule (30-minute bolus followed by 4-hour infusion) of flavopiridol was more effective than bolus administration in refractory chronic lymphocytic leukemia. Our phase 1 trial “hybrid FLAM” in 55 adults with relapsed/refractory acute leukemias began at a total flavopiridol dose of 50 mg/m2 per day 3 times (20-mg/m2 bolus, 30-mg/m2 infusion). Dose-limiting toxicity occurred at level 6 (30-mg/m2 bolus, 70-mg/m2 infusion) with tumor lysis, hyperbilirubinemia, and mucositis. Death occurred in 5 patients (9%). Complete remission occurred in 22 (40%) across all doses. Overall and disease-free survivals for complete remission patients are more than 60% at more than 2 years. Pharmacokinetics demonstrated a dose-response for total and unbound plasma flavopiridol unrelated to total protein, albumin, peripheral blast count, or toxicity. Pharmacodynamically, flavopiridol inhibited mRNAs of multiple cell cycle regulators, but with uniform increases in bcl-2. “Hybrid FLAM” is active in relapsed/refractory acute leukemias, with a recommended “hybrid” dose of bolus 30 mg/m2 followed by infusion of 60 mg/m2 daily for 3 days. This clinical trial is registered at www.clinicaltrials.gov as #NCT00470197