bortezomib & radiation in head/neck
Abstract No: 5592
Author(s): B. A. Conley, E. Donovan, C. Muir, J. Morris, A. D. Colevas, D. Gius, E. Trehu, E. Sausville, J. Wright, C. Vanwaes
Abstract:
Background: We & others have previously shown that bortezomib inhibits activation of NF?B & other survival effectors & has cytotoxic, radiosensitizing, & anti-tumor activity in preclinical HNSCC models. A previous NCI phase 1 study evaluating dose escalation of bortezomib with rRT established a maximum tolerable dose (MTD) of 0.6 mg/m2 twice weekly IV with daily rRT for 6.6 weeks (60-70 Gy) based on dose limiting toxicity (DLT) of grade 3 hyponatremia & hypotension in 2 pts. The current study was designed to further explore the MTD & best tolerated schedule of bortezomib with rRT by introducing a treatment break.
Methods: Pts with recurrent or metastatic HNSCC in whom rRT was feasible, who were > 6 months since prior RT, had PS <2, AST/ALT <2.5x normal, bilirubin <1.5x normal, adequate hematopoetic & renal function were enrolled. Bortezomib was administered twice weekly as an IV bolus at the 0.6 mg/m2 dose level with rRT 1.8 Gy/day to 60-72 Gy. A 2-week break in both drug & rRT was given halfway through planned rRT.
Results: 5 of 6 pts enrolled are currently evaluable for toxicity (CTC v 2.0) & response. Median ECOG PS was 1 (0-1). DLT was seen in 1 pt (hyponatremia & orthostatic hypotension), who died unexpectedly less than a week after treatment completion. Other grade 3 toxicities (non-DLT) included mucositis, vomiting, hyperglycemia & pain. Four pts completed treatment at the 0.6 mg/m2 dose without DLT & are alive 3-9 months after treatment; one pt remains on treatment.
Conclusions: The combination of bortezomib & rRT is feasible with this novel schedule employing a treatment interruption. Bortezomib MTD with rRT is <0.6 mg/m2. A future study will explore continuous rRT with 2-week midcourse bortezomib treatment break.
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SUMOylation's Role in Apoptosis
Abstract No: 9650
Author(s): V. Chung, B. Zhou, X. Liu, L. Zhu, H. Nguyen, D. K. Ann, Y. Yen
Abstract:
Background: The ubiquitin-proteosome pathway plays a critical role in cellular homeostasis. Some studies have shown a synergistic effect between gemcitabine and bortezomib; however, the molecular mechanisms are not fully understood.
Methods: Human oropharyngeal carcinoma KB wild-type (KBwt) and gemcitabine resistant (KBGem) cells were treated with gemcitabine and bortezomib in a time and sequence manner.
Results: Gemcitabine followed by bortezomib 4-8 hours later synergistically induced apoptosis in both cell lines and increased the percentage of subG1 cells seen by flow cytometry. Utilizing a luciferase reporter vector, NF-?B activity was found to be five fold greater in KBGem cells and drug treatment decreased NF-?B activity by 44% in KBwt cells and 28% in KBGem cells. By western blot analysis, treating KBwt cells with gemcitabine and bortezomib resulted in cleavage of NF-?B which was not seen in KBGem cells. Cyclin A and B1 expression decreased in KBwt cells following drug treatment; whereas, only cyclin B1 expression decreased in KBGem cells. Since the KBGem cells had higher expression of small ubiquitin modified protein (SUMO), its role was further explored by transducing KBwt and KBGem cells with HIV-based lenti-SUMO-1 and the unconjugatable lenti-SUMO-1aa, respectively, followed by drug treatment. The expression of cyclins A, D1 and E was markedly reduced in KBGem SUMO-1 cells and restored in SUMO-1aa transduced cells.
Conclusions: Drug resistance has been a hurdle in the treatment of malignancies and the inducible activation of NF-?B is one pathway that has been implicated. Bortezomib is a novel agent that works on many pathways. For KBwt cells, the NF-?B pathway plays a significant role in inducing apoptosis after drug treatment, whereas in KBGem cells, the role of SUMOylation appears more significant. Sequential drug treatment overcoming gemcitabine resistance through SUMOylation is a unique mechanism. Thus, we have currently developed a phase I clinical trial of fixed dose rate gemcitabine followed by bortezomib (supported by NCI 72767).
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Proteasome inhibition as therapy
Abstract No: 3122
Author(s): D. Chauhan, L. Catley, G. Li, T. Hideshima, P. Richardson, M. Palladino, K. C. Anderson
Abstract:
Multiple Myeloma (MM) remains fatal despite all available therapies, and novel approaches which target mechanisms regulating MM cell growth, survival, and apoptosis are urgently needed. Proteasome is a promising target in the treatment of MM. The proteasome inhibitor Bortezomib/PS-341/Velcade has shown potent preclinical activity in vitro as well as therapeutic activity in MM and is the first treatment in more than a decade to be FDA approved for patients with MM. However, prolonged exposure is associated with attendant toxicity and development of bortezomib-resistance. Here we demonstrate that novel proteasome inhibitor NPI-0052, distinct in its chemical structure from bortezomib, induces apoptosis even in MM cells resistant to bortezomib, dexamethasone (Dex), melphalan and thalidomide without significantly affecting the viability of normal cells. NPI-0052 overcomes growth/survival and drug-resistance in MM cells conferred by bone marrow (BM) microenvironment, and/or altered expression of anti-apoptotic proteins Bcl2, Hsp27, and IAPs. Mechanistic studies demonstrate that NPI-0052-triggered MM apoptosis is associated with loss of mitochondrial membrane potential; Superoxide generation; release of mitochondrial proteins cytochrome-c/Smac; and activation of caspase-8/9/3. Alternatively, pretreatment of MM cells with the pan-caspase inhibitor Z-VAD-fmk blocks NPI-0052-induced cell death. In vivo studies in animal models show that oral administration of NPI-0052 decreased proteasome activity in packed whole blood lysates in a dose dependent fashion. Moreover, studies using a human plasmacytoma xenograft mouse model show that NPI-0052 is orally active and has significant anti-MM activity at doses that are not only well tolerated, but also inhibit MM cell growth and prolong survival. Collectively, these data provide the framework for clinical evaluation of NPI-0052 to trigger apoptosis in MM cells, reduce bortezomib-associated toxicity, overcome bortezomib-resistance, and improve patient outcome in MM.
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Bortezomib and pancreatic cancer
Abstract No: 3161
Author(s): B. Yeung, E. N. Freeburg, D. T. Huang, F. A. Sinicrope
Abstract:
Background: Bortezomib (Velcade) is a potent and reversible proteosome inhibitor that has shown in vitro and in vivo activity against tumor types including pancreatic cancer. Inhibition of the 26S proteosome induces apoptosis that is blocked by Bcl-2, an inhibitor of oxidative stress and mitochondrial injury. We determined the effect of a small molecule Bcl-2 inhibitor (HA14-1) plus bortezomib on mitochondrial membrane potential [delta psi(m], generation of reactive oxygen species (ROS), and apoptosis induction.
Materials & Methods: BxPC-3 cells, grown in RPMI 1640 medium with 10% FBS, were incubated with bortezomib (25, 50, 100 nM), HA14-1 (15 µM) or their combination for 48 h. Sequential treatment (second drug added after 24 h) was also performed. For analysis of delta psi(m), cells (5 x 106) were incubated in dihexyloxacarbocyanine iodide [40 nM] or for ROS, in dihydroethidium (2 µM) at 37°C for 15 min. Floating and attached cells were harvested and FACS analysis was performed. Apoptosis was measured by Annexin V-FITC labeling. Bcl-2 and Bcl-xL expression were determined by immunoblotting.
Results: BxPC-3 cells overexpressed Bcl-2 and Bcl-xL proteins. Incubation of these cells with bortezomib (48 hr) markedly changed mitochondrial membrane potential (delta psi(m) and generated ROS, whereas HA14-1 produced minimal changes compared to untreated cells. Co-administration or sequential treatment with bortezomib followed by HA14-1 changed delta psi(m) and generated ROS to a greater extent than did bortezomib alone or the reverse sequence. By Annexin V FITC labeling, bortezomib triggered a dose-dependent increase in apoptosis and a 3-fold increase over control cells at 100 nM. HA14-1 did not induce apoptosis over control cells. Bortezomib followed by HA14-1 induced apoptosis to a greater extent than did bortezomib alone, their co-administration, or the reverse sequence.
Conclusion: Bortezomib induces oxidative stress and mitochondrial injury that are coupled with induction of apoptosis. These events were synergistically enhanced by co-administration or subsequent treatment with a Bcl-2 inhibitor, suggesting a novel therapeutic strategy against pancreatic cancer. |
