Molecular characterization of PS-341 (bortezomib) resistance:: implications for overcoming resistance using lysophosphatidic acid acyltransferase (LPAAT)-β inhibitors

PS-341 (bortezomib, Velcade(TM)) is a promising novel agent for treatment of advanced multiple myeloma ( MM); however, 65% of patients with relapsed refractory disease in a phase II study do not respond to PS-341. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-beta inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (i.e., dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we therefore determined whether CT-32615 could also overcome resistance to PS- 341. We first characterized molecular mechanisms of resistance to PS- 341 in DHL-4 cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase ( PARP), or DNA fragmentation factor 45 was triggered by PS- 341 treatment. We have previously shown that PS- 341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS- 341-induced apoptosis. We here show that phosphorylation of SEK-1, JNK, and c-Jun are not induced by PS-341 treatment, suggesting that PS- 341 does not trigger a stress response in DHL-4 cells. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time-and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggered swelling and lysis of DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-beta inhibitor CT-32615 triggers necrosis, even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS- 341 resistance and improve patient outcome.