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

Abstract

PS-341 (bortezomib, Velcade™) 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)-β 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-β 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.

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Abbreviations

MM:

multiple myeloma

LPA:

lysophosphatidic acid

LPAAT:

LPA acyltransferase

PARP:

poly (ADP)-ribose polymerase

Dex:

dexamethasone

Dox:

doxorubicin

BM:

bone marrow

BMSC:

BM stromal cell

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyl tetrazolium bromide

PI:

propidium iodine

Hsp:

heat–shock protein

JNK:

c-Jun NH2-terminal kinase

DFF:

DNA fragmentation factor

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Acknowledgements

This study is supported by National Institutes of Health SPORE IP50 CA10070-01, PO-1 78378, and RO-1 CA 50947 Grants; the Doris Duke Distinguished Clinical Research Scientist Award (KCA); the Multiple Myeloma Research Foundation (TH); and the Cure for Myeloma Research Fund (KCA).

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Correspondence to Kenneth C Anderson.

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Hideshima, T., Chauhan, D., Ishitsuka, K. et al. Molecular characterization of PS-341 (bortezomib) resistance: implications for overcoming resistance using lysophosphatidic acid acyltransferase (LPAAT)-β inhibitors. Oncogene 24, 3121–3129 (2005). https://doi.org/10.1038/sj.onc.1208522

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