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. 2011 Jan 27;117(4):1311-4.
doi: 10.1182/blood-2010-08-303099. Epub 2010 Nov 16.

Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma

Ioanna Papandreou  1 , Nicholas C Denko, Michael Olson, Heleen Van Melckebeke, Sofie Lust, Arvin Tam, David E Solow-Cordero, Donna M Bouley, Fritz Offner, Maho Niwa, Albert C Koong
Affiliations

Identification of an Ire1alpha endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma

Ioanna Papandreou et al. Blood. .

Abstract

Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimyeloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM.

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Figures

Figure 1
Figure 1
Identification of an Ire1α endonuclease inhibitor. (A) Chemical structure of inhibitor STF-083010. (B) STF-083010 inhibited endogenous XBP1 mRNA splicing. RPMI 8226 cells were treated with 300nM thapsigargin (Th), 60μM STF-083010, or both for the indicated amount of time, and the relative XBP1 splicing was determined by reverse-transcribed polymerase chain reaction. Solid arrow indicates the unspliced form; and broken arrow, the spliced form. (C) STF-083010 inhibits the production of sXBP1 protein but not the autophosphorylation of Ire1α. The indicated MM cell lines were treated for the indicated times with 300nM Th and 60μM STF-083010, and sXBP1 protein was detected by immunoblotting (left panel). RPMI 8226 cells were treated with 300nM Th and 60μM STF-083010 for the indicated times, and the levels of phosphorylated and total Ire1α were detected using specific antibodies (right panel). (D) STF-083010 effect on cell-free Ire1α RNase (endonuclease) activity. Upper panel: hIre1 was incubated with uniformly labeled (32P) HAC1 508-nt transcript for 30 minutes in the presence of increasing concentrations of STF-083010 (1-100μM). HAC1 mRNA cleavage reaction was analyzed by separation of products on denaturing polyacrylamide gels, followed by autoradiography. Lower panel: Quantitation of HAC1 mRNA processing showing half-maximal inhibition at approximately 25μM. Error bars represent SEM of 3 independent experiments. (E) Effect of STF-083010 on cell-free Ire1α kinase activity. Upper panel: hIre1α was incubated with 32P-γATP and increasing concentrations (0-100μM) of STF-083010. Ire1α autophosphorylation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography to determine the amount of 32P incorporation (32P-hIRE1). Lower panel: Kinase activity showed no significant change during coincubation with STF-083010. Error bars represent SEM of 3 independent experiments.
Figure 2
Figure 2
In vivo and ex vivo effects of STF-083010. (A) STF-083010 blocks bortezomib-induced XBP1 activity in vivo. Transgenic XBP1-luc mice were injected intraperitoneally with drug vehicle (16% chremophor), 1 mg/kg bortezomib, or 1 mg/kg bortezomib and 60 mg/kg STF-083010, and bioluminescence was measured after 24 hours. Graph represents the average change in the number of photons per animal 24 hours after treatment. Error bars represent SEM of at least 4 animals. (B) Images of representative animals before and after treatment. (C) In vitro cytotoxicity of STF-083010. RPMI 8226, MM.1S, and MM.1R MM cells were treated with 0, 30, or 60μM of STF-083010, and viable cell number was measured daily by the trypan blue exclusion method. (D) Antitumor activity of STF-083010 in vivo. RPMI 8226 MM cells were established as subcutaneous tumor xenografts in NOD/SCID/IL2Rγ null mice. When tumors reached an average volume of 150 mm3, 2 groups of 5 mice each were treated with 30 mg/kg STF-083010 or drug vehicle once weekly for 2 weeks. (E) STF-083010 is preferentially cytotoxic against human MM cells. MM cells were obtained by CD138+ selection from bone marrow samples from MM patients; lymphocytes were obtained by Ficoll density-gradient centrifugation of peripheral blood samples from control patients, followed by staining with anti-CD3, anti-CD19, and anti-CD56 monoclonal antibodies to differentiate between T, B, and NK cells. Cells were cultured with the indicated concentrations of STF-083010 for 24 hours, and cell viability was measured by flow cytometric analysis of annexin V/propidium iodide (MM) or 7-amino-actinomycin D (peripheral blood lymphocytes)-stained samples.
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