In vitro activity: STF-083010 (formerly IRE1 Inhibitor I) is a novel and specific small-molecule inhibitor of IRE1α endonuclease. Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). 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. In RPMI 8226, MM.1S, and MM.1R MM cell lines, STF-083010 exhibits cytostatic and cytotoxic activity in a dose and time dependent manner. In MiaPaCa2, Panc0403, and SU8686 cell lines, STF-083010 inhibits XBP1 splicing and blocks IRE1α's endonuclease activity without affecting its kinase activity. In Eμ-TCL1 CLL cells, STF-083010 exhibits about 70% growth inhibition after 3 days culture. In MEC1 and MEC2 cells, STF-083010 produces 20% growth inhibition in 48 h. WaC3 cells respond to treatments with STF-083010 with gradually decreased growth.

Kinase Assay: Autophosphorylation activity is determined by the addition of 32P-γ ATP. Endonuclease activity is determined by the addition of radiolabeled HAC1 508-nt RNA substrate synthesized in vitro using α32P-UTP. STF083010 is incubated with recombinant hIRE1 protein, radiolabeled HAC1 508 nt RNA, and appropriate buffers. Kinase activity is quantitated by polyacrylamide gel electrophoresis. RNAsecleavage products are quantitated by 32P-γATP or 32P-UTP autoradiography.

Cell Assay: Three thousand cells are seeded in 96-well plates overnight and drug treatment started the next day. After 48 h incubation, MTT is added to cells and cultured at 37℃ for 4 h followed by stop solution (4 mM HCl, 0.1% Nondet P40 in isopropanol) which is added to dissolve MTT. The plates are read with a spectrophotometer at 590 nm absorbance with reference at 630 nm. IC50 values are calculated using GraphPad Prism.