Fig. 6.

LDN192960 bypasses bortezomib resistance. (A) DYRK2 differential gene expression in human relapsed MM specimens and human normal tissue as available from a public database GSE6477 (relapsed vs. normal tissue, P value derived from empirical Bayes estimation on linear models of gene expression in limma package) (see also SI Appendix, Fig. S3). (B and C) Bortezomib EC₅₀ for parental MM.1S and bortezomib-resistant MM.1S.BR cells (B) and for parental RPMI8226 and bortezomib-resistant 8226.BR cells (C). (D) LDN192960 EC₅₀ for parental MM.1S, parental RPMI8226, MM.1S.BR, and 8226.BR cells. (E) Proteasome activity in total cell lysates from the indicated bortezomib-resistant cells with or without 10 μM LDN192960 treatment for 2 h was measured with Suc-LLVY-AMC and normalized to total protein content. **P < 0.01 (compared to control treated for each cell line, ordinary 1-way ANOVA, mean ± SD from n = 3 independent experiments). (F) Experimental flow for myeloma xenograft study in G and H. (G and H) The 8226 parental (G) or 8226.BR (H) cells were injected s.c. into J:NU nude mice. Palpable tumor-bearing mice were randomized (16 d for parental and 23 d for 8226.BR) into 2 equal groups each and treated with vehicle control or LDN192960 3 times a week by i.p. injection, and tumor volume was measured twice a week (n = 5 per condition). **P < 0.01 (compared to vehicle treated, 2-way ANOVA, mean ± SD, from n = 5 mice). (I) Proteasome activity in total cell lysates from 8226 WT or 8226.BR was measured with Suc-LLVY-AMC. **P < 0.01 (8226 WT vs. 8226.BR, unpaired Student’s t test, mean ± SD from n = 3 independent experiments). Immunoblotting of the cell lysates was carried out with indicated antibodies.