A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms.

Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS.

Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy.

Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.