P-189: Indirubin Derivative Acts as Dual Inhibitor Targeting Proteasome and Autophagy for Treating Multiple Myeloma

Introduction: Multiple myeloma (MM) remains incurable despite the availability of various therapies due to high rates of relapse and recurrence. Previous studies have highlighted the effectiveness of indirubin-3-monoxime (I3MO) as a proteasome inhibitor against MM cell proteasome activity. Histone deacetylase 6 (HDAC6) inhibitors have also shown promise in inhibiting the aggresome-autophagy pathway. Here, a novel I3MO derivative 8b was synthesized by coupling an HDAC6 inhibitor with the I3MO structure.

Methods: We investigated the anti-MM effects of 8b both in vivo and in vitro. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. Dual-luciferase reporter gene assay was performed to identify downstream targets of 8b treatment. We further explored the function of TRIM28, the target of 8b, through shRNA knock-down (KD) and over-expression (OE) experiments. ChIP-seq and IP-MS were examined to clarify the potential target of TRIM28.

Results: The novel derivative 8b efficiently inhibited myeloma cell proliferation and induced apoptosis in a dose- and time-dependent manner. Treatment with 8b caused cell cycle arrest in the S phase. In vivo studies confirmed the anti-MM effects of 8b treatment. Furthermore, 8b decreased proteasome activity and inhibited the formation of aggresomes and autophagosomes. Combining 8b with bortezomib (BTZ) enhanced apoptosis in MM cells both in vitro and in vivo.
Mechanistically, the dual-luciferase reporter gene assay revealed that 8b treatment significantly down-regulated TRIM28 promoter activity, resulting in transcriptional repression of TRIM28. Bioinformatics analysis showed a positive correlation between the levels of TRIM28 and multiple proteasome subunit genes, including PSMB1 and PSMB2, in MM. ChIP-qPCR assay confirmed that TRIM28 directly bound to the promoter region of PSMB1 and PSMB2. Knock-down of TRIM28 led to decreased PSMB1 and PSMB2 expression and reduced proteasome activity, increasing MM cell sensitivity to proteasome inhibitors (PIs). Knockdown of TRIM28 also suppressed autophagosome formation, promoting sensitivity to PIs treatment in multiple MM cell lines. IP-MS and Co-IP demonstrated that TRIM28 interacts with 14-3-3ζ and mediates its ubiquitination and degradation. 14-3-3ζ is a well-known negative regulator of autophagy, and its degradation induced by TRIM28 activates autophagy in MM cells.

Conclusions: Our study identified a novel indirubin derivative targeting proteasome and autophagy for treating myeloma. This represents a promising therapeutic approach that could assistant overcoming the PIs resistance in myeloma. Additionally, our study for the first time clarified the role of TRIM28 in MM pathogenesis, which should be considered as a potential therapeutic target for MM.