Targeting proliferating cell nuclear antigen via a miRNA-CSC axis as a new therapeutic approach in multiple myeloma

Multiple myeloma (MM) is a plasma cell cancer, which is ranked as the second most common hematologic malignancy. Despite advances in treatment MM remains incurable. Proliferating cell nuclear antigen (PCNA) is an essential protein which plays a key role in regulation of cell growth and proliferation. A post-translationally modified isoform of PCNA plays a critical role in the pathogenesis of a wide variety of malignant diseases including MM. Increased expression of PCNA has been shown to be associated with an aggressive MM phenotype, making it an attractive therapeutic target. We tested the anti-tumor activity of the novel small molecule PCNA inhibitor AOH-1996 (alone and in combination with either bortezomib or venetoclax) against 5 different sub-types of MM cell lines. We assessed the cytotoxic anti-MM effects of varying doses AOH-1996 using standard viability assays and also 3D spheroid degradation assays. Changes in gene expression relevant to cell growth, cell-cycle progression, apoptosis, methylation status, and cytokine production following AOH-1996 exposure were analyzed using RT-qPCR and Western blotting, as appropriate. We also evaluated the impact of AOH-1996 exposure on miRNA levels. The anti-MM effect of AOH-1996 monotherapy and combinations versus the same cell lines was then further evaluated in xenograft animal model. AOH-1996 demonstrated dose-dependent reduction of cell growth (2D) and CSC-like sphere formation (3D) of different subtypes of MM cell lines. Cell cycle assay revealed that AOH1996 treatment resulted in the delayed mitosis in MM cells. AOH-1996 treatment also significantly down-regulated the gene expression of selected anti-apoptotic markers (PCNA, CDK4/6, BCL2), cancer stem cell markers (EZH2, Sox2), and inflammatory and pro-survival cytokines (IL-6, FGF2). AOH-1996 treatment down-regulated oncogenic miRNA (miR-21, miR-30a, miR-99a, miR-100, miR-142, miR-191 and miR-222) levels and up-regulated anti-oncogenic miR-145 along with the reduced cytokine production of VEGF. There was a synergic effect on cell growth and CSC-like sphere formation when either bortezomib or venetoclax was added to AOH-1996. AOH-1996 and its combination with venetoclax or velcade showed decreased the protein expressions of EZH2, c-Myc, and PARP in MM cells, and significantly inhibited MM.1S cell line-derived xenograft tumor growth. Re-expression of miR-145 by its mimic transfection decreased cell growth and increased the drug sensitivity of AOH-1996 along with the down-regulation of EZH2, CDK4, and CDK6 genes in MM cells, suggesting an important role of AOH1996-mediated miR-145 in MM cells. These findings clearly suggest that PCNA is a potential therapeutic target in MM. We have demonstrated for the first time that the PCNA inhibitor AOH-1996 shows potent anti-myeloma activity through its effects on several key tumor-associated genes/proteins and miRNAs.