SIRT Regulates the Molecular Interaction Between c-MYC and HIF-1α in Multiple Myeloma

Abstract 574

Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the accumulation of malignant plasma cells. Dysregulation of MYC by rearrangement or translocation are common somatic events described either in early or late stage of the disease, and transcriptional profiling of MYC pathway activation is observed in more than 60% of MM cell lines. Hypoxia Inducible Factor-1α (HIF-1α) overexpression has been described in several MM cell lines and in about 30% of MM patients samples. In solid tumours, deregulation of c-MYC has been associated with HIF-1α upregulation: under physiologic conditions HIF-1α inhibits c-MYC activity by direct interaction and stimulation of a proteasome-dependent pathway.

In the present study we explored the interaction between c-MYC and HIF-1α in a panel of MM cell lines (MM1S, OPM2, RPMI8226, U266). We had previously shown that treatment with EZN2968, a locked nucleic acid antisense oligonucleotide directed against HIF-1α, resulted in a significantly reduction of HIF-1α protein level after 24h of incubation. The reduction of HIF-1α was specific and lasted over 96h. To confirm the inhibition of HIF-1α activity, MM1S cells were treated with EZN2968 for 24h, lysed, co-precipitated with p300, and incubated with anti-HIF-1α antibody. We showed that HIF-1α was no longer associated p300 in EZN-treated compared to untreated samples, suggesting an inhibitory effect of HIF-1α activity. We next observed that treatment with EZN2968 induced a progressive accumulation of cells in S-phase with concomitant reduction of G₂/M phase. By western blot analysis, we observed that p21 and p27, cell cycle check points negatively regulated by c-MYC, were up-regulated in treated samples. We further verified the effect of HIF-1α inhibition on c-MYC protein level by western blotting analysis. After treatment with EZN2968, c-MYC protein expression was reduced in a time dependent manner (c-MYC protein was almost undetectable after 72h of incubation), suggesting that c-MYC protein level is associated with inhibition of HIF-1α. To examine whether HIF-1α and c-MYC regulate each other promoter activity, we performed Chromatin Immunoprecipitation (ChIP) assays with HIF-1α or c-MYC antibodies. HIF-1A and MYC promoter amplification signals, were present in the controls samples, and increased after EZN2968 exposure, suggesting that these proteins can play a direct role in regulating each other's activity.

Recently, it has been shown that SIRT1, a transcription factor involved in a development, cellular stress responses, and metabolism, can modulate HIF-1α and c-MYC activity. By Immunoblotting assay, we observed that SIRT1 physically interacts with both proteins and that, after 24h of exposure to EZN2968, c-MYC and HIF-1α were no longer associated to SIRT1. These results were also confirmed at the transcriptional level, by ChIP assay using an anti-SIRT1 antibody. After 24h of treatment with EZN2968, we observed a significant increase of HIF-1A and MYC promoter amplification signals in treated compared to untreated samples, suggesting that SIRT1 recruitment at both promoters is dependent on HIF inhibition.

We showed that in MM cell lines the expression of HIF-1α and c-MYC are linked and mediated by SIRT1 deacetylase protein. The data suggests a new regulatory mechanism for controlling c-MYC and HIF-1α activity by SIRT1. The identification of a HIF-c- MYC–SIRT 1 interaction in MM cell lines suggests a novel therapeutic target for MM patients.