Quantitative Proteomic Profiling Unravels Dynamic Changes in the Myeloma Cell Proteome Treated with Valproic Acid (VPA)

Abstract 1847

Multiple Myeloma (MM) is a post germinal center B-cell malignancy characterized by abnormal proliferation of terminally differentiated plasma cells in the bone marrow. Despite significant advances in multiple myeloma treatment, the disease remains incurable. Earlier studies have shown that HDAC inhibitors are effective in inducing antitumor effects in many hematological malignancies, but the exact mechanism through which they act is unclear. Since Valproic acid (a FDA approved HDAC inhibitor to treat epilepsy) is cost effective and suitable for old patients, we investigated VPA in this study. Our study aims to unravel the mechanism by which VPA induces antitumor effects in MM cell lines using both biochemical and proteomics approaches.

In our experiments, MM cell lines (RPMI8226, NCI H929 and MM1R) when treated with VPA showed a block in the G1-S transition (decrease in the S-phase and G2M-phase, increase in the Sub-G0 phase) and decrease in the cell viability in a dose and time dependent fashion. Also, Annexin V staining by FACS and PARP cleavage by immunoblot analysis confirmed the induction of apoptosis through Caspases. We then analyzed the expression pattern of several genes, including those involved in transcription, cell cycle regulation and signaling pathway by quantitative RT PCR and by immunoblot analysis. Our study showed an induction of H3 acetylation, p21 and an increase in the NOTCH1 target genes (Hes1 and Hey1) in the myeloma cells treated with VPA.

To further understand the key signaling pathways involved in the pathogenesis of MM and also to characterize how VPA regulates growth arrest, we investigated the differentially expressed proteome in the RPMI 8226 cell line treated with and without VPA using SILAC (Stable Isotope Labelling with Amino acids in Cell culture) based quantitative proteomics approach. Briefly, one population of RPMI 8226 cells was grown in medium with heavy (isotopic) amino acids (¹³C₆ L-Lysine and ¹³C₆ L-Arginine), while the other population was grown in medium containing naturally abundant isotopic form of (normal) amino acids (¹²C₆ L-Lysine and ¹²C₆ L-Arginine). Cells grown in heavy medium were left untreated, while the cells grown in light medium were treated with 1mM VPA for 24 hrs. Cell lysates were pooled, resolved on SDS-PAGE, protein bands excised, trypsin digested and analyzed on LTQ orbitrap velos mass spectrometer.

Using SILAC approach, we identified and quantified 2,032 proteins in myeloma cells treated with VPA. We found that several proteins including protein kinases, Cell cycle regulators, transcription factors, membrane proteins, mitochondrial proteins and metabolic enzymes were regulated by VPA. Checking for the presence of known cell surface markers for plasma cell in the proteome data, we found Syndecan (CD138) with 3 unique peptides and noted that its expression was decreased upon VPA treatment. This was confirmed by FACS analysis. Interestingly, we also found that CCND2, an important regulator of plasma cell development and the one that is often implicated in myeloma pathogenesis, was significantly down regulated by VPA treatment. This was confirmed by Immunoblot analysis in a dose and time dependent manner. We also found that VPA is more effective in regulating CCND2 promoter activity in combination with Dexamethazone.

Based on the above results, we then reasoned if NOTCH1 induction could regulate CCND2 expression. Interestingly, our study showed that active form of intracellular NOTCH1 down regulated CCND2 promoter activity. NOTCH1 being a membrane bound transcriptional activator, we then hypothesized that increased NOTCH1 signaling pathway could down regulate CCND2 expression by inducing a transcriptional repressor. Our preliminary results showed that NOTCH1 mediated Hes1 induction repressed the promoter activity of CCND2.

Overall, our global quantitative proteomic analysis demonstrates that Valproic acid treatment induces dynamic changes in the myeloma proteome. In addition, we have shown that VPA may control the proliferation of myeloma cells at least in part via a NOTCH-Hes1-CCND2 regulatory axis. These results provide an invaluable starting point to design and use Valproic acid in combination with Dexamethazone and/or with Bortezomib as an effective therapy for myeloma.