Selective Inhibition of HDAC1 and HDAC3 Inhibits Survival of Diffuse Large Cell Lymphoma Via Epigenetic Modulation of Death Associated Protein (DAPK)

Histone deacetylases (HDACs) are key players of epigenetic regulation of gene expression and are therefore attractive targets for cancer therapy. Many epigenetic drugs including HDAC inhibitors (HDACi) have shown anti-tumor activity in several types of cancers. Recently SAHA and belinostat Pan-HDACi have been FDA-approved for the treatment of T cell lymphoma. However, the capacity of Pan-HDACi for distinguishing different classes and isoforms of HDACs is limited, and may produce profound side effects. Therefore, it is important to identify cancer-relevant HDACs to promote selective HDAC inhibition for therapy in other malignancies such as diffuse large B cell lymphoma (DLBCL), the most aggressive non-Hodgkin lymphoma. Recent studies have shown that HDAC1 (HDACs class I) are commonly overexpressed in multiple cancer types and contribute to poor overall survival. However, the role of HDAC3 is not well known. The goal of this study was to determine the mechanistic role of HDAC3 and HDAC1 in DLBCL.

Immunohistochemistry performed on DLBCL tumors (n=29) demonstrate 42% positivity for HDAC3 and 100% positivity for HDAC1. Ectopically expressed HDAC3 led to increased thymidine uptake, whereas HDAC3 knockdown using siRNA led to a significant decrease in thymidine uptake (p<0.001) suggesting that HDAC3 could be necessary for DLBCL cell growth. Moreover, inhibition of HDAC3 using a selective pharmacological inhibitor RGFP966 suppressed cell proliferation in a dose dependent manner in several DLBCL cell lines (SUDHL6, OCILY1, OCILy10 and OCILy3). However, the effect of RGFP966 on cell survival as determined by annexin V/ propidium iodide was underwhelming. We sought to target both HDAC1 and HDAC3 using the pharmacological inhibitor RG283 and assessed biological effect of dual inhibition. Interestingly, dual inhibition of HDAC1 and HDAC3 with RG2833 significantly suppressed both cell proliferation (<0.0001) and survival (p<0.001) in most of the DLBCL cell lines tested. As expected, treatment with RG2833 increased acetylation of H3K9, H3K14, H3K27 and H4K5 histone marks and led to P21 upregulation in DLBCL cells. Interestingly, exogenously overexpressed HDAC3 but not HDAC1 increased sensitivity of DLBCL cells to Pan-HDAC inhibitors vorinostat and belinostat, which suggest that endogenous HDAC3 in DLBCL cells may exist in a protein complex that may evade targeting by Pan-HDACi. In order to identify a potential mechanism of action for dual HDAC3 and HDAC1 inhibition, we have focused on tumor suppressor gene DAPK (Death associated protein), which has been previously shown to be silenced in DLBCL patients via DNA methylation. DAPK is a serine/threonine kinase and possesses pro-apoptotic and tumor-suppressive functions. We have confirmed by RT-PCR that DAPK was suppressed/silenced in DLBCL cell lines (n=8) as compared to CD19+ (n=3) normal B cells. HDAC inhibition alone or in combination with DNA methyltransferase inhibitors (DNMTi) can induce apoptosis by modulating expression of tumor suppressor genes. We next asked whether HDAC3 and HDAC1 dual inhibition through RG2833 alone or its combination with DNMTi, azacytidine (AZA) will be able to restore expression of DAPK in DLBCL cells. Quantitative RT-PCR analysis in OCILy3 and OCILY10 cells demonstrated that treatment with RG2833 alone modestly increased DAPK expression; however, co-treatment with RG283 and AZA led to marked increase in DAPK expression. As expected, the combination of RG2833 and Aza had significantly greater pro-apoptotic effect in DLBCL cells than either drug alone suggesting that DAPK expression may be an important determinant of response to HDAC1/HDAC3 dual inhibitor in DLBCL cells.

In summary, our study provides first evidence of antitumor activity of dual HDAC3 and HDAC1 targeted agent therapy in DLBCL patients. Moreover, we have identified epigenetic modulation of DAPK as potential modulator of cellular response to HDAC1/HDAC3 dual inhibition. This study suggests for further clinical trials and/or development of selective inhibitors of HDAC3 or HDAC1/HDAC3 in DLBCL patients.