HDAC Inhibitors As Potential New Agents Improving the Efficacy of Monoclonal Antibodies

HDAC (histone deacetylase) inhibitors belong to a novel class of antitumor agents widely explored in hematology. So far two of them - romidepsin and vorinostat have been registered in cutaneous T-cell lymphoma, while panobinostat awaits FDA decision concerning its registration in multiple myeloma. Several other agents are currently being tested in clinical trials in lymphoma and myeloma patients and preliminary results suggest that they may be most effective when used in combinations with other anti-cancer drugs. Reports from others (Sugimoto et al. Biochem Biophys Res Commun 2009 Shimizu et al. Leukemia 2010) and the results from our group show that HDAC inhibitors augment the efficacy of anti-CD20 monoclonal antibodies (mAbs) in complement-dependent cytotoxicity (CDC) by up-regulating CD20 level. In our previous reports we have shown that HDAC6 isoform is involved in the regulation of CD20 degradation and trafficking. Since the efficacy of mAbs depends not only on the level of target molecule but is also regulated by the expression of complement regulatory proteins (mCRPs) - CD46, CD55 and CD59, we sought to investigate the influence of HDAC inhibitors on the expression of those molecules. Moreover, given the fact that HDAC inhibitors have been reported to regulate NK cells activity, we investigated their influence on antibody-dependent cell-mediated cytotoxicity (ADCC) which is one of the mechanisms of action of mAbs in vivo.

The purpose of this study was to thoroughly investigate the influence of HDAC inhibition on the level of surface molecules – CD20 and CD38 – targets already used and tested in clinical trials in immunotherapy of B-cell malignancies as well as the expression of mCRPs. Using non-selective pan-inhibitors as well as selective inhibitors of particular HDAC classes and isoforms we sought to show the inhibition of which HDAC enzyme is the most effective in potentiating the outcome of mAbs based anti-tumor therapies. Since the results of clinical trials show that the use of HDAC inhibitors has several non-specific adverse effects, we hypothesize that selective inhibition of particular isoforms may considerably diminish these toxicities.

Using flow cytometry we performed experiments assessing the level of surface CD20 CD38 and mCRPs – CD46, CD55 and CD59. We used established lymphoma, chronic lymphocytic leukemia (CLL) and myeloma cell lines as well as primary cells isolated from the patients suffering from CLL. We observed that the use of HDAC pan-inhibitors – trichostatin A (TsA) and vorinostat (Fig.1A) and selective HDAC6 inhibitors – tubacin and ACY1215 (ricolinostat) (Fig.1B) considerably improves the outcome of anti-CD20 mAbs – rituximab and ofatumumab in CDC in several B-cell lymphoma cell lines and in CLL primary samples (Fig. 1C). In our experiments we show that selective inhibition of HDAC6 in Raji cells does not influence the expression of mCRPs (Fig. 1D). However, we observed that pre-incubation with tubacin considerably increases the expression of CD38 (Fig. 1 D). Therefore, in our further studies we aim at expanding our observations to primary samples from multiple myeloma patients, especially since novel anti-CD38 mAbs are currently intensively explored in the therapy of this malignancy.

Moreover, using flow cytometry we determined the influence of HDAC inhibition on degranulation of NK cells isolated from healthy donors in ADCC in vitro assay with rituximab and ofatumumab. Contrary to the reports of others, we observed no negative regulation of NK cells activity and their ability to kill target tumor cells. Therefore, since HDAC inhibitors seem to influence mainly CDC, we investigated the influence of specific HDAC inhibitors on the level of mCRPs. Those results suggest that specific inhibition of HDAC3 (Fig. 1E) and HDAC1/2 (Fig. 1F) are effective therapeutic strategies as they considerably decreased the expression of complement inhibitors and concomitantly increased the level of CD20 antigen. However, further studies including silencing of particular HDAC isoforms with shRNA are needed to confirm these findings. Altogether, our results strongly suggest that HDAC inhibitors potentiate the outcome of mAbs used in hematological malignancies.

Figure 1

View largeDownload slide

Figure 1

View largeDownload slide

Close modal