HDAC Inhibition Involves CD26 Induction on Multiple Myeloma Cells Via the c-Myc/Sp1-Mediated Prompter Activation and Overcomes Therapeutic Resistance By Humanized Antibody

Background: CD26, a 110-kDa transmembrane glycoprotein, which is expressed on several tumor cells including malignant lymphoma has been implicated in tumorigenesis, whereas, little is known about its roles in plasma cell malignancies. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, while its expression in plasma cells of patients with multiple myeloma (MM) reveals heterogenous due to its cellular diversity or immune escape, leading to a marked heterogeneity of response to humanized anti-CD26 monoclonal antibody (mAb) therapy in MM. Decreased expression levels of CD26 in MM cells are one of the mechanisms underlying innate and acquired resistance to CD26mAb therapy in MM.

Aim: Here, we clarify the potential impact of epigenetic modification by HDAC inhibition (HDACi) with isoform selective- or broad- inhibitors on the regulation of CD26 expression in MM cells and elucidate its mechanisms, thereby affecting the performance of CD26mAb in refractory MM.

Methods and Results: Immunostaining of bone marrow tissues of MM showed that CD26^pos/CD138^pos plasma cells were detected in several patients but not in others. First, we investigated the impact of HDACi on CD26 expression of MM cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 MM cell lines (KMS26, KMS27, KMS28, KMS11, RPMI8226), cultured alone, the increased expression in CD26 levels of MM cells was detectable within 24 hr of the treatment with HDAC1i; FK228, HDAC3i; BG45, MS-275, RG2833 or HDAC6i; nexturastat A, tubastatin A, ACY-1215 as well as broad HDACi; LBH-589, SAHA by flow cytometry. Its expression increased further during the exposure to each HDACi and its maximum increase was observed at 72 hr of the treatment. Then, subsequent removal of HDACi for 48 hrs resulted in a decline of CD26 expression on MM cells to reduced or baseline levels. In addition, the levels of CD26mRNA were concomitantly augmented, which was paralleled with an increase in the induction of CD26 protein and its DPPIV enzymatic activity in MM cells. We also examined the effect of each HDACi on the viability of CD26^neg MM cells in the absence or presence of CD26mAb.CD26mAb alone did not induce lysis of CD26^neg MM cell lines at any doses, whereas, combining each HDACi plus CD26mAb at 10 μg/ml synergistically facilitated lysis of CD26^neg MM cells via direct effects as well as NK cell- mediated ADCC by CD26mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26^neg cell populations, refractory to HDACi or CD26mAb alone.

Next, gene expression profiles in KMS11, KMS27 and RPMI8226, left untreated or treated with LBH589 or RG2833 revealed that Myc is one of the overlapped genes, altered in its expression. The 5'-flanking region of human CD26 gene, representing the promoter activity contains potential binding sites of several transcriptional factors including Sp1 and c-Myc. So, further to elucidate the mechanisms by which CD26 expression is induced in MM cells by HDACi, we examined the epigenetic status in histone and non-histone at the CD26 promoter of MM cells with the treatment of HDACi. First, ChIP-qPCR demonstrated that the acetylation in histone 3 lysine 27 is increased at the CD26 promoter of 5 MM cell lines after exposure to each HDACi, suggesting parts of mechanisms in the CD26 promoter activation. Moreover, the expression level of c-Myc was time-dependently decreased in KMS11, KMS27 and RPMI8226 with the treatment of LBH-589 or RG2833 both at mRNA and protein levels and was significantly down-regulated after 12 to 24 hr of exposure. On the other hand, the acetylation of c-Myc on K323 was significantly increased after 12 to 24 hr of exposure to each HDACi. ChIP-qPCR revealed that the recovery % IP/INPUT showing the binding of c-Myc to the CD26 promoter was exposure time-dependently reduced in KMS11, KMS27 and RPMI8226 by the exposure to LBH589 or RG2833. Namely, in the absence of HDACi, c-Myc was present on the CD26 promoter via binding to Sp1 on the proximal G-C box and repressed CD26 promoter in MM cells, whereas, in the presence of HDACi, c-Myc was highly detached from Sp1 on the CD26 promoter after 12 to 24 hr with its increased acetylation, leading to activate CD26 promoter and initiate CD26 transcription in MM cells.

Conclusion: HDACi not only shows anti-MM activity itself but sensitizes MM cells with CD26 antigen loss to CD26mAb via c-Myc/Sp1-mediated CD26 induction and augments its cytotoxicity.

No relevant conflicts of interest to declare.