Abstract
Background: Histone deacetylases (HDACs) are potential novel therapeutic targets for multiple myeloma (MM) treatment. A pan-HDAC inhibitor (HDI) panobinostat was approved by the FDA in 2015 to treat relapsed/refractory MM patients, and several other HDIs are currently in different phases of clinical trials. However, unfavorable side-effects of the non-selective HDIs necessitate further dissection of the roles of individual HDAC isoforms to best target plasma cell malignancies with minimal toxicity. HDAC11 was recently found to regulate function in key immune cell populations including regulatory T cells, effector T cells, neutrophils, and myeloid-derived suppressor cells (MDSC). Though HDAC11 expression is confirmed in B cells and plasma cells, its functions in these cells remain largely unknown. In this study, we attempted a functional analysis of HDAC11 in plasma cell development along with its pro-tumorigenic function in MM cells.
Methods: Mouse models, including a transgenic mouse strain expressing eGFP under the regulation of the HDAC11 promoter (Tg-HDAC11-eGFP), and also an HDA11-deficient mouse (B6.HDAC11-/-) were studied to establish the importance of HDAC11 in plasma cell biology. Pharmacologic inhibition of HDAC11 in MM cell lines was accomplished by using elevenostat, a new HDAC11-selective inhibitor in comparison with pan-inhibitors quisinostat and panobinostat. Impact on viability in human-derived MM cell lines was assessed using the CCK-8 assay, while induction of cell death was measured via detection of activated Caspase-3 and annexin/propidium iodide staining by flow cytometry. Synergy studies were performed by following the Chou-Talalay method for drug combinations. Post-translational modifications and subcellular localization changes induced by HDIs exposure were assessed by western blotting of fractionated cell lysates, while immunoprecipitation and proximity ligation assays (in situ PLA) were used to identify a binding partner for HDAC11.
Results: Studies in Tg-HDAC11-eGFP mice reveal that HDAC11 expression in B cell lymphopoiesis is minimally detectable prior to B cell activation but demonstrates strong induction upon maturation into a plasma cell. Consistent with this, plasma cell development is markedly impaired in the absence of HDAC11. The HDAC11-selective inhibitor elevenostat showed significant cytotoxic potential in different MM cell lines that express moderate to high level of HDAC11, with IC50 values ranging 0.6-2.0 µM. Consistently, MM cell lines expressing null/very low level of HDAC11 were insensitive to elevenostat. Moreover, combining elevenostat with proteasome inhibitors bortezomib (BTZ) and carfilzomib resulted in significant synergistic effects evident from combination index (CI) and dose-reduction index (DRI) values measured by CompuSyn software. Elevenostat was also able to re-sensitize BTZ-resistant sub-clones (e.g., RPMI-8226-B25, KAS-6-V10R, and ANBL6-V10R) to BTZ and exhibited superior synergistic effects. Furthermore, elevenostat-treated cells showed a time-dependent alteration in the subcellular localization of HDAC11. HDAC11 gradually disappeared from the nuclear fractions with simultaneous upregulation in cytoplasmic fractions; similar observations were made from pan HDIs (quisinostat and panobinostat) treatment. However, unlike pan HDIs, the elevenostat treatment caused global downregulation of HDAC11 in some MM cell lines at the later time points (72 or 96 hrs), suggesting differential effects of various HDIs. Inhibition of HDAC11 also caused downstream suppression of several pro-tumorigenic factors of MM cells including IRF4 and c-Myc. Additionally, a novel interaction between HDAC11 and IRF4, an essential regulator of PC differentiation and MM survival, was identified by using PLA. HDAC11 dynamically interacts with IRF4 which can be induced by LPS stimulation and inhibited by HDIs, indicating the involvement of HDAC11 in the IRF4-mediated regulatory circuit.
Conclusions: We observe that targeted inhibition of HDAC11 can impair MM cell survival and overcome acquired resistance to proteasome inhibitors. Furthermore, we identify IRF4 as a nuclear binding partner of HDAC11 and propose this interaction as a candidate mechanism regulating PC maturation and survival.
Disclosures
No relevant conflicts of interest to declare.
Functional analysis of the involvement of apurinic/apyrimidinic endonuclease 1 in the resistance to melphalan in multiple myeloma