Background: Histone deacetylase (HDAC) inhibitors (HDI), although only marginally active as single agent therapy in multiple myeloma (MM), demonstrate an ability to salvage proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs) responsiveness in refractory patients, thus raising interest in this class of targeted therapeutics. Selective inhibition of particular HDAC isoforms may further improve therapeutic efficacy. HDAC11, the newest member of the HDAC family, is expressed in a progressive manner during B cell development with greatest expression in mature plasma cells (PC).
Methods: B cell lymphopoiesis was evaluated using Tg-HDAC11-eGFP mice expressing eGFP regulated by the HDAC11 promoter and congenic mouse strains deficient in HDAC11 expression globally (B6.HDAC11-/-) or targeted to the B cell lineage (CD19Cre.HDAC11-/-). In vitro assays were completed using established human myeloma cell lines that include RPMI-8226, RPMI-8226-B25, OPM2, H929, KMS11, KMS28, KAS6, KAS6R10R, MM1.S, and MM1R10R (kind gift from Dr. Orlowski). Inhibition of HDAC11 in MM cell lines was accomplished by both molecular and pharmacologic approaches. Viability was measured according to activated caspase-3 and Annexin/PI staining by flow cytometry, and by CCK-8 viability assay. Subcellular localization changes induced by HDI exposure were assessed by Western Blot of fractionated cell lysates, while immunoprecipitation and confocal microscopy, using both immunofluorescence and proximity ligation assay techniques, were used to identify IRF4 as a novel nuclear binding partner for HDAC11.
Results: Profound eGFP increases in PC of Tg-HDAC11-eGFP mice suggest HDAC11 influences late stage B cell development. Consistent with this, HDAC11 deficiency results in dramatically reduced PC in the bone marrow and periphery. PC depletion in CD19Cre.HDAC11-/- mice, where HDAC11 ablation is restricted to the B cell lineage, suggests activity inherent in B cells rather than via externally derived signals. Initial studies evaluating the effects of quisinostat, a second-generation HDI with increased selectivity that includes HDAC11, showed dose-dependent cytotoxicity versus 10 myeloma cell lines with EC50 measurements in the 1-10nM range. This activity was synergistic with BTZ and carfilzomib (CFZ) in RPMI-8226 cells and degree of synergism was amplified in the BTZ-resistant RPMI-8226-B25 cell line. Subsequent experiments using HDTK-010 (FORMA Therapeutics), an HDAC11-selective HDI, showed dose-dependent cytotoxicity in 21 MM cell lines (EC50 1-10mM). Targeted siRNA-mediated silencing of HDAC11 in RPMI-8226 cells yielded a reduction in cell viability, as measured by Annexin/PI staining and detection of activated caspase-3. An important interaction between HDAC11 and IRF4, an essential regulator of PC differentiation and MM survival, was first identified by immunoprecipitation assay in 8226 cells. This interaction has been confirmed by confocal microscopy using immunofluorescent co-localization and proximity ligation techniques in 8226, MM1.S, OPM2 and H929 cell lines as well as in CD138-enriched MM cells from patient marrow aspirates. Furthermore, this interaction is disrupted by exposure to quisinostat, panobinostat, and HDTK-010. The identification of IRF4 as a putative binding partner for HDAC11 unveils a potential mechanism by which HDAC11 may regulate MM cell survival.
Conclusions:We show that MM cell survival can be impaired by targeted inhibition of HDAC11. Furthermore, we identify IRF4 as a nuclear binding partner for HDAC11 and propose this interaction as a candidate mechanism regulating PC maturation and MM cell survival.
Baz: Sanofi: Research Funding; BMS: Research Funding; merck: Research Funding; takeda: Research Funding; celgene: Honoraria, Research Funding; karyopharm: Research Funding. Pinilla-Ibarz: ARIAD: Consultancy, Honoraria; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.
Asterisk with author names denotes non-ASH members.