Epigenetic Modifications of the Bone Marrow (BM) Niche in Multiple Myeloma (MM) - a Three-Dimensional (3D) in Vitro Approach

Introduction:Since the discovery of histone modifications by Allfrey et al. in 1964 and their impact on gene transcription, numerous epigenetic mechanisms and potential targets, including writer, eraser and reader proteins, have been identified, which has consequently led to a variety of current clinical "epidrug" applications. In particular hematological neoplasia, e.g. T-cell lymphomas, myelodysplastic syndrome and MM, show good responses to epigenetic modulation. To foster this progress, while fulfilling the crucial role of the BM microenvironment in MM pathogenesis in early preclinical drug development, we are testing novel epigenetic modifying agents provided by the resident Institute of Pharmaceutical Sciences (M.Jung) in MM using an established agarose-based 3D matrix (FAM) platform.

Methods: For our purpose semi-adherent MM cell lines as well as primary MM patient (pt) samples are seeded into conical 2mm deep microcavities of the FAM (see Figure 1), whereby the culture period can be extended up to at least 3 weeks in either distance or contact co-culture to HS-5 or pt-derived MSP-1 stromal cell lines. The well-defined conical shape and transparency of the agarose matrices allows straightforward qualitative and quantitative cluster monitoring via non-destructive transmitted light scanner analysis, confocal imaging or histological examinations. In addition, the matrices can be cut and divided precisely to perform viability assays, Western blots and multicolor flow cytometry (CD138, CD38, SlamF7, CXCR4, CXCR7, PD-L1, VLA-4) in parallel and at different time points on days (d) 2 and 4 with (w) or without (w/o) epigenetic treatment or stromal support. To conclusively assess our FAM model in terms of more in-vivo like prolonged proliferation and diminished treatment response, we also performed comparative studies with the widely used 3D liquid overlay technique (LOT).

Results: In contrast to LOT, the well-defined, steep microwells of our FAM enabled semi-adherent plasma cells (PCs) to form solid 3D-aggregates and mediated drug resistance to 6nM bortezomib and 3µM auranofin treatment. In this context qualitative confocal analysis of mCherry-transduced RPMI 8226 cells and fluorescent dye staining confirmed uniform drug distribution within the FAM. Moreover, PCs seeded into the microcavities showed prolonged cluster growth in accordance with intratibial MM engraftment in our NOD/SCID mouse model, underlining the more in vivo -like growing conditions within the FAM and its superiority over LOT.

To date, our screening approach has comprised 6 lysine deacetylase inhibitors (KDACi) [selective KDAC6i: JS08, JS28 and JS46 as well as entinostat, panobinostat and vorinostat] and three JARID1-selective lysine demethylase inhibitors (KDMi) [SYN22327689, Z425307602 and KDM5-C49]. With regard to cytotoxicity using ATP-dependent assay and flow cytometry, we achieved the most promising results for JS28 and KDM5-C49 in low micromolar concentrations after 2d and 7d, respectively. Additionally, for JS28 we could demonstrate synergism, if combined with bortezomib and a high selectivity for KDAC6 inhibition via Western blot in both 2D and 3D cultures.

Whilst 3D stromal co-culture led per se to lower SlamF7 and CXCR4 surface levels, increased PD-L1 expression and the persistence of CD138 and CD38, our data also suggest, that additional treatment with KDACi further modulates expression patterns concerning diminished VLA-4, SlamF7, PD-L1 and CD138 levels.

Conclusions: Our FAM platform provides various benefits to MM cells compared to the widely employed LOT, namely 1) solid cluster formation of semi-adherent PCs including more in vivo -like proliferation and drug resistance, 2) examination of contact vs. distance co-culture, 3) simultaneous, non-destructive observation of approx. 880 spheroids per matrix over time, 4) cultivation of proliferating and viable primary MM-pt samples for at least 3 weeks, but most importantly 5) displaying an easy to use high throughput screening platform, which meets the requirement for an in vitro BM model. Our ongoing analysis, including selective KDACi, like Sirtuin-1 or -2-inhibitors and lysine methyltransferase or lysine acetyltransferase inhibitors, will generate more data on rational drug combinations between epidrugs and adhesion-based therapies, including NOX-A12, elotuzumab, daratumumab, indatuximab-ravtansine and durvalumab.

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