The Molecular Landscape and Essential Pathways Involved in Bone Marrow-Mediated Carfilzomib Resistance of Multiple Myeloma

Abstract

Background Multiple myeloma (MM) remains an incurable plasma cell malignancy where virtually all patients develop drug resistance, relapse, and ultimately die from the disease. Anti-myeloma drugs, such as the proteasome inhibitors (PIs) bortezomib (BTZ) and carfilzomib (CFZ) constitute an essential part of contemporary treatment regimens in clinical practice and have considerably improved MM patient prognosis. Despite therapeutic advances in the past years, clonal evolution of myeloma cells, which is characterized by heterogeneous genomic aberrations, cell-intrinsic proteomic and metabolomic adaptations, as well as cell-extrinsic tumor-promoting alterations within the bone marrow (BM) microenvironment still pose major challenges in MM treatment. Understanding the underlying mechanisms responsible for PI resistance provide a key to targeting both, PI-resistant minimal residual disease that drives relapsed MM after prolonged PI-containing frontline therapy, as well as PI-refractory, aggressive advanced MM. While potential drug resistance mechanisms have been described in vitro, we still lack understanding of PI resistance in vivo, where clonal heterogeneity and the tumor microenvironment (TME) within the BM add additional levels of complexity. Thus, in this study we aim to identify molecular alterations occurring during MM progression under the long-term selective pressure of CFZ in vivo as well as in the context of the TME to identify essential molecular pathways and discover novel therapeutic options for the treatment of relapsed/refractory MM. In addition, confocal imaging of MM-harboring mouse femur cross-sections showed MM and/or PI-induced morphological changes within the TME in vivo. Methods NSG mice bearing intrafemorally injected human RPMI-8226 cells were either left untreated or were treated long-term with 4 mg/kg i.v. CFZ until they eventually became drug resistant. At this point, CFZ-naïve and CFZ-resistant cells, together with other cells from the TME, were isolated and processed for single-cell RNA sequencing (scRNA-seq, 10x Genomics) with the aim to characterize a transcriptional CFZ resistance signature in refractory myeloma cells. Concomitantly, non-injected femurs from the same mice were processed for confocal imaging of femur cross-sections to investigate myeloma-induced morphological changes in and without the presence of CFZ. Results ScRNA-seq analysis showed a distinct transcriptional profile for CFZ-refractory RPMI-8226 cells when compared with CFZ-naïve cells isolated from the BM of untreated mice. Unsupervised clustering analysis, using UMAP, revealed several clusters that were present only in the samples derived from refractory mice. Those clusters were then compared with clusters from the untreated samples which disappeared upon exposure to CFZ to characterize the transcriptional changes characteristic for the emerging clusters. The gene list obtained from this comparison was used as input for gene set enrichment analysis (GSEA), which showed high normalized enrichment scores (NES) for gene sets related to oxidative phosphorylation and interferon signaling whereas low negative NES were obtained for gene sets related to cell cycling, when compared to CFZ-naïve cells. Confocal images revealed the occurrence of tumor-induced neovascularization as well as the presence of a tumor-associated network of CXCL12 abundant reticular stromal cells which are in close proximity of the myeloma tumor mass. Conclusion In conclusion, MM cells that acquired CFZ resistance within the TME differ significantly from CFZ-naïve cells. CFZ resistance is likely occurring due to various cell-intrinsic and cell-extrinsic alterations, which are yet to be determined. However, the data suggests that increased OXPHOS and reduced cell cycling are likely to be key factors with regards to cellular adaptation towards CFZ exposure in vivo. Confocal imaging of mouse femur cross-sections showed an altered morphology within the TME which is induced presumably by the presence of myeloma cells. Moreover, the formation of new blood vessels in combination with mutual interaction with other BM homing cells is likely to play an important role in PI-resistance in vivo.