A Potential Role of NK Cells and ABCB9 in Modulating the Response to Melflufen in Multiple Myeloma

Introduction Melphalan flufenamide (melflufen), is a novel peptide-drug conjugate that delivers alkylating agents in tumours upon hydrolysis by aminopeptidases. Melflufen was recently approved by the EMA for the treatment of relapsed/refractory multiple myeloma (MM). Considering the challenges in treating this group of patients and their low overall survival, information that can support treatment selection and success is urgently needed. To discover possible biomarkers of melflufen sensitivity, we applied a multiparametric drug sensitivity assay to MM patient samples ex vivo and analysed the samples by single cell RNA sequencing (scRNAseq) and flow cytometry. Drug testing identified groups of MM samples with high and low sensitivity to melflufen, while differential gene expression and flow cytometry analysis revealed markers associated with response and cytotoxicity.

Methods Bone marrow mononuclear cells from 12 newly diagnosed (ND) and 12 relapsed/refractory (RR) patients were used for multi-parametric flow cytometry-based drug sensitivity and resistance testing (DSRT) evaluation to melflufen and melphalan, and for scRNAseq after written informed consent following approved protocols in compliance with the Declaration of Helsinki. Based on the results from the DSRT assay and drug sensitivity scores (DSS), we divided the samples into three groups - high sensitivity (HS, DSS > 40 (melflufen) or DSS > 16 (melphalan)), intermediate sensitivity (IS, 31 ≤ DSS ≤ 40 (melflufen) or 10 ≤ DSS ≤ 16 (melphalan)), and low sensitivity (LS, DSS < 31 (melflufen) or DSS < 10 (melphalan)). To identify genes, responsible for the general sensitivity to we conducted differential gene expression (DGE) analyses between HS and LS samples for both drugs ("HS vs. LS melphalan” and "HS vs. LS for melflufen", respectively). In addition, we conducted flow cytometry analysis to evaluate the levels of HLA-B protein, associated with increased cytotoxicity of NK cells.

Results We analyzed the DSRT and scRNAseq data for the MM samples of different melflufen sensitivity groups (HS, IS, LS). DGE analysis showed significantly lower levels of the ABCB9 gene in plasma cells populations of the HS compared to LS group (2.2-fold, HS vs. LS groups for melflufen, p < 0.01). ABCB9 belongs to a family of proteins known for drug efflux and multidrug resistance. A similar pattern was detected for the melphalan HS vs. LS (3.2-fold, p < 0.01) comparison suggesting that this alteration might be a common indicator of sensitivity to melflufen and melphalan. As a confirmation, we found that MM cell lines treated with melphalan (Fig. 1) also tend to express higher levels of ABCB9, as compared to control cells (1.2-fold, p<0.001), indicating its potential role in drug resistance. Based on scRNAseq and flow cytometry analysis, we also discovered that NK cells may be associated with melflufen cytotoxic effect. Our results demonstrated higher levels of NK and NKT subpopulations, as well as lower surface HLA-B protein level (p<0.01) and activation of NFKB signalling in the melflufen HS group in comparison to the LS group (Fig. 2). The latter two features are associated with increased NK cells cytotoxicity, thus, suggesting synergistic effect of innate immunity with melflufen.

Conclusion In summary, we demonstrate the association of ABCB9 expression with resistance both to melphalan or melflufen. Furthermore, we observed that MM samples that were highly sensitive to melflufen exhibited markers associated with NK-mediated cytotoxicity, suggesting a potential role of NK cells for enhancing the efficacy of melflufen.

Slipicevic:Oncopeptides: Ended employment in the past 24 months. Nupponen:Oncopeptides: Ended employment in the past 24 months. Acs:Oncopeptides: Current Employment. Lehmann:Oncopeptides AB: Current Employment, Current equity holder in private company. Heckman:Oncopeptides: Research Funding; Novartis: Research Funding; WntResearch: Research Funding; IMI2 projects HARMONY and HARMONY PLUS: Research Funding; Orion: Research Funding; Kronos Bio: Research Funding; Celgene: Research Funding.