P-251: Bone Marrow Immune Ecosystem Shapes Acquired Daratumumab Resistance in Plasma Cell Myeloma

Introduction: Daratumumab (dara), a monoclonal antibody to CD38, is an effective therapy of multiple myeloma. However, most initial responders relapse or progress. Several possible mechanisms underlying this have been described, but the potential impact of changes in the bone marrow immune ecosystem are still poorly-defined.

Methods: Here, we studied the cellular composition of the bone marrow immune ecosystem associated with loss of response to daratumumab therapy, using scRNA-seq, DSP technology, validated by PrimeFlow RNA flow cytometry and in vitro and in vivo experiments.

Results: We compared the differences in the percentage of T-cells before therapy and after acquired resistance. Variations were further examined using PrimeFlow in another cohort. Higher GZMK MFI expression in CD8+ T-cells was correlated with anti-CD38 treatment resistance but decreased expression in subjects with a complete response, while CCR7 showed the opposite trend. Three of the predominantly terminal CD8+ T-cell subsets identified by trajectory analysis had higher transcriptional signatures for cytotoxicity and exhaustion. CD8+ T-cells had an increased exhausted signature, increased cytotoxic signature and decreased naïve signature upon resistance. Expression levels of most checkpoint markers increased after acquiring resistance. We also analyzed B-cells, myeloid cells and NK/NKT-cells phenotype alterations. Then we focused more on plastic plasma cells. Neoplastic plasma cells clusters 2, 5, 7 and 8 increased in numbers after acquiring dara resistance. Combined with GO/KEGG enrichment analysis, we found cluster 2, 5, 7 and 8 characterized by different pathways. CD138+ and CD45+ cells marked in the DSP were also evaluated and adjusted based on H&E and IHC staining. The ssGSEA algorithm based on the altered immune subset signature showed signatures of various cells decreased in the cancer centre regions. Plasma cell cluster 8 was elevated in parallel with acquiring dara resistance. Resistance associated neoplastic plasma cell cluster 8 signatures were also prognostic in 4 databases. We integrated module analysis and GO/KEGG enrichment analysis and found that MYC regulon was identified as the key. We further studied whether IFN-γ produced by consistently activated cytotoxic immune cells activates MYC associated with acquired resistance. IFN-γ exposure stimulated MYC expression and increased phosphorylation of MYC in two plasma cell myeloma cell lines. MYCi975 reversed resistance to dara-mediate ADCC. After giving dara and NK-cells mean FLuc signal of MYC OE cohort mice was significantly higher compare with NC cohort. Combining MYCi975 and dara reversed acquired dara resistance in MYC OE mice.

Conclusions: Mechanisms underlying acquired daratumumab resistance are complex involving crosstalk between immune cells and neoplastic plasma cells. Increased activation of MYC may be an important mechanism promoting acquired daratumumab resistance.