(OA-64) Evidence for Pre-Existing Myeloma Cells with a Gene Expression Pattern Associated with Resistance to BCMA CAR T Cells.

Introduction: Targeting BCMA has shown promise in multiple myeloma (MM), but the durability of responses remains limited. To identify mechanisms of resistance to BCMA CAR T cells, we performed Spatiotemporal Genomic Analysis (SaGA) on myeloma cells engaged by CAR T cells but not killed in culture, followed by RNAseq.


Methods:

We introduced Dendra2 into KMS12PE, KMS18, KMS26, KMS27, RPMI8226 and cultured them with BCMA- or CD19-CAR T cells (1:1) for up to 20 h. Cells that were engaged for 16 hours and alive, were photoconverted and sorted for RNAseq.  Differentially expressed gene gene sets from these data as well as from differentially expressed genes on day 28 post CAR T infusion (Dhodapkar, BCD, 2022) were then used to find pre-existing cells.



Results:

Engagement times leading to cell death in the 5 cell lines ranged from 5.48-9.42h, but a proportion of cells survived (24.7% on average, range 3.8-42.9%). We repeated the experiment with CD19 CAR T cells in KMS18 and KMS12PE and the total time of engagement of live cells was reduced from 12.49 and 14.63h to 5.39 and 2.74h respectively. Importantly with BCMA CAR T the average and total time of engagement was nearly identical for all cells, indicating stable engagement. In contrast for CD19 CAR T the average time of engagement was less than 30min. To explore the role of resistance in engagement time, KMS18 cells lacking CD95 and overexpressing Serpin B9 were tested and showed increased viability (28.8% to 88.3%) and longer engagement (53.3% to 83.0% engaged for 16h). RNAseq of photoconverted cells from KMS12PE and KMS26 revealed 1101 commonly upregulated genes (padj< 0.01, FC >2), and top gene ontology hits included "integrin signaling" and "inflammation by chemokine and cytokine signaling." The latter was related to upregulation of IFNg-responsive genes (e.g., CXCL10, GBP4, IL6). Down-regulated genes were associated with cell death (e.g., TNFRSF10B, PMAIP1, BBC3) and unfolded protein response (e.g., ATF4, ERN1, DDIT3).  This is consistent with reports of residual cells post-BCMA CAR T (Dhodapkar, BCD, 2022) and in vitro selection of resistance to BCMA T cell engagers (Lee, ASH, 2024). In these studies, the selection time was 4 weeks, in this present study it was 16 h, suggesting selection of pre-existing cells.  To determine if similar cells existed in patients pre-CAR T infusion, we determined the top 100 down-regulated genes 28 days post-CAR T and if there were cells at day 0 where gene expression correlated (>0.8).  We found that a subset of day 0 cells in each patient had a day 28-like expression pattern.  We performed a similar analysis using the top 100 down-regulated genes from the resistant KMS12PE (correlation >0.85) and the findings reflected the day 28 signature.



Conclusions:

These data suggest that MM cells resistant to BCMA-targeted therapies are pre-existing in patients. These cells can be detected by single cell analysis and could be used to inform treatment decisions.