P-234: Autocrine IL6 signaling in stromal cells in multiple myeloma influences the bone marrow microenvironment

Introduction:
The bone marrow microenvironment can have a profound influence in multiple myeloma (MM) and interleukin-6 is a well-established mediator of these effects. To further investigate the role of IL6 in the interactions between stromal cells (MSCs) and MM cells we used CRISPR/Cas9 to delete IL6 from the HS5 MSC line and determine effects on co-culture-induced protection.

Methods:
IL6 gene editing was confirmed by NGS and ELISA. In co-culture (CC) and conditioned media (CM) assays, MM cells were preincubated for 30-60 min before addition of drug or T cells. Cell death was measured using Annexin V staining. MM cells were identified by CD38 staining, HS5 by mCherry and T cells via CD3. RNA isolated from HS5 Cas9 and IL6KO cells was analyzed by RNAseq. STAT3 phosphorylation was determined by Western blot.

Results: IL6KO HS5 cells do not offer protection against venetoclax-induced cell death in tested human myeloma cell lines (HCML). We also tested the effects of CC on BCMA CAR-T cell killing. We found that CC could protect HCMLs from CAR-T killing and this protection was not significantly different with CC of IL6KO cells. Results with CM from Cas9 and IL6KO cells were similar, suggesting that soluble factors independent of IL6 can protect MM cells from BCMA CAR-T killing. To identify these soluble factors, we performed RNAseq comparing the HS5 Cas9 and IL6KO cells. We found 1637 genes were differentially expressed (633 significantly up- and 1004 down-regulated in IL6KO cells, FDR< 0.01). GSEA indicated that gene sets associated with inflammatory and IFNα signaling were upregulated in IL6KO cells and several growth and survival cytokines were downregulated. Interestingly IL6R is upregulated. We confirmed that HS5 cells express IL6R by flow cytometry and that it is upregulated in IL6KO cells. We also observed IL6R on MSCs cultured from a patient sample. Expression of IL6 signaling component genes IL6ST, JAK1 and STAT3 along with target gene SOCS3 was also detected, suggesting that IL6R may signal in HS5. Control and IL6KO cells express similar levels of total STAT3 protein, however phospho-STAT3 was lost in IL6KO cells. For validation in patient samples we queried bulk RNAseq from MSCs purified from MM patients and confirmed that IL6R, IL6ST, JAK1 and STAT3 were expressed in cells isolated from normal, newly diagnosed and post-induction therapy patients. Finally, previous scRNAseq has indicated that MSCs fall into 5 unique transcriptional clusters, with 2 of these clusters unique to MM patients (De Jong et al., Nature Immunology, 2021). We find that like IL6 itself, IL6-regulated genes are enriched in myeloma-induced MSCs.

Conclusions:
These data indicate that myeloma-induced MSCs utilize IL6 in an autocrine-dependent fashion to produce factors that can influence the bone marrow microenvironment, suggesting blocking IL6 or IL6R could influence therapeutic response by reprogramming the microenvironment.