P-001: The bone marrow stroma influences extrinsic apoptotic signaling and results in resistance to BCMA CAR-T cell induced cell death

Introduction: BCMA targeted CAR-T therapy is effective at inducing disease remission in heavily pretreated relapsed refractory Multiple Myeloma (MM) patients with a cumulative overall response rate of 85.2%, however, duration has been disappointing. Bone marrow microenvironment (BMM)-derived IL6 is a survival signal for MM cells and is known to promote drug resistance. CAR-T therapy induces an immune response, resulting in increased IL6. With this in mind, we investigated the impact of the BMM and IL6 on CAR-T induced cell death in MM.

Methods: MM cell lines were exposed to BCMA-CAR-T cells or CD95L for 24 hours and cell death measured via Annexin V staining. CD3, CD38, and mCherry expression were used to distinguish T cells, MM cells, and HS-5 stromal cells respectively. In stromal coculture (SCC) and conditioned media (SCM) assays, MM cells were preincubated for 1 hour before T cell addition. Caspase 8 activity was measured with an IETD-FMK fluorometric assay.

Results: SCC protected all 3 MM cell lines tested from CAR-T induced cell death while SCM protected 2 of the 3 cell lines. CAR-T CD107a surface expression remained consistent between control and SCC/SCM groups indicating no change in T cell activation. Gene editing to ablate CD95 expression protected 3 of 4 MM cell lines demonstrating its importance in CAR-T induced cell death. Therefore, to determine the mechanism of protection we focused on the effects of SCC, SCM, and IL6 on rCD95L killing. All were sufficient to protect myeloma cells from rCD95L. Loss of mitochondrial-mediated apoptosis through the deletion of BID or BAX/BAK protected 2 of 4 cell lines from rCD95L, indicating that rCD95L induces type 1 (mitochondria-independent) or type 2 (mitochondria-dependent) death in a cell line specific manner. When type 2 cells deficient in mitochondrial apoptosis were cultured in SCM and exposed to rCD95L, there was no enhancement of protection beyond SCM alone. In contrast, when type 1 cells deficient in mitochondrial apoptosis were exposed to rCD95L, the combination of SCM and BAK/BAX DKO protected the type 1 cells beyond SCM alone suggesting an effect upstream of the mitochondria. Consistent with this possibility, SCM inhibits CD95L induced activation of caspase 8 in both type 1 and type 2 cells lines.

Conclusions: These results demonstrate that the BMM can induce MM cell intrinsic protection against CAR-T therapy in part due to soluble stromal factors. This protection may be due in part to inhibition of the extrinsic apoptotic pathway. Stromal factors including IL6 inhibit rCD95L induced type 1 induced cell death through a reduction in Caspase 8 activity. We hypothesize that reduced caspase 8 activity changes a type 1 cell death signal to a type 2 death signal that can be inhibited by anti-apoptotic BCL2 family.