Targeted deletion of liver myeloid derived suppressor cells by inhibition of STAT3 dependent survival pathways to rescue CAR-T function

Abstract

Abstract Myeloid derived suppressor cells (MDSC) subvert anti-tumor immunity. Previously we reported that the GM-CSF/ JAK2/STAT3 axis drives liver MDSC (L-MDSC) proliferation and CAR-T suppression. We hypothesized that STAT3 supports L-MDSC survival and suppressive function by inhibiting apoptosis. We treated liver metastasis (LM) in mice with STAT3 inhibitors (STATTIC or BBI) or with vehicle control. STAT3 inhibition caused a significant reduction in tumor burden (p<0.05) and L-MDSC frequency (DMSO 41±3% vs. STATTIC 29±3%/BBI 20±3%, p<0.0001, n=10) in association with lower pSTAT3 levels (DMSO 33±4% vs. STATTIC 11±3%/BBI 9±2%, p<0.0001, n=10). L-MDSC isolated from STATTIC or BBI treated mice were co-cultured with CAR-Ts and corresponding target tumor cells at 1:1:1 ratio. There was a significant decrease in tumor cell density (DMSO 100% vs. STATTIC 71±5%/BBI 20±3%, p<0.05, n=5) and enhancement of tumor cell killing (DMSO 28±4% vs. STATTIC 54±4%/BBI 52±7%, p<0.01, n=5). Rescue of CAR-T tumor killing function correlated with enhanced L-MDSC apoptosis signaling. We detected upregulation of pro-apoptotic proteins Bax, caspase 3, and Fas. Signaling molecules downstream of Fas, JNK and p38 MAPK (p<0.05), were also activated in L-MDSC. In contrast, L-MDSC pro-survival Bcl2, pErk, and pAkt (p<0.05) were downregulated in response to STAT3 inhibition. Microarray results confirmed the STAT3-induced changes in apoptotic and survival gene expression, which was validated by RT-PCR (p<0.05). Within LM, STAT3 inhibition drove L-MDSC apoptosis via the Fas/Fas- L pathway with downstream pro-apoptotic signaling through p38 MAPK. Blocking STAT3 may have clinical application for enhancing immunotherapy for LM.