Unexpected PD-L1 Immune Evasion mechanism in TNBC, Ovarian and other Solid Tumors by DR5 Agonists (2020)
Figure 4

Figure 4. DR5 agonist activated ROCK1 functions to help PD-L1 surface mobilization

(A, B) KMTR2, KMTR2+Z-VAD and lexatumumab treated MDA-MB-436 and OVCAR3 (respectively) lysates for indicated early time points were analyzed for caspase-8, caspase-3, ROCK1, pMLC, PARP, PD-L1 and CMTM6 etc. as indicated.

Red arrows indicate sequential kinetics of caspase-8, ROCK1 and caspase-3 activation, blue arrows indicate cleaved and activated caspase-8, ROCK1 and caspase-3 and black arrows indicates nonspecific (n.s.) band by ROCK1 antibody.

(C) OVCAR3 cells were treated with pMLC activating ionophore A23187 (positive control) and lexatumumab ± ROCK1 inhibitors or ± rDR5 or ± rFOLR1. Lysates were later analyzed for ROCK1, pMLC, caspase-3 and PARP with GAPDH as loading control. Blue arrows indicate cleaved and activated ROCK1 and caspase-3.

(D) MDA-MB-436 and OVCAR3 cells were treated with indicated ROCK1 inhibitors 2 hours prior to DR5 agonist KMTR2 treatment (20nM). After 6 hours flow cytometry was used to analyze surface PD-L1. (GSK269: GSK269962A, GSK429: GSK429286A)

(E) MDA-MB-436 cell survival assay after treatment with DR5 agonist antibody KMTR2 ± ROCK1 inhibitors, error bars indicate SD (n=2). (GSK269: GSK269962A, GSK429: GSK429286A)

(F) Schematic of immunoprecipitation assay shown in G and H. Cultured MDA-MB-436 cells were treated with KMTR2 (IgG1-Fc) or IgG1 control ± ROCK1i (GSK269962) for 6 hrs. After 6 hrs, 500nM anti-PD-L1 avelumab (IgG4-Fc) was added to the media for additional 1 hr. Cellular lysates were pulled down with anti-hu-IgG4 specific beads.

(G) Immunoprecipitated lysates and leftover supernatant after various treatments as described in (F) were run at the same time followed by blotting with ROCK1, PD-L1 and CMTM6. Total lysates as a control were also generated in exactly similar conditions and run next to supernatant and IP samples. Blue arrows indicate cleaved and activated ROCK1.

(H) Immunoprecipitation assays with anti-PD-L1 (avelumab) same as G using OVCAR-3 cell lines. Blue arrow indicates cleaved and activated ROCK1.

(I) After treatment with IgG1 control and indicated DR5 agonists (KMTR2, Lexa, Tigatuzumab) ± ROCK1 inhibitors (GSK269962A, GSK429286A) tumor cells were co-cultured with anti-CD3 stimulated PD1+ effector Jurkat cells (stably expressing luciferase under NFAT-RE promoter). Relative luciferase signal was quantified and plotted after subtraction of background signal from untreated cells.

(J) Relative luciferase activity from assay as described in (I). Data is from 4 different DR5 agonist antibodies used in combination of two different ROCK1i inhibitors (n=3).

(K) Tumor regression efficacy of MD5-1 (50μg), GSK269962A (<2mg/kg) and MD5-1+ GSK269962A (50μg + 2mg/kg) 4T1 tumors (n=4-7). 6-8 weeks old BALB/c mice bearing 4T1 tumors (~100mm3) were intraperitoneally (i.p.) injected with 50μg of indicated antibody every third day (n=4-6). GSK269962A (ROCK1i) was injected directly into the tumors. Tumor volumes were quantified at indicated days by caliper measurements.

Data information: Error bars in (J) represent SD. Statistical significance in (J) was determined by unpaired student t-test and in (K) using two-tailed paired Wilcoxon Mann-Whitney test. (*p <0.05, **p <0.005)