Combined MEK and STAT3 inhibition reprograms stromal inflammation to overcome immunotherapy resistance in pancreatic cancer

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by immune exclusion, stromal desmoplasia, and resistance to immune checkpoint inhibition (ICI). We have previously demonstrated that reciprocally activated RAS/RAF/MEK/ERK and JAK/STAT3 pathways mediate therapeutic resistance, while combined MEK and STAT3 inhibition (MEKi/STAT3i) overcomes this resistance in PDAC. Herein, we show that combined MEKi/STAT3i alters stromal architecture and remodels the tumor-infiltrating innate and adaptive immune compartments by downregulating immunosuppressive myeloid populations and promoting T-cell enrichment and activation in Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mice. MEKi/STAT3i-mediated reprogramming of the PDAC tumor microenvironment primes tumor-infiltrating CD8+ T-cells for PD-1 blockade to augment their activation and functional cytotoxicity. As such, the addition of MEKi/STAT3i to PD-1 blockade dramatically inhibits tumor growth and prolongs survival, overcoming resistance to ICI, in PKT mice. Importantly, treatment of a patient with chemotherapy-refractory metastatic PDAC with MEKi (Trametinib), STAT3i (Ruxolitinib), and PD-1 inhibitor (Nivolumab) was not only well-tolerated but also yielded significant clinical benefit. These data uncover a novel paradigm in which combined MEKi/STAT3i reprograms the stromal and immune microenvironment to overcome immunotherapy resistance in PDAC. The clinical efficacy of combined MEKi, STAT3i, and anti-PD1 treatment in chemotherapy-refractory PDAC provides encouraging signals for its translatability, and is being currently pursued in a clinical trial.

Neutrophil-Mediated Stromal-Tumor IL-6/STAT-3 Signaling Underlies the Association between Neutrophil-to-Lymphocyte Ratio Dynamics and Chemotherapy Response in Localized Pancreatic Cancer: A Hybrid Clinical-Preclinical Study

“…C57BL/6 mice were then orthotopically injected with 50×10 3 KPC6694c2 cells and randomized into four groups starting 10 days after tumor inoculation (n=8-10/arm): vehicle control, NLR-attenuating anti-Ly6G alone (25 μg/dose) q3 days starting day 10, gemcitabine (100 mg/kg) and paclitaxel (10 mg/kg) once weekly starting day 14, and gemcitabine/paclitaxel treatment (day 14) following a “priming” phase of anti-Ly6G attenuation starting day 10 (for details, see Appendix 1 ). Mice were sacrificed following 3 weeks of treatment, tumor burden and metastatic outgrowth evaluated, and tumor samples subjected to histological analysis, immunohistochemistry (Ly6G/Gr1, phosphorylated STAT3, cleaved caspase-1, and CD31), flow cytometric CAF and immune phenotyping, and enzyme-linked immunosorbent assay (ELISA; IL-6 and IL-1β) [12].…”

“…Quantitative polymerase chain reaction (qPCR) was performed using incubation with II6 and Cxcl1 mouse gene-specific predesigned primers (RT2 qPCR Primer Assay, Qiagen) and iQ SYBR Green Supermix (Bio-Rad). Gene expression was normalized to the housekeeping gene GAPDH using the ΔΔCT method and reported as fold change relative to control. KPC6694c2 tumor cells were incubated with conditioned media harvested from ex vivo co-cultures of intratumoral neutrophils and CAFs as described above, either alone or with anti-IL-1β or anti-IL-6 neutralizing antibodies (Thermofisher, Waltham, MA), and ensuing protein lysates blotted for phosphorylated STAT-3 (pSTAT3) and total STAT-3, as described previously [12]. Total STAT3 (124H6) and pSTAT3 (Y705) antibodies were purchased from Cell Signaling Technology (Denver, MA).…”

“…The following day, slides were washed and developed using VECTASTAIN R Elite ABC-HRP based kit (Vector) as per the manufacturer’s protocol with diaminobenzidine (DAB) as the chromogen. Tissue sections were counterstained with Mayer’s hematoxylin, mounted, and imaged. Quantitative histological analysis was performed by sampling multiple random, non-overlapping 20x fields in each tissue section (n=5 mice per group) and quantified using ImageJ Fiji software (NIH) to measure the number of positive cells per field, as described previously [12].…”

“…Cell suspension of whole pancreas obtained from KPC orthotopic mice (same procedure as described above) underwent magnetic column separation to obtain Ly6G + cells as per mouse Myeloid-Derived Suppressor Cell Isolation Kit manufacture’s protocol (Miltenyi Biotec). Ly6G + cells were then co-cultured with KPC CAFs [9,12] in standard 6-well plates. The conditions of the experiment were: (1) CAFs alone (2×10 5 cells per well); (2) CAFs + intra-tumoral Ly6G + cells (10 6 cells per well)’ (3) CAFs pre-treated with Anakinra (α-IL-1R1 inhibitor; with license from SOBI Pharmaceuticals, Sweden) 50 μg/mL + intra-tumoral Ly6G + cells; and (4) CAFs + intra-tumoral Ly6G + cells treated with anti-IL-1β neutralizing antibody 10 μg/mL (R&D Systems cat# AF-401-NA).…”

“…Flow cytometric analysis was performed as described previously [9,12]. Briefly, whole pancreata harvested from C57BL/6 mice were digested enzymatically utilizing a solution containing 0.6 mg/ml of collagenase P (Roche), 0.8 mg/ml Collagenase V (Sigma Aldrich), 0.6 mg/ml soybean trypsin inhibitor (Sigma Aldrich), and 1800 U/ml DNase I (Thermo Scientific) in RPMI medium for 20-30 minutes at 37°C.…”