Angiogenin predicts an exceptional response to TGFβ inhibition in pancreatic ductal adenocarcinoma by sustaining the recruitment of Tnf-alpha-secreting M2-polarized macrophages

The inhibitor of TGFβ receptor galunisertib demonstrated a positive signal of efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the randomized phase 2 H9H-MC-JBAJ study. However, the identification of biomarkers that could predict which patients would benefit more from this strategy remains of unique importance. We recently used a 279 multi-analyte panel to measure plasma proteins at baseline and during treatment in patients enrolled in the H9H-MC-JBAJ study. We identified different factors as predictive markers useful to select patients who may benefit from TGFβ receptor inhibition. Among them, Angiogenin was one the most significant biomarkers for chemoresistance in the population of patients receiving single agent gemcitabine [mOS (95%CI), high vs low, 5.7 (3.0-7.7) vs 12.5 (5.5-23.8) months, HR (95% CI) = 2.73 (1.50-23.8); p<0.05]. On the contrary, evidence showed galunisertib dramatically reverted this resistance and Ang resulted as a positive predictive marker for the combination of this agent plus chemotherapy [mOS (95% CI), galunisertib + gemcitabine vs. placebo + gemcitabine, 9.0 (5.5-13.7) vs 5.7 (3.00-7.7) months, HR (95% CI) = 0.55 (0.33-0.90); p<0.05]. Here, we explored the mechanisms responsible for the exceptional response to the inhibition of TGFβ signaling pathway of PDAC characterized by this prognostically negative ecotype sustained by elevated levels of Angiogenin. Among five different murine PDAC orthotopic models, mice bearing high-Ang PDAC tumors had a shorter mOS duration and a significantly higher infiltration of M2-polarized tumor-associated macrophages (TAMs) if compared with tumors from mice with undetectable levels of serum Ang. By using co-culture models of PDAC cells with a murine macrophage cell line (RAW264.7), we showed that tumor-derived Ang binds to the receptor PlexinB2 on macrophages, inducing their recruitment. The expression of Tgfβ1 ligand and the phosphorylation of Smad2 were triggered only in TAMs if co-cultured with Ang-high FC1245scr or DT4313Ang, and not with Ang-low FC1245shAng or DT4313NTC controls. We also demonstrated that autocrine TGFβ triggered the M2-polarization and induced the secretion of the cytokine Tumor Necrosis Factor (Tnf)-a from macrophages. In orthotopic models, inhibition of TGFβ signaling by using the second-generation TGβRI inhibitor LY3200882 acted on TAMs by suppressing their autocrine TGFβ signaling, which redirected their polarization toward an M1 phenotype and inhibited their production of Tnf-a. By combining in vitro and preclinical models, we also provided evidence that Tnf-α-secreting macrophages sustain the resistance to classical chemotherapeutic drugs. Indeed, administration of recombinant Tnf-a was able to fully revert the positive response of high-Ang PDAC cells to the combination of LY3200882 plus chemotherapy in co-culture models. To strengthen our observations, we measured the circulating levels of TNF-α in the plasma of 20 patients of the randomized phase 2 H9H-MC-JBAJ study collected in our clinical unit at baseline and after 60 days of treatment with gemcitabine plus placebo or gemcitabine plus galunisertib. We found a significant correlation between plasmatic ANG and TNF-α (Pearson coefficient R=0.469; p=0.0368). Nonetheless, we measured a significant difference in median progression-free survival duration between high-ANG patients with decreased levels of TNF-α and those who had no difference in plasmatic level of TNF-α within the experimental arm of gemcitabine plus galunisertib (mOS TNF-α decrease vs TNF-α not decrease, 14 vs 7 months; HR (95%CI)= 2 (0.579-6.909) vs 0.5 (0.1447 vs 0.727); p=0.037). In conclusion, we demonstrate the existence of a paracrine Ang-TGFβ axis that sustains the recruitment of Tnf-a-secreting M2-polarized TAMs in the tumor site, leading to chemoresistance. This mechanism could help to explain the exceptional response to galunisertib observed in patients with high-Ang PDAC.