Mechanistic Investigation Of Nap1051, A Lipoxin A4 Biomimetic, In Treating Colorectal Cancer.

Abstract Purpose: Resolving tumor-associated inflammation in the tumor microenvironment (TME) can restore the immune response against malignant tissue. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. We developed a LXA4 biomimetic, NAP1051, with increased stability and longer half-life, and demonstrated its properties using in vitro assays and in vivo xenograft tumor models and explored the underlying mechanism of action (MoA). Methods: Differentiated HL-60 and THP-1 induced by DMSO and PMA, respectively, were used as models. dHL-60 was used to determine the effect of NAP1051 on fMLP-induced neutrophil chemotaxis. NAP1051-induced dTHP-1 efferocytosis of apoptotic dHL-60 was measured using fluorescent microscopy. Concentration escalation and time of NAP1051 exposure were used to probe the molecular mechanism using Western Blot and RT-PCR. The efficacy of oral NAP1051 was evaluated in CT26 (CRC) xenograft model established in immunocompetent Balb/c mice with a dosage escalation design. Results: NAP1051 inhibited neutrophil chemotaxis towards fMLP by > 40% at 1, 10 and 100 nM (p < 0.05). NAP1051 dose-dependently promoted dTHP-1 efferocytosis (p < 0.05) and was equipotent to ATLA4. In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT from 10 nM to 1 µM. When compared to ATLA4 and W-peptide, NAP1051 caused strong phosphorylation at both S473 and T308 of AKT. The NAP1051-induced p-ERK1/2 and p-AKT were in parallel and in a dose-dependent and time-dependent way. MoA studies revealed that such p-ERK1/2 was mediated by MEK1/2. However, NAP1051 led to p-AKT at both S473 and T308 despite PI3K inhibition, indicating a PI3K-independent pathway was involved. In the mouse xenograft CRC model, we demonstrated that NAP1051 significantly inhibit tumor growth when given p.o. at 5 mg/kg/day. Flow cytometry on splenic cells showed that NAP1051 reduced splenic neutrophil and MDSC populations, which correlated to the tumor sizes. IHC analyses revealed that NAP1051 decreased the intratumoral Ly6G+ neutrophils most significantly at 5 mg/kg/day (p<0.1). In combination with low-dose 5-FU and CTX, NAP1051 significantly decreased splenic neutrophils and MDSCs compared to chemotherapies alone (p<0.05). Conclusions: NAP1051 has LXA4-like activity including inhibiting fMLP-mediated neutrophil chemotaxis and promoting macrophage efferocytosis of apoptotic cells. The molecular MoA of NAP1051 involves ERK1/2 and AKT signaling pathways which are interrelated closely. A PI3K-independent pathway may be activated and resulted in AKT activation. NAP1051 inhibited tumor growth significantly at 5 mg/kg/day in a CRC model with significant changes in splenic and intratumoral immune cell populations. With these properties, NAP1051 may be an effective component in the treatment of CRCs by modulating the inflammatory immune cells in the TME. Citation Format: Tiange Dong, Priyal Dave, Brandon Ebright, Kabir Ahluwalia, Eugene Zhou, Isaac Asante, Malika Salimova, Hua Pei, Tracey Lin, Andrew Mead, Zeyang Li, Nicos Petasis, Stan Louie. Mechanistic investigation of NAP1051, a lipoxin A4 biomimetic, in treating colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1436.