SUMMARY Autotaxin (ATX; ENPP2 ) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1–6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8 + T cells ex vivo , with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα 12/13 -coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8 + T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8 + T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival (OS) and a dramatic improvement in patient quality of life. Despite the success of this approach, the number of benefitting patients is limited and there is a need for predictive biomarkers as well as a deeper mechanistic analysis of the cellular populations involved in clinical responses. With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients receiving pembrolizumab or nivolumab treatment at Karolinska University Hospital. Blood samples were collected before treatment and before administration of the second and fourth doses. Peripheral blood mononuclear cells were isolated and stained for flow cytometric analysis within 2 h of sample collection. Overall survival and progression-free survival (PFS) were inversely correlated with CD69 expression NK cells. In the myeloid compartment, high frequencies of non-classical monocytes and low frequencies of monocytic myeloid derived suppressor cells (MoMDSCs) correlated with response rates and OS. A deeper characterization of monocytic subsets showed that PD-L1 expression in MDSCs, non-classical and intermediate monocytes was significantly increased in patients with shorter PFS in addition to correlating inversely with OS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of activated NK cells and monocytic subsets are inversely correlated with survival and clinical benefit, suggesting that their role as predictive biomarkers should be further evaluated.

To improve immunotherapy efficacy, a better understanding of the factors that regulate Tcell migration into tumors is essential. Here we uncover a role for autotaxin (ATX) in this process. ATX (encoded by ENPP2) produces lysophosphatidic acid (LPA) that activates G protein-coupled receptors (LPAR1-6) to regulate multiple (patho)physiological processes, including tumor progression via LPAR1 and lymphocyte homing via LPAR2.Unexpectedly, we find that melanoma cell-secreted ATX is a major chemorepellent for tumor-infiltrating lymphocytes ex vivo through Gα12/13-coupled LPAR6, with ATX functioning as an LPA-producing chaperone. Using an anti-cancer vaccination model, we provide proof-of-concept that secreted ATX opposes tumor infiltration of CD8+ T cells.Additionally, ENPP2 expression in melanoma tumors correlates with reduced CD8+ T-cell infiltration as inferred from single-cell transcriptomics. Hence, by counteracting T-cell infiltration while activating tumor cells via different LPA receptors, the ATX/LPA complex exerts dual actions in the tumor immune microenvironment, which may provide new therapeutic approaches.

Cancer stem cells (CSCs) are located in dedicated niches, where they remain inert to chemotherapeutic drugs and drive metastasis. Although plasticity in the CSC pool is well appreciated, the molecular mechanisms implicated in the regulation of cancer stemness are still elusive. Here, we define a fucosylation-dependent reprogramming of colon cancer cells towards a stem cell-like phenotype and function. De novo transcriptional activation of Fut9 in the murine colon adenocarcinoma cell line, MC38, followed by RNA seq-based regulon analysis, revealed major gene regulatory networks related to stemness. Lewisx, Sox2, ALDH and CD44 expression, tumorsphere formation, resistance to 5-FU treatment and in vivo tumor growth were increased in FUT9-expressing MC38 cells compared to the control cells. Likewise, human CRC cell lines highly expressing FUT9 displayed phenotypic features of CSCs, which were significantly impaired upon FUT9 knock-out. Finally, in primary CRC FUT9+ tumor cells pathways related to cancer stemness were enriched, providing a clinically meaningful annotation of the complicity of FUT9 in stemness regulation and may open new avenues for therapeutic intervention.

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Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival, and a dramatic improvement in patient quality of life. Despite the success of this therapeutic approach, the number of responding patients is limited and there is a need for predictive biomarkers and a deeper mechanistic analysis of the cellular populations involved in a clinical response. With this objective in mind, an in-depth immune monitoring study was conducted in advanced melanoma patients undergoing treatment with pembrolizumab or nivolumab. 29 patients receiving pembrolizumab (n=5) or nivolumab (n=24) treatment at Karolinska University Hospital were included in this study. Blood samples were collected from patients at the following time points: Before treatment and at the time of the second and fourth doses. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained for flow cytometric analysis within two hours of sample collection. In the 29 patients included, median OS was 884 days from the start of treatment. Adverse events were observed in 18 patients (62%), including three grade 3-4 (10%). Patients were classified according to their response as PD (progressive disease, 45%), SD (stable disease, 3%) MR (mixed response, 7%) PR (partial response, 31%) and CR (complete response, 14%). For analytic purposes, patients were divided into two groups: 19 (59%) patients with clinical benefit (includes responders and patients with stable disease) and no clinical benefit (12 patients with progressive disease). Patients with clinical benefit had a median overall survival (MOS) of 1013 days, significantly longer than the 303 day MOS in the no clinical benefit group. After analyzing the patients PBMC with a broad flow cytometry panel that included major immune subsets (T cells, NK cells, dendritic cells and myeloid derived suppressor cells (MDSCs)), our results showed that, before treatment, the frequency of two populations is related to clinical benefit: On one hand, patients with clinical benefit had significantly lower frequencies of monocytic MDSCs. On the other hand, NK cell frequencies were higher in these patients. Furthermore, Kaplan Meier analysis showed that patients with high frequencies of NK cells had significantly longer MOS times when compared to patients with low NK cell frequencies. Similarly, patients with low frequencies of monocytic MDSCs, showed significantly better MOS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of NK cells and monocytic MDSCs are correlated with survival and clinical benefit and their role as predictive biomarkers should be further evaluated. Citation Format: Yago Pico De Coaña, Maria Wolodarski, Irene van der Haar Avila, Giuseppe V. Masucci, Johan Hansson, Rolf Kiessling. Myeloid derived suppressor cells and NK cells are correlated with clinical benefit and survival in advanced melanoma patients treated with PD-1 blocking antibodies nivolumab and pembrolizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-116.