Targeting TREM1 augments antitumor T cell immunity by inhibiting myeloid-derived suppressor cells and restraining anti–PD-1 resistance

The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.


Targeting TREM1 augments antitumor T-cell immunity by inhibiting myeloidderived suppressor cells and restraining anti-PD-1 resistance
Ashwin Ajith, Kenza Mamouni, Daniel D. Horuzsko, Abu Musa, Amiran K. Dzutsev, Jennifer R. Fang, Ahmed Chadli, Xingguo Zhu, Iryna Lebedyeva, Giorgio Trinchieri, Anatolij Horuzsko  screening of potential TREM1 inhibitors from the NCI Diversity Set was carried out using molecular docking analysis with AutoDock Vina v1.1.12(Supplemental Figure S2A).A scoring system was used to discriminate compounds with the highest estimated affinity for the TREM1 binding site as described previously (1).The top 25 candidate compounds with higher binding mode to TREM1 were selected for additional screening using the TREM1 reporter cell lines stably transfected with TREM1 together with DAP12 and NFAT-driven GFP.In these reporter cells, TREM1 activation induces Ca 2+ -driven signaling for NFAT nuclear translocation and subsequent NFAT-promoted synthesis of GFP, which in turn is detected by flow cytometry (Supplemental Figure S2B).The TREM1 reporter cells were activated by the anti-TREM1 agonistic antibody AF1278 (R&D Systems, AF1278) at a concentration of 4μg/ml for 24 hrs and served as positive control.For TREM1 blocking/inhibition studies, reporter cells were preincubated with each test compound at 50 μM for 6 hrs followed by stimulation with the anti-TREM1 agonistic antibody AF1278 for an additional 24 hrs.TREM1 activation in each blocking and agonist combination could be measured as a GFP signal acquired by an Attune NxT Acoustic Focusing flow cytometry platform (Supplemental Figure S2C).Based on the reporter cell line screening, we identified several candidate compounds exhibiting a TREM1 inhibitory effect and TREM1 agonist activity.From the TREM1 antagonistic compounds, NCI118818 was selected as the most effective TREM1 inhibitor.Additionally, the blocking potency of TREM1 inhibitors (morin hydrate and NCI118818) were assessed via functional assay by measuring the reduction in cytokine production by TREM1-activated human neutrophils during inhibition treatment (Supplemental Figure S2D).For the functional assay, peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and stimulated with anti-TREM1 agonist antibody for 24 hours at 4 μg/ml or were pretreated with the indicated inhibitory compounds (at 50 μM) for 6 hrs followed by activation with anti-TREM1 agonist antibody for 24 hrs (Supplemental Figure S2D).Neutrophils were identified by staining with cell surface markers CD15 (BioLegend, clone: W6D3, 323005, 1:200 dilution) and CD16 (BioLegend,clone: 3G8,980104,1:200 dilution).Intracellular staining for proinflammatory cytokines IL-8 (BioLegend, clone: BH0814, 514604, 1:200 dilution), IL-6 (BioLegend, clone: MQ2-13A5, 501112, 1:200 dilution) and IL-12 (BioLegend, clone: C8.6, 508807, 1:300 dilution) were performed using the Cyto-Fast Fix perm buffer set (BioLegend, 426803) as per the manufacturer's instructions.The percent of cytokine-producing neutrophils from TREM1-activated cells treated with morin hydrate and NCI188818 were acquired on an Attune NxT Acoustic Focusing flow cytometry platform and analyzed using FlowJo v10.0.From the functional assays, we determined that NCI 118818 and morin hydrate exhibited relatively high cytotoxicity on human neutrophils.To decrease the cytotoxic effect of NCI 118818, new chemical modifications had to be implemented onto the compound; the resulting novel small molecular inhibitor VJDT was synthesized by LeadGeneLabs, NJ (Supplemental Figure S2E).Additional details are provided in the patent application.The TREM1 inhibitory effect of VJDT was demonstrated in the reporter cells (Supplemental Figure S2F) and in liver injury experiments (detailed in patent application).Extensive titration studies with these models were carried out to find the optimal concentration of VJDT to be 50 μM for effectively blocking TREM1 activation (detailed in patent application).

Table of Contents
We used this concentration of VJDT for the functional assay in human neutrophils (Supplemental Figure S2G) and all further in vitro and in vivo experiments.The graphical summary determined that VJDT exhibited the strongest TREM1 inhibition in comparison to other inhibitors such as NCI 118818 and morin hydrate.Cell viability assays of VJDT were performed on HepG2, B16F10, U87 and U251 cells as described below (Supplemental Figure S2H).Oral administration of VJDT to Trem1 +/+ mice (Supplemental Figure S2I) exhibited limited liver toxicity as shown by minimal infiltration of immune cells (Supplemental Figure S2J).VJDT administration did not alter body weight (Supplemental Figure S2K) nor induce significant elevation in liver functional enzymes (Supplemental Figure S2L).
Cell viability assay: Cell viability was determined using the 3-( 4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT) and the CellTiter-Glo viability (Promega, G7570) assays (Supplemental Figure S2H).B16F10 and HepG2 cells (10 5 cells/well) were plated in 96-well tissue culture plates in complete medium (100µl/well).The multi-well plates were incubated at 37 0 C, 5% CO2 for 24 hrs.After cell adhesion, the culture medium was removed and equal volumes (100µl/well) of the treatments were added to each well.In vehicle control wells, 100µl of DMSO was added.72 hrs later, proliferative cells were detected by MTT by recording its absorbance at 450nm and via luminescence for the CellTiter assay using a Biotek Synergy multi-well spectrophotometer.The IC50 was calculated using GraphPad Prism 9.
Liver toxicity study: Toxicity studies for VJDT treatment with either intraperitoneal or oral administration for 12 days were carried out as described (Supplemental Figure S2I).Liver tissues were fixed in 4% paraformaldehyde and embedded with paraffin.7 µm sections were stained with hematoxylin and eosinophil (H & E) (Thermo Fisher Scientific).All images were captured by a Keyence BZ-9000 microscope (Keyence).
Serum ALT and AST levels were measured with the calorimetric assays Liquid ALT and Liquid AST (Pointe Scientific, 2366087) according to the manufacturer's recommendation.
Wound healing assay: HepG2 or B16F10 cells (3 × 10 5 cells/well) were seeded in 24-well plates and allowed to grow to monolayers for 72 hrs and 24 hrs, respectively.
A sterile 20-200 μL pipette tip was held vertically to scratch a cross in each well.The detached cells were removed by washing with 500 μL PBS.500 μL of fresh medium with either VJDT (10 μM or 50 μM) or the vehicle (DMSO) were added, and cultures incubated.For HepG2 cells after 24 hrs of treatment, cells were washed with 500 μL PBS, pre-warmed medium added and images collected at 72 hrs.Before image acquisition, the plate was washed with 500 μL PBS before adding pre-warmed medium.The scratch closure was monitored and imaged using a Keyence BZ-9000 microscope (Keyence).
Fluorescent multiplexed immunohistochemistry assay, imaging, and quantitation: Human tumor and corresponding control tissue sections were purchased from US Biomax.These included paired cancer and normal brain (NCT001), cancer and normal liver (NCT046), cancer and normal breast (NCT121) and cancer and normal skin (NCT246) tissues.The sections were deparaffinized for multiplexed staining using the Opal protocol.Per the manufacturer's instructions, paraffin-embedded tumor tissue specimens were heated at 57°C overnight, residual paraffin was removed using xylene, and tissues were rehydrated in a series of graded alcohols to distilled water.Antigen retrieval was performed according to the manufacturer's protocol (Perkin Elmer).Following cooling and washing with TBST, tissue sections were blocked by 30 min incubation with 10% normal goat serum (Jackson ImmunoResearch, 005-000-121).Subsequent multiplexed staining of the tissue sections was carried out using the manual Opal 7-Color IHC Kit (Akoya-Perkin Elmer, NEL811001KT) per the manufacturer's instructions.Tissue sections were then incubated for 1 hr with primary antibody, followed by incubation with Opal polymer HRP Ms + Rb (as secondary antibody).The sections were washed, and the tyramide signal amplification (TSA)-dye (Akoya-Perkin Elmer, NEL811001KT) was applied.The process was repeated for staining with all primary antibodies: CD11b (Novus Biological, NB11089474, 1:500 dilution), CD68 (BioLegend, clone: BL13756, 375602,

Cell proliferation assay:
The five stably transfected HepG2-shTREM1 cell lines and the shControl clones were assayed for cell proliferation with the WST-1 reagent (Sigma-Aldrich, 05015944001) per kit instructions.Briefly, HepG2 cells (4x10 3 cells/well in 200 µl culture medium) were seeded into a 96-well flat-bottom tissue culture plate.Cell proliferation reagent WST-1 (Sigma, 5015944001, 20 µl/well) was added, and cells were incubated at 37 o C in a humidified CO2 incubator.Absorbance was measured repeatedly using a microplate (ELISA) reader at 440 nm wavelength and reference wavelength at 600 nm.The first absorbance was taken 4 hours after incubation and counted as day 0; the subsequent measurements were taken every 24 hours until day 5.
Cell cycle analysis: 10 6 of the stably transfected HepG2-shTREM1 cells and the shControl clones were seeded onto a 6-well plate containing the appropriate culture media.Each clone was treated with VJDT at 10µg or 50µg or DMSO for 24 hours.The cells were harvested by trypsinization, washed twice with PBS, and fixed in ice-cold 70% ethanol.The cells were washed again, subjected to RNase A at a final concentration of 100µg/ml (Qiagen, 19101) and incubated with 50µg/ml propidium iodide (BioLegend, 421301) at 4°C for 15 min in the dark.Samples were acquired on a FACSCanto (BD Biosciences) platform, and cell cycle analysis was performed with FlowJo v10.0.
RNA isolation and reverse transcription-quantitative PCR (RT-qPCR): Total RNA was isolated from tumor specimens and the transfected HepG2 cell cultures using TRIzol reagent (ThermoFisher Scientific, 15-596-026) followed by purification with the RNeasy Mini kit (Qiagen, 74004) per the manufacturers' instructions.Total RNA concentration was measured by NanoDrop spectrophotometer (ThermoFisher Scientific, ND-2000).cDNA was synthesized from 750 ng of total RNA using RT 2 First Strand kit (Qiagen, 330401) per the manufacturer's instructions.RT-qPCR mixes were set up using SYBR Green qPCR Master Mix (Bimake, B21203), and the reactions were carried out in duplicate in a StepOnePlus TM real-time PCR system (Applied Biosystems, 4376600) at 95 o C (3 min) followed by 40 cycles of 95 o C (15 sec), 64 o C (45 sec).Fold change was determined by calculating the ratio of mRNA levels to control values using the Δ threshold cycle (Ct) method (2 -ΔΔCt ).All data were normalized based on the average of three housekeeping genes, ACTB, GAPDH, and HPRT1, and the values are expressed as fold induction in comparison to the analyzed group.Gene expression analysis was performed using specific sets of primers for

Microarray gene expression profiling:
Freshly harvested tumors from the PDX or shTREM1 HepG2 xenografts were processed into single-cell suspensions as described previously, and RNA purity and concentration were evaluated by spectrophotometry using a NanoDrop spectrophotometer (ThermoFisher Scientific).