anti-PD1 Efficacy Research Articles

Radiotherapy-activated NBTXR3 nanoparticles promote ferroptosis through induction of lysosomal membrane permeabilization

PurposeRadiotherapy-activated NBTXR3 (NBTXR3 + RT) has demonstrated superior efficacy in cancer cell destruction and tumor growth control, compared to radiotherapy (RT), in preclinical and clinical settings. Previous studies highlighted the immunomodulatory properties of NBTXR3 + RT, such as modification of tumor cell immunogenicity/adjuvanticity, producing an effective local tumor control and abscopal effect, related to an enhanced antitumor immune response. Furthermore, NBTXR3 + RT has shown potential in restoring anti-PD1 efficacy in a refractory tumor model. However, the early events leading to these results, such as NBTXR3 endocytosis, intracellular trafficking and primary biological responses induced by NBTXR3 + RT remain poorly understood.MethodsWe analyzed by transmission electron microscopy endocytosis and intracellular localization of NBTXR3 nanoparticles after endocytosis in various cell lines, in vitro and in vivo. A kinetic of NBTXR3 endocytosis and its impact on lysosomes was conducted using LysoTracker staining, and a RNAseq analysis was performed. We investigated the ability of NBTXR3 + RT to induce lysosomal membrane permeabilization (LMP) and ferroptosis by analyzing lipid peroxidation. Additionally, we evaluated the recapture by cancer cells of NBTXR3 released from dead cells.ResultsNBTXR3 nanoparticles were rapidly internalized by cells mainly through macropinocytosis and in a less extend by clathrin-dependent endocytosis. NBTXR3-containing endosomes were then fused with lysosomes. The day following NBTXR3 addition, we measured a significant increase in LysoTracker lysosome labeling intensity, in vitro as in vivo. Following RT, a significant lysosomal membrane permeabilization (LMP) was measured exclusively in cells treated with NBTXR3 + RT, while RT had no effect. The day post-irradiation, a significant increase in lipid peroxidation, a biomarker of ferroptosis, was measured with NBTXR3 + RT compared to RT. Moreover, we demonstrated that NBTXR3 nanoparticles released from dead cells can be recaptured by cancer cells.ConclusionsOur findings provide novel insights into the early and specific biological effects induced by NBTXR3 + RT, especially LMP, not induced by RT in our models. The subsequent significant increase in lipid peroxidation partially explains the enhanced cancer cell killing capacity of NBTXR3 + RT compared to RT, potentially by promoting ferroptosis. This study improves our understanding of the cellular mechanisms underlying NBTXR3 + RT and highlights its potential as an agnostic therapeutic strategy for solid cancers treatment.Graphical

Exosomal PDL1 Suppresses the Anticancer Activity of CD8+ T Cells in Hepatocellular Carcinoma.

Tumor microenvironment (TME) is essential for the development and progression of hepatocellular carcinoma (HCC). Exosomes participate in constructing TME by passing biological information, but the regulatory effect of PDL1 in exosomes on anticancer activity of CD8+ T cells in HCC still needs to be further explored. In this study, high level of PDL1 was found in plasma exosomes of HCC patients, which turned out to be significantly associated with the increased number of tumor nodules, the upregulated level of serum AFP, the raised tendency of TNM stage, and the poor prognosis of HCC. The expression of CD8 may be inhibited in HCC that is characterized with high level of PDL1, and the protein level of exosomal PDL1 was determined by intracellular PDL1 abundance. High level of exosomal PDL1 inhibited the proliferation and activation of CD8+ T cells, but exhibited limited effect on the proliferation of hepatic cancer cells. Moreover, the growth of tumors formed by hepatic cancer cells Hepa1-6 in C57L mice was significantly promoted by the exosomal PDL1, which might be caused by the inhibitory effect of exosomal PDL1 on CD8+ T cells. Thus, exosomal PDL1 promotes the development and progression of HCC through inhibiting the anticancer activity of CD8+ T cells. This study provides sights for understanding the oncogenic role of PDL1 and a reasonable explanation for the low efficacy of anti-PD1/PDL1 immunotherapies in HCC.

Galectin-7 Induction by EHMT2 Inhibition Enhances Immunity in Microsatellite Stability Colorectal Cancer

Although immunotherapy shows substantial advancement in colorectal cancer (CRC) with microsatellite instability high, it has limited efficacy for CRC with microsatellite stability (MSS). Identifying combinations that reverse immune suppression and prime MSS tumors for current immunotherapy approaches remains an urgent need. An invitro CRISPR screen was performed using coculture models of primary tumor cells and autologous immune cells from MSS CRC patients to identify epigenetic targets that could enhance immunotherapy efficacy in MSS tumors. We revealed EHMT2, a histone methyltransferase, as a potential target for MSS CRC. EHMT2 inhibition transformed the immunosuppressive microenvironment of MSS tumors into an immunomodulatory one by altering cytokine expression, leading to T-cell-mediated cytotoxicity activation and improved responsiveness to anti-PD1 treatment. We observed galectin-7 up-regulation upon EHMT2 inhibition, which converted a "cold" MSS tumor environment into a T-cell-inflamed one. Mechanistically, CHD4 repressed galectin-7 expression by recruiting EHMT2 to form a cotranscriptional silencing complex. Galectin-7 administration enhanced anti-PD1 efficacy in MSS CRC, serving as a potent adjunct cytokine therapy. Our findings suggest that targeting the EHMT2/galectin-7 axis could provide a novel combination strategy for immunotherapy in MSS CRC.

Clinical efficacy and safety of anti PD-1/PD-L1 antibodies as monotherapy in patients with non-small-cell lung cancer

ObjectiveTo evaluate the efficacy and safety of anti-PD-1 and anti-PD-L1 immunotherapy agents as monotherapy in patients with non-small cell lung cancer. MethodThis was a four-year retrospective observational study that included all patients with non-small cell lung cancer treated with nivolumab, pembrolizumab, and atezolizumab in a third level hospital. Demographic, clinical (ECOG status, stage, PD-L1 expression level), therapeutic (drug, start date, line of treatment and number of cycles), efficacy (date and status at the end of follow-up) and toxicity variables were collected. Data was extracted from the patient's electronic medical record. Overall survival and progression-free survival rates for different monitoring times were calculated. ResultsThe study included 80 patients, 35 on nivolumab, 32 on pembrolizumab and 13 on atezolizumab. The median overall survival was not achieved. Overall survival at 6, 12, 18 and 49 months in patients treated with nivolumab was 79.7%, 74.0%, 65.8% and 65.8%, respectively. Median progression-free survival was 15 months. Adverse events were observed in 85.7% of cases, the most common being asthenia (45.7%), hypothyroidism (25.7%) and cough (20.0%). For pembrolizumab, the overail survival rate at the end of follow-up for first- and second-line treatment was 100% and 70.9%, respectively. Median progression-free survival was 17 months in the first-line and 24 months in the second-line setting. Adverse events were observed in 84.4% of subjects, the most common ones being dyspnea (31.3%), arthralgia (28.1%) and asthenia (25.0%). The overall survival rate from 3 to 7 months remained at 75.8% for atezolizumab. Median progression-free survival could not be determined. At 3 and 6 months, 49.5% of subjects had made some progress. The most frequent adverse events included toxicity (69.2%), asthenia (30.8%), and cough, dyspnea, and skin toxicity (15.4% each). ConclusionsSubjects showed a trend toward stabilization and chronification of the disease. A positive and considerable survival rate was observed, as compared with previous studies. Further studies are required with larger sample sizes and longer follow-up times to confirm these findings.

1473P Efficacy of anti-PD1/PDL1 antibody monotherapy in patients with advanced non-small cell lung cancer with increased hepcidin expression

1473P Efficacy of anti-PD1/PDL1 antibody monotherapy in patients with advanced non-small cell lung cancer with increased hepcidin expression

1062P Anti-PD1 efficacy in European patients with advanced MSI-H/MMRd non-colorectal cancers

1062P Anti-PD1 efficacy in European patients with advanced MSI-H/MMRd non-colorectal cancers

Focused Ultrasound for Blood-Brain Barrier Opening and Delivery of Anti-PD1 in Diffuse Midline Gliomas

Diffuse midline glioma with H3K27 mutation is a fatal pediatric brain tumor, most commonly arising in the brainstem. This tumor remains universally fatal, despite a multitude of clinical trials, with a median overall survival of only 9-12 months. While immune-checkpoint inhibitors (ICIs) have transformed the treatment landscape of multiple solid tumors, delivery past the blood brain barrier (BBB) remains challenging. Programmed cell death protein 1 (PD1) is an immune checkpoint protein expressed on the surface of activated T cells; interaction with its ligand, PDL1, is tumor-protective, dampening T cell response. Recent phase I clinical trials have shown that ICIs targeting proteins along the PD1/PDL1 axis are well tolerated in patients with DMG; however, efficacy remains low. The blood-brain barrier (BBB) poses a major challenge to the efficacious delivery of therapeutic agents with large molecular size, such as anti-PD1. We hypothesize that BBB opening (BBBO) using focused ultrasound (FUS), a form of non-ionizing acoustic radiation, can enhance delivery and efficacy of anti-PD1 for treatment of DMG. We established a syngeneic mouse DMG model with intracranial injection of cell line 4423 (PDGFB+, H3.3K27M, p53-/-). Magnetic resonance imaging (MRI) was utilized to evaluate BBBO and tumor progression. We measured delivery of anti-PD1 after BBBO using Western Blot and 3D in vivo optical fluorescent imaging/CT (OI/CT) of Cy7 labeled anti-PD1. We demonstrate that delivery of anti-PD1 can be enhanced over 3.5-fold after reversible BBBO with FUS and concurrent microbubble administration. OI/CT revealed enhanced real-time antibody distribution peritumorally. Furthermore, we demonstrate that combined treatment of FUS and anti-PD1 led to benefit in local control of tumor growth using volumetric analysis of MRI. Preliminary survival studies suggest a positive trend for overall survival. Our results support that FUS-mediated BBBO can increase treatment efficacy of anti-PD1 in a DMG murine model, due to improved targeted delivery to the tumoral region after systemic antibody administration. We consider these findings strong rationale for further investigation of the therapeutic effects of combinatorial treatment using FUS-mediated BBBO and ICIs for the treatment of DMG.

Lactobacillus gallinarum-derived metabolites boost anti-PD1 efficacy in colorectal cancer by inhibiting regulatory T cells through modulating IDO1/Kyn/AHR axis

ObjectiveGut microbiota is a key player in dictating immunotherapy response. We aimed to explore the immunomodulatory effect of probiotic Lactobacillus gallinarum and its role in improving anti-programmed cell death protein...

Myeloid-specific KDM6B inhibition sensitizes glioblastoma to PD1 blockade.

Glioblastoma (GBM) tumors are enriched in immune-suppressive myeloid cells and are refractory to immune checkpoint therapy (ICT). Targeting epigenetic pathways to reprogram the functional phenotype of immune-suppressive myeloid cells to overcome resistance to ICT remains unexplored. Single-cell and spatial transcriptomic analyses of human GBM tumors demonstrated high expression of an epigenetic enzyme-histone 3 lysine 27 demethylase (KDM6B)-in intratumoral immune-suppressive myeloid cell subsets. Importantly, myeloid cell-specific Kdm6b deletion enhanced proinflammatory pathways and improved survival in GBM tumor-bearing mice. Mechanistic studies showed that the absence of Kdm6b enhances antigen presentation, interferon response and phagocytosis in myeloid cells by inhibition of mediators of immune suppression including Mafb, Socs3 and Sirpa. Further, pharmacological inhibition of KDM6B mirrored the functional phenotype of Kdm6b-deleted myeloid cells and enhanced anti-PD1 efficacy. This study thus identified KDM6B as an epigenetic regulator of the functional phenotype of myeloid cell subsets and a potential therapeutic target for enhanced response to ICT.

TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using Tcell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. Tcell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of Tcell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

Effect of MDSC-targeted TFF2-MSA with PD-1 blockade therapy in advanced gastric cancer models.

e16037 Background: Recent studies revealed chemotherapy increases anti-PD1 response of gastric cancer (GC) by reducing myeloid-derived suppressor cells (MDSCs). However, a more specific MDSC-targeted treatment is needed to further improve anti-PD1 efficacy in advanced GC. Trefoil factor family 2 (TFF2), a partial agonist of CXCR4 and a secreted anti-inflammatory peptide, can suppress MDSC expansion. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 in syngeneic GC mouse models. Methods: Murine serum albumin (MSA) was fused to murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice then received either TFF2-MSA or anti-PD-1 antibody or both when tumors reached 150 mm3. At the endpoint, flow cytometry analysis was performed to examine immune profiles. In parallel, a group of the s.c. tumor bearing mice were followed up for survival differences in response to the treatments. In the orthotopic model, ACKP-luc cells were implanted to stomach submucosa, and recipient mice received the same treatments started after 1 week. In the metastasis model, subcutaneous flank tumors were resected in ACKP-grafted mice once tumors reached 500 mm3 to permit spontaneous lung metastasis, and mice then received the same treatments. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p>0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p<0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) in a synergistic manner. The combination therapy efficiently reduced tumor HDC-GFP+ MDSCs by 67%, and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells by 18 fold. The myeloid/lymphoid progenitor and output ratio in the bone marrow were decreased by the combination, to a similar level of tumor-free mice. While standard chemotherapy (5-fluorouracil and oxaliplatin) showed no efficacy, adding chemotherapy to the combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, in the orthotopic model, the TFF2-MSA/PD-1 antibody combination eradicated GC in 80% mice without recurrence compared to 0% with either monotherapy. Finally, the combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p<0.0001), compared to minimal inhibition with either monotherapy (p>0.05). Conclusions: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of gastric cancer.

Abstract LB284: CLAUDIN-1 targeting antibody ALE.C04 drives single activity and restores anti-PD1 efficacy in solid tumors

Abstract Claudin 1 (CLDN1) is a protein confined within the normal epithelial tight junctions of different tissues. Upon malignant transformation, CLDN1 is overxpressed and epitopes become exposed outside the tight junctions (non-Junctional CLDN1). ALE.C04 is a highly specific humanized monoclonal antibody that recognizes a unique CLDN1 exposed epitope in different solid tumors. Herein we show that non-Junctional CLDN1 (NJ-CLDN1) is frequently overexpressed in solid tumors. Pre-clinically, ALE.C04 drives tumor growth inhibition in different CDXs and PDX in vivo tumor models. Notably, CLDN1 expression on tumor cells positively correlates with T cell exclusion, a mechanism described to hinder the efficacy of Checkpoint inhibitors (CPIs). On this line, the overexpression of Cldn1 in mouse tumor cells promoted T-cell exclusion and resistance to anti-PD1 treatment. Importantly, ALE.C04 restored both T-cell infiltration and anti-PD1 efficacy in tumors. Mechanistically, NJ-CLDN1 interacts with different components involved in extracellular matrix remodeling, thus establishing a physical barrier that exclude immune cells from the tumor nest. ALE.C04 perturbs the interface between CLDN1+ tumor cells and the stroma, thus restoring immune cell infiltration. Our pre-clinical data showed that: i) NJ-CLDN1 is a novel and druggable target in solid tumors ii) ALE.C04 is a selective anti-CLDN1 antibody capable of driving anti-tumor activity as both single agent and in combination with anti-PD1. Citation Format: Alberto Toso, Geoffrey Teixeira, Tina Zimmermann, Stefanie Gill Gill, Daniela Schmitter, Markus Meyer, Marion Muller, Laurent Mailly, Thomas Baumert, Luigi Manenti, Roberto Iacone. CLAUDIN-1 targeting antibody ALE.C04 drives single activity and restores anti-PD1 efficacy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB284.

Abstract 5080: TU2218, a novel ALK5/VEGFR2 dual inhibitor, overcomes tumor endothelial cell anergy and enhances anti-PD1 immunotherapy efficacy

Abstract Immune tolerance by TGF-β and VEGF is inextricably related with poor outcomes of approved anti-PD-(L)1 therapy. Accordingly, a dual target for ALK5 and VEGFR2 via single or combination treatments can be an unequivocal tactic to tune tumor-microenvironment (TME) favorable to ICI, and to essentially overcome immune evasion against TGF-β- and VEGF-enriched tumors. Specifically, several reports from clinical data suggest that VEGF-induced endothelial cell anergy (ECA) acts as a vascular immune checkpoint in TME immune response, and the activation of ECA is associated with worse outcomes. Herein, we demonstrate that TU2218, a first-in-class, orally available inhibitor against ALK5 and VEGFR2 can recover the downregulated endothelial adhesion molecules, i.e., ICAM-1 and VCAM-1, and suppress ECA. In this work, TU2218 completely recovered the expression of ICAM-1 and VCAM-1 on VEGF-induced ECA in HUVECs. The restored level of ICAM-1 and VCAM-1 at 1 μM TU2218 was equivalent to the activity of combined treatment of 1 μM Vactosertib (ALK5 inhibitor) and 25 μg/ml Ramucirumab (VEGFR2 inhibitor). 1 μM of Vactosertib alone, however, did not show such restoration. These results indicate that VEGF-induced ECA is mediated by both VEGFR2 and TGF-β signal, thereby validating the superiority of dual target strategy for ALK5 and VEGFR2 over a single target in overcoming ECA. We further tested if TU2218 could restore VEGF-induced decrease of Jurkat adhesion to HUVECs, considering the close relationship between the expression of adhesion-molecules of endothelial cell surface and the adhesion of lymphocytes to endothelium. TU2218 recovered the number of Jurkat adhering to VEGF-elicited HUVEC monolayer in a dose-dependent manner, but Vactosertib did not. Furthermore, the activity of TU2218 on Jurkat adhesion was reversed by VCAM-1 neutralizing antibody. Therefore, our results demonstrate that TU2218 improves Jurkat adhesion by restoring VCAM-1 expression. Finally, the in vivo translatability of TU2218 in overcoming ECA was confirmed with B16F10-bearing mice, a well-defined immune desert model, after treatments of anti-PD1 antibody, TU2218, or combined regimen for 15 days. TU2218 combined with an anti-PD1 antibody significantly suppressed tumor growth by c.a. 74 % compared to vehicle, thus being superior to a single treatment (e.g., tumor growth inhibition (TGI) 44% for TU2218, TGI 45% for anti-PD1). In this combination, TU2218 increased the number of both CD31+VCAM-1+ and IFNγ+CD8+ T cells in the tumor. We conclude that TU2218 leads not only to the enhancement of T cell-traffic toward TME, but also to the conversion of immune balance favorable to anti-PD1 therapy. The Phase 1b trial of TU2218 combined with pembrolizumab is underway for advanced solid cancers (NCT05204862). Citation Format: Jihyun Lee, Nam-hoon Kim, Hun-Taek Kim. TU2218, a novel ALK5/VEGFR2 dual inhibitor, overcomes tumor endothelial cell anergy and enhances anti-PD1 immunotherapy efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5080.

Abstract 4038: Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells

Abstract Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) has emerged as a promising cancer immunotherapy target. Previously, we reported on the functional role of PTPN2 in the pathogenesis of colorectal carcinoma (CRC). We demonstrated increased PTPN2 phosphatase activity correlates with disease progression and decreased immune responses in tumor tissues, while loss of PTPN2 in T-cells or in dendritic cells (DCs) reduces tumor burden in several CRC models and potentiates anti-PD1 efficacy (Katkeviciute et al., 2021). Others reported that loss of PTPN2 in tumor cells sensitizes to immune-mediated tumor killing (Manguso et al., 2017; Goh et al., 2022). Thus, PTPN2 was proposed as a critical node to modulate anti-tumor immunity, and PTPN2 inhibition is expected to enhance anti-tumor immunity by sensitizing tumor cells and by activating immune cells. Recently, also the closely related phosphatase PTPN1 (PTP1B) was demonstrated to restrain T-cell mediated tumor killing (Wiede et al., 2022). Inhibition of Protein Tyrosine Phosphatases has historically been a demanding area for drug discovery. However, both allosteric PTP inhibitors (eg targeting SHP2/PTPN11) and catalytic-site inhibitors (targeting PTPN2/N1) have recently progressed to clinical studies. We now identified a chemical series of novel dual PTPN2/N1 inhibitors. These inhibitors bind to the catalytic site of PTPN2, as confirmed by crystallography. The best molecules of the series are low nM inhibitors of PTPN2 and PTPN1 enzymes with excellent selectivity across other phosphatases. As an immediate consequence of PTPN2/N1 inhibition, these compounds augment phosphorylation of STAT1 and/or STAT5 isoforms in myeloid and T-cell lines, and in primary T-cells, macrophages and DCs. This results in immune-cell activation as evidenced by increased production of effector cytokines (e.g. IFNγ), immune-activation, levels of cytotoxicity markers in T-cells (e.g. granzyme B) as well as upregulation of antigen presentation and co-stimulatory markers in macrophages and DCs. Furthermore, inhibition of PTPN2/N1 sensitizes murine and human cancer cell lines to IFNγ, and enhances immune-mediated tumor killing in co-culture assays in vitro. In vivo evaluation of selected examples is in progress and results will be presented at the conference. Our data indicate that inhibition of PTPN2/N1 phosphatase activity is a powerful pharmacologic strategy to promote anti-tumor immunity, with strong potential to be exploited for cancer immunotherapy both as a single agent treatment in anti-PD1 refractory cancers, and in combination with anti-PD1 therapy. Citation Format: Kalliopi Pervolaraki, Egle Katkeviciute, Dominique Lambin, Sandro Boland, Amuri Kilonda, Vincent Pericolle, Marnik Nijs, Wanda Haeck, Kristine Metzger, Hugo Klaassen, Arnaud Marchand, Patrick Chaltin, Matthias Versele, Marianne Spalinger, Michael Scharl. Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4038.

Abstract 2414: Early biological impacts of radiotherapy-activated NBTXR3 nanoparticles

Abstract Since many decades, radiotherapy (RT) is worldwide used and crucial therapeutic approach for the treatment of cancers. Nowadays, about 50% of cancer patients will receive radiotherapy during their treatment. Nevertheless, the use of RT is limited by the damage induced by ionizing radiation on healthy tissues. To overcome this constraint, we have developed high electron density of functionalized hafnium oxide nanoparticles (NBTXR3), allowing a high probability of interaction with incoming ionizing radiation, increasing energy dose deposit within cells. Many preclinical data have shown that RT-activated NBTXR3 (NBTXR3+RT) increase cancer cell destruction as well as better control treated tumor growth, compared to RT alone. In clinic, the benefit of NBTXR3+RT compared to RT alone has been demonstrated for patients with locally advanced Soft Tissue Sarcoma, in the randomized controlled phase II/III Act.in.Sarc study (NCT02379845). We also demonstrated in preclinical settings that, beyond the destruction of tumor cells, NBTXR3+RT was also able to induce an antitumor immune response capable to produce a significant abscopal effect, linked to the increase of CD8 infiltrates in tumors. It has been recently reported that NBTXR3+RT can also restore anti-PD1 efficacy in an anti-PD1 resistant mouse model. At the cancer cell level, we reported that NBTXR3+RT could modulate the immunopeptidome of cancer cells, induce the activation of the cGAS-STING pathway and increase the expression of ICD biomarkers. All these data indicate that NBTXR3+RT has the ability to transform the tumor into an in situ vaccine more efficiently than RT alone and could have important implications for the use of NBTXR3+RT in combination with immunotherapy. Nevertheless, we currently have relatively few elements on the early cellular events that could trigger or explain the above reported performances of NBTXR3+RT, compared to RT alone. To address this question, we have carried out a series of in vitro studies (cytometry, microscopy) on different cancer models (human and murine). We have thus identified that the vast majority of NBTXR3 nanoparticles are located in lysosomes after endocytosis by tumor cells. Once activated by RT, we detected lysosomal membrane permeabilization (LMP) only in cells treated with NBTXR3+RT, but not in cells only treated by RT. Following LMP, we measured a significant increase of in lipid peroxidation. These first elements allow a better understanding of the early biological consequences induced by NBTXR3+RT, compared to RT alone. Citation Format: Jordan Da Silva, Célia Bienassis, Sebastien Paris. Early biological impacts of radiotherapy-activated NBTXR3 nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2414.

Therapeutic potential of Clostridium butyricum anticancer effects in colorectal cancer

ABSTRACT Probiotic roles of Clostridium butyricum (C.B) are involved in regulating disease and cancers, yet the mechanistic basis for these regulatory roles remains largely unknown. Here, we demonstrate that C.B reprograms the proliferation, migration, stemness, and tumor growth in CRC by regulating pivotal signal molecules including MYC. Destabilization of MYC by C.B supplementation suppresses cancer cell proliferation/metastasis, sensitizes 5-FU treatment, and boosts responsiveness of anti-PD1 therapy. MYC is a transcriptional regulator of Thymidylate synthase (TYMS), a key target of the 5-FU. Also MYC is known to impact on PD-1 expression. Mechanistically, C.B treatment of CRC cells results in MYC degradation by enhancing proteasome-mediated ubiquitination, thereby mitigating MYC-mediated 5-FU resistance and boosting anti-PD1 immunotherapeutic efficacy. Together, our findings uncover previously unappreciated links between C.B and CRC cell signaling, providing insight into the tumorigenesis modulating mechanisms of C.B in boosting chemo/immune therapies.

Supplementation with α-ketoglutarate improved the efficacy of anti-PD1 melanoma treatment through epigenetic modulation of PD-L1

Patients with advanced melanoma have shown an improved outlook after anti-PD1 therapy, but the low response rate restricts clinical benefit; therefore, enhancing anti-PD1 therapeutic efficacy remains a major challenge. Here, our findings showed a significantly increased abundance of α-KG in healthy controls, anti-PD1-sensitive melanoma-bearing mice, and anti-PD1-sensitive melanoma patients; moreover, supplementation with α-KG enhanced the efficacy of anti-PD1 immunotherapy and increased PD-L1 expression in melanoma tumors via STAT1/3. We also found that supplementation with α-KG significantly increased the activity of the methylcytosine dioxygenases TET2/3, which led to an increased 5-hydroxymethylcytosine (5-hmC) level in the PD-L1 promoter. As a consequence, STAT1/3 binding to the PD-L1 promoter was stabilized to upregulate PD-L1 expression. Importantly, single-cell sequencing of preclinical samples and analysis of clinical data revealed that TET2/3-STAT1/3-CD274 signaling was associated with sensitivity to anti-PD1 treatment in melanoma. Taken together, our results provide novel insight into α-KG’s function in anti-PD1 treatment of melanoma and suggest supplementation with α-KG as a novel promising strategy to improve the efficacy of anti-PD1 therapy.

Targeting m6A reader YTHDF1 augments antitumour immunity and boosts anti-PD-1 efficacy in colorectal cancer

ObjectiveThe role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC)...

Determinants of activity and efficacy of anti-PD1/PD-L1 therapy in patients with advanced solid tumors recruited in a clinical trials unit: a longitudinal prospective biomarker-based study

Immune-checkpoint inhibitors (ICI) have revolutionized the therapeutic landscape of cancer. However, optimal patient selection is still an unmet need. One-hundred-forty-six patients with metastatic cancer candidates to ICI at the Hospital Clinic of Barcelona Clinical Trials Unit were prospectively recruited in this observational study. Blood samples were collected at different timepoints, baseline LIPI score calculated and pre-ICI archived tissues retrieved to evaluate PD-L1, tumor-infiltrating lymphocytes (TILs) and PD1 mRNA levels. Tumor assessments were centrally reviewed by RECIST 1.1 criteria. Associations with overall response rates (ORR), durable clinical benefit (DCB), progression-free survival (PFS) and overall survival (OS) were performed with univariable/multivariable logistic and Cox regressions, where appropriate. At a median follow-up of 26.9 months, median PFS and OS were 2.7 and 12.9 months. Response rates were 17.8% with duration of response (DOR) of 4.4 months. LIPI score was independently associated with PFS (p = 0.025) and OS (p < 0.001). Immunotherapy-naïve status was independently associated with better PFS (p = 0.005). Time-to-best response (TTBR) and ORR (p < 0.001 both) were associated with better OS at univariate analysis. PFS and DOR were moderately correlated with OS (p < 0.001 both). A PD-L1 10% cut-off detected worse/best responders in terms of ORR (univariate p = 0.011, multivariate p = 0.028) and DCB (univariate p = 0.043). PD1 mRNA levels were strikingly associated to complete responses (p = 0.021). To resume, in our prospective observational pan-cancer study, baseline LIPI score, immunotherapy-naïve status, cancer type and RT before starting ICI were the most relevant clinical factors independently correlated with immunotherapy outcomes. Longer TTBR seemed to associate with better survival, while PD1 mRNA and PD-L1 protein levels might be tumor-agnostic predictive factors of response to ICI and should be furtherly explored.

KLF5 inhibition potentiates anti-PD1 efficacy by enhancing CD8+ T-cell-dependent antitumor immunity.

Background: Immune checkpoint blockers (ICBs) are revolutionized therapeutic strategies for cancer, but most patients with solid neoplasms remain resistant to ICBs, partly because of the difficulty in reversing the highly immunosuppressive tumor microenvironment (TME). Exploring the strategies for tumor immunotherapy is highly dependent on the discovery of molecular mechanisms of tumor immune escape and potential therapeutic target. Krüppel-like Factor 5 (KLF5) is a cell-intrinsic oncogene to promote tumorigenesis. However, the cell-extrinsic effects of KLF5 on suppressing the immune response to cancer remain unclear. Methods: We analyzed the immunosuppressive role of KLF5 in mice models transplanted with KLF5-deleted/overexpressing tumor cells. We performed RNA sequencing, immunohistochemistry, western blotting, real time-PCR, ELISA, luciferase assay, chromatin immunoprecipitation (ChIP), and flow cytometry to demonstrate the effects of KLF5 on CD8+ T cell infiltration and related molecular mechanism. Single-cell RNA sequencing and spatial transcriptomics analysis were applied to further decipher the association between KLF5 expression and infiltrating immune cells. The efficacy of KLF5/COX2 inhibitors combined with anti-programmed cell death protein 1 (anti-PD1) therapy were explored in pre-clinical models. Finally, a gene-expression signature depending on KLF5/COX2 axis and associated immune markers was created to predict patient survival. Results: KLF5 inactivation decelerated basal-like breast tumor growth in a CD8+ T-cell-dependent manner. Transcriptomic profiling revealed that KLF5 loss in tumors increases the number and activated function of T lymphocytes. Mechanistically, KLF5 binds to the promoter of the COX2 gene and promotes COX2 transcription; subsequently, KLF5 deficiency decreases prostaglandin E2 (PGE2) release from tumor cells by reducing COX2 expression. Inhibition of the KLF5/COX2 axis increases the number and functionality of intratumoral antitumor T cells to synergize the antitumorigenic effects of anti-PD1 therapy. Analysis of patient datasets at single-cell and spatial resolution shows that low expression of KLF5 is associated with an immune-supportive TME. Finally, we generate a KLF5/COX2-associated immune score (KC-IS) to predict patient survival. Conclusions: Our results identified a novel mechanism responsible for KLF5-mediated immunosuppression in TME, and targeting the KLF5/COX2/PGE2 axis is a critical immunotherapy sensitizer.