Cell-mediated Antitumor Responses Research Articles

Comparative Analysis of Systemic Immune Responses and Metastatic Risks in Tumor Ablation: An Animal Study of Radiofrequency Ablation and Irreversible Electroporation with Immune Modulation.

This study aimed to compare systemic immune responses and metastatic effects induced by radiofrequency ablation (RFA) and irreversible electroporation (IRE) in murine tumor models. We assessed cytokine production, growth of treated and untreated metastatic tumors, and synergy with immune checkpoint inhibitors (ICIs). Hep55.1c murine hepatoma cells were implanted in C57BL/6N mice to establish primary tumors. In Experiment 1 (n = 50), RFA or IRE was applied to primary tumors, followed by CD8+ T cell depletion in some groups to assess anti-tumor immune responses. Experiment 2 (n = 45) tested RFA and IRE combined with anti-PD-1 therapy for enhanced abscopal effects. In Experiment 3 (n = 28), anti-IL-6 antibody was administered in IRE-treated mice to examine IL-6's role in secondary tumor growth. Tumor volumes and cytokine/chemokine levels were monitored. Both techniques induced significant CD8+ T cell-mediated anti-tumor responses, with abscopal effects observed in untreated secondary tumors. CD8+ T cell depletion abolished these effects, confirming their role in systemic tumor control. Anti-PD-1 therapy combination further suppressed secondary tumor growth. However, IRE uniquely elevated IL-6 and other inflammatory cytokines, unexpectedly accelerating secondary tumor growth. Administration of an anti-IL-6 antibody mitigated this effect, reducing secondary tumor progression. The results of this animal study indicate that both techniques promote systemic anti-tumor immunity, though IRE uniquely induces an inflammatory response that risks exacerbating micro-metastases through IL-6. Combining IRE with IL-6 blockade may offer a promising strategy for nonthermal tumor ablation therapies. Further studies are warranted to refine ablation-immune therapy combinations for optimal therapeutic outcomes.

Napabucasin-loaded PLGA nanoparticles trigger anti-HCC immune responses by metabolic reprogramming of tumor-associated macrophages

BackgroundJAK/STAT3 is one of the critical signaling pathways involved in the occurrence and development of hepatocellular carcinoma (HCC). BBI608 (Napabucasin), as a novel small molecule inhibitor of STAT3, has shown previously excellent anti-HCC effects in vitro and in mouse models. However, low bioavailability, high cytotoxicity and other shortcomings limit its clinical application. In this study, PLGA was selected to prepare Napabucasin PLGA nanoparticles (NPs) by solvent evaporation method, overcoming these limitations and improving the passive targeting effect that nanoparticle mediated. Base on this, we systematically evaluated the anti-HCC effect of Napabucasin-PLGA NPs and explored the underlying mechanisms.MethodsNapabucasin-PLGA NPs were prepared by solvent evaporation method. CCK-8 assay, Annexin V/PI double staining, RT-qPCR, colony formation assay, and Western blotting were performed to evaluate the anti-HCC effect of Napabucasin-PLGA NPs in vitro. Proliferation assay and migration assay were used to detect the effects of Napabucasin-PLGA NPs-treated HCC-TAMs on tumor biological characteristics of HCC cells. Flow cytometry was used to detect anti-HCC immune responses induced by Napabucasin-PLGA NPs in vivo.ResultsOur results demonstrated that Napabucasin-PLGA NPs could improve the bioavailability of Napabucasin and enhance Napabucasin-mediated the anti-HCC effects in vitro and in vivo with no significant drug toxicity. In addition to the direct inhibitory effects on the tumor biological characteristics of HCC cells, Napabucasin-PLGA NPs could promote the polarization of macrophages from tumor-promoting M2-type to anti-tumor M1-type, improving the tumor immune microenvironment and augmenting T cell-mediated anti-tumor responses. The underlining mechanisms showed Napabucasin-PLGA NPs suppressed the STAT3/FAO signaling axis in HCC-induced tumor-associated macrophages (TAMs).ConclusionsThese findings demonstrated Napabucasin-PLGA NPs is a potential therapeutic candidate for HCC, and provided a new theoretical and experimental basis for further development and clinical application of Napabucasin.

Novel biomarkers: the RUNX family as prognostic predictors in colorectal cancer

While biomarkers have been shown to enhance the prognosis of patients with colorectal cancer (CRC) compared to conventional treatments, there is a pressing need to discover novel biomarkers that can assist in assessing the prognostic impact of immunotherapy and in formulating individualized treatment plans. The RUNX family, consisting of RUNX1, RUNX2, and RUNX3, has been recognized as crucial regulators in developmental processes, with dysregulation of these genes also being implicated in tumorigenesis and cancer progression. In our present study, we demonstrated a crucial regulatory role of RUNX in CD8+T and CD103+CD8+T cell-mediated anti-tumor response within the tumor microenvironment (TME) of human CRC. Specifically, RUNXs were significantly differentially expressed between tumor and normal tissues in CRC. Patients with a greater proportion of infiltrating CD8+RUNX1+, CD103+CD8+RUNX1+, CD8+RUNX2+, CD103+CD8+RUNX2+, CD8+RUNX3+, or CD103+CD8+RUNX3+ T cells demonstrated improved outcomes compared to those with lower proportions. Additionally, the proportions of infiltrating CD8+RUNX1+T and CD8+RUNX3+T cells may serve as valuable prognostic predictors for CRC patients, independent of other clinicopathological factors. Moreover, further bioinformatic analysis conducted utilizing the TISIDB and TIMER platforms demonstrated significant associations between the members of the RUNX family and immune-infiltrating cells, specifically diverse subpopulations of CD8+TILs. Our study of human colorectal cancer tissue microarray (TMA) also revealed positive and statistically significant correlations between the expressions of RUNX1, RUNX2, and RUNX3 in both CD8+T cells and CD103+CD8+T cells. Our study comprehensively revealed the varied expressions and prognostic importance of the RUNX family in human colorectal cancer tissues. It underscored their potential as vital biomarkers for prognostic evaluation in colorectal cancer patients and as promising targets for immunotherapy in treating this disease.

Microwave Ablation Combined with Flt3L Provokes Tumor-Specific Memory CD8+ T Cells-Mediated Antitumor Immunity in Response to PD-1 Blockade.

For medically inoperable non-small cell lung cancer, microwave ablation (MWA) represents a superminimally invasive alternative treatment. However, tumor recurrence remains a concern. Here, it is demonstrated that the combination of MWA with Flt3L significantly inhibits tumor recurrence by CD8+ central memory T (TCM)-like cell-dependent antitumor immune responses within the tumor-draining lymph nodes (TdLN). TdLN-TCM-like cells encompassedboth tumor-specific memory T (TTSM) and progenitor-exhausted T (TPEX) cells. The expansion of these cells markedly altered the differentiation of exhausted T cells within the tumor microenvironment (TME). TPEX predominantly differentiated into transitory effector-like exhausted T cells (TEX-int). The expansion of TTSM cells elicited by the combined therapy was reliant on conventional dendritic cells (cDCs) and was likely specifically dependent on the migratory cDC1s (Mig cDC1s) within the TdLN. The upregulation of ICOSL on migratory cDC1s was pivotal in initiating TTSM-like cell-mediated antitumor responses. Slc38a2 may be a critical gene responsible for the upregulation of ICOSL in Mig cDC1s following combined treatment. Finally, the combined treatment significantly enhanced the antitumor efficacy of immunotherapy based on PD-1 blockade. The research thereby afforded a novel strategic approach to forestall tumor recurrence after MWA therapy, while also providing the foundational proof-of-concept for impending clinical investigations.

CWF19L1 promotes T-cell cytotoxicity through the regulation of alternative splicing

Enhancing host anti-tumor immunity is paramount for advancing cancer immunotherapy. In this study, we identify CWF19-like cell cycle control factor 1 (CWF19L1) as a novel splicing regulator that enhances T cell-mediated cytotoxicity. CWF19L1 interacts prominently with key splicing factors within the nucleus, including components of the U5 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA processing factor 19 (PRPF19) complex. Deficiency of CWF19L1 disrupts alternative splicing of immune-related genes, resulting in diminished expression of cytotoxic molecules. Furthermore, CWF19L1 plays a critical role in promoting T cell-mediated anti-tumor responses by upregulating the expression of effector cytokines. Our findings unveil previously undocumented functions of CWF19L1 in alternative splicing and its involvement in the regulation of anti-tumor immunity, highlighting its potential as a therapeutic target for novel cancer immunotherapies.

A CXCR4 partial agonist improves immunotherapy by targeting polymorphonuclear myeloid-derived suppressor cells and cancer-driven granulopoiesis.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that potently impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target1. Here, we fused a secreted CXCR4 partial agonist TFF2 to mouse serum albumin (MSA) and demonstrated that TFF2-MSA peptide synergized with anti-PD-1 to induce tumor regression or eradication, inhibited distant metastases, and prolonged survival in multiple gastric cancer (GC) models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track PMN-MDSC in vivo , we found TFF2-MSA selectively reduced the immunosuppressive Hdc-GFP + CXCR4 hi tumor PMN-MDSCs while preserving proinflammatory neutrophils, thereby boosting CD8 + T cell-mediated anti-tumor response together with anti-PD-1. Furthermore, TFF2-MSA systemically reduced PMN-MDSCs and bone marrow granulopoiesis. In contrast, CXCR4 antagonism plus anti-PD-1 failed to provide a similar therapeutic benefit. In GC patients, expanded PMN-MDSCs containing a prominent CXCR4 + LOX-1 + subset are inversely correlated with the TFF2 level and CD8 + T cells in circulation. Collectively, our studies introduce a strategy of using CXCR4 partial agonism to restore anti-PD-1 sensitivity in GC by targeting PMN-MDSCs and granulopoiesis.

Dendritic Cell-Targeted Nanoparticles Enhance T Cell Activation and Antitumor Immune Responses by Boosting Antigen Presentation and Blocking PD-L1 Pathways.

Dendritic cells (DCs) within the tumor microenvironment (TME) have an insufficient capacity to activate T cells through antigen presentation. Furthermore, the programmed cell-death ligand 1 (PD-L1), abundantly expressed on tumor-associated DCs, binds the programmed cell-death 1 (PD-1)-positive T cells and suppresses their immune function. The binding of PD-L1 to CD80 (B7.1) on the same DC via cis-interactions further prevents T cell costimulation through CD28. Here, we present a strategy to simultaneously promote antigen cross-presentation and block the inhibitory interactions of PD-L1 on DCs to amplify T cell-mediated antitumor responses within the TME. Mesoporous silica nanoparticles (MSNPs) were loaded with clotrimazole (CLT) to boost MHC II-mediated antigen presentation by DCs, surface-modified with mannose to target CD206 on DCs, and then decorated with PD-L1 binding peptide (PDL1bp) to block PD-L1-mediated interactions. PDL1bp was cleaved from the mannosylated and CLT-loaded MSNPs (MSNP-MaN/CLT) under conditions simulating the TME and tethered to PD-L1 to reverse CD80 sequestration on DC2.4 cells. The blocking of PD-L1 by PDL1bp-decorated NPs (MSNP-MaN-PDL1bp) increased the cellular interactions between DC2.4 and EL4 T cells and the amount of IL-2 secretion. The MSNP-MaN/CLT were taken up rapidly by DC2.4 cells, promoted MHC II presentation of hen egg lysozyme (HEL), and increased IL-2 production from HEL antigen-primed 3A9 T cells, which was further enhanced by PDL1bp. In vivo investigation revealed that administration of the CLT-loaded and PDL1bp-functionalized MSNPs remarkably inhibited subcutaneous B16-F10 melanoma tumor growth when compared with anti-PD-L1 therapy. MSNP-MaN-PDL1bp/CLT treatment upregulated the levels of effector molecules such as granzyme B and proinflammatory cytokines (IFNγ and INFα) in the tumor tissue, indicating antitumoral T cell responses. This strategy of utilizing nanoparticles to trigger DC activation while promoting T cell stimulation can be used to amplify the antitumor T cell responses and represents a promising alternative to anti-PD-L1 immunotherapy.

DSCI: a database of synthetic biology components for innate immunity and cell engineering decision-making processes

Although significant progress of clinical strategy has been made in gene editing and cell engineering in immunotherapy, it is now apparent that design and modification in terms of complex signaling pathways and motifs on medical synthetic biology are still full of challenges. Innate immunity, the first line of host defense against pathogens, is critical for anti-pathogens immune response as well as regulating durable and protective T cell-mediated anti-tumor responses. Here, we introduce DSCI (Database of Synthetic Biology Components for Innate Immunity, https://dsci.renlab.cn/), a web-accessible and integrative database that provides better insights and strategies for innate immune signaling circuit design in biosynthesis. Users can interactively navigate comprehensive and carefully curated components resources that presented as visualized signaling motifs that participate in innate immunity. The current release of DSCI incorporates 1240 independent components and more than 4000 specific entries contextually annotated from public literature with experimental verification. The data integrated into DSCI includes the components of pathways, relationships between regulators, signal motifs based on regulatory cascades, and loop graphs, all of which have been comprehensively annotated to help guide modifications to gene circuits. With the support of DSCI, users can easily obtain guidance of gene circuits construction to make decision of cell engineering based on innate immunity. DSCI not only provides comprehensive and specialized resource on the biological components of innate immune synthesis, but also serves as a useful tool to offer modification or generation strategies for medical synthetic biology.

Intrinsic ADRB2 inhibition improves CAR-T cell therapy efficacy against prostate cancer

Chimeric antigen receptor (CAR)-T cell therapy has shown limited success in patients with solid tumors. Recent invitro and invivo data have shown that adrenoceptor beta-2 (ADRB2) is a novel checkpoint receptor that inhibits Tcell-mediated anti-tumor responses. To inhibit ADRB2-mediated inhibitory signaling, we downregulated ADRB2 in CAR-T (shβ2-CAR-T) cells via RNA interference, assessed different parameters, and compared them with conventional second-generation CAR-T cells. ADRB2 knockdown CAR-T cells exhibited enhanced cytotoxicity against prostate cancer cell lines invitro, by increasing CD69, CD107a, GzmB, IFN-γ, T-bet, and GLUT-1. In addition, ADRB2 deficiency led to improved proliferation, increased CD8/CD4 Tcell ratio, and decreased apoptosis in CAR-T cells. shβ2-CAR-T cells expressed more Bcl-2 and led to the generation of more significant proportions of T central memory cells. Finally, the ZAP-70/NF-κB signaling axis was shown to be responsible for the improved functions of novel CAR-T cells. In tumor-bearing mice, shβ2-CAR-T cells performed better than conventional CAR-T cells in eradicating prostate tumors. The study provides the basis for future clinical and translational CAR-T cell research to focus on adrenergic stress-mediated challenges in the tumor microenvironment of stressed tumors.

Safety and efficacy of belantamab mafodotin with pembrolizumab in patients with relapsed or refractory multiple myeloma.

Belantamab mafodotin (belamaf) has shown promising antimyeloma activity in relapsed or refractory multiple myeloma (RRMM) as a single agent. It was hypothesized that its multimodal activity may be enhanced by programmed cell death protein 1 pathway inhibition and activation of T cell-mediated antitumor responses. This study investigated the efficacy and safety of belamaf with pembrolizumab in patients with RRMM. DREAMM-4 (NCT03848845) was an open-label, single-arm, phase 1/2 study divided into dose-escalation (part 1) and dose-expansion (part 2) phases. Patients were ≥18 years old with ≥3 prior lines of therapy including a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 agent. Patients received belamaf (2.5 or 3.4 mg/kg, part 1; 2.5 mg/kg, part 2) and 200 mg pembrolizumab for ≤35 cycles. Of 41 enrolled patients, 34 (n=6 part 1, n=28 part 2) who received 2.5 mg/kg belamaf plus pembrolizumab were included in this final analysis. Sixteen patients (47%) achieved an overall response. Minimal residual disease negativity was achieved in three of 10 patients who had very good partial response or better. Five of eight patients who had prior anti-B-cell maturation antigen therapy achieved partial response or better, including two who had B-cell maturation antigen-refractory disease. Common grade ≥3 adverse events were keratopathy (38%) and thrombocytopenia (29%). Despite belamaf-related ocular events, quality-of-life measures remained stable over time. No new safety signals were observed. The results of DREAMM-4 demonstrated clinical activity and a favorable safety profile of belamaf plus pembrolizumab in patients with RRMM. This trial is registered at www. gov as NCT03848845.

Novel insights into paclitaxel’s role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment

BackgroundTriple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed...

NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody

NK cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9-ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared to anti-NKG2A antibody blockade, NKG2A-knockout NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2A-deficient NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody-coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

Deciphering CD4+ T cell-mediated responses against cancer.

It'sbeen long thought that CD8+ cytotoxic T cells play a major role in T cell-mediated antitumor responses, whereas CD4+ T cells merely provide some assistance to CD8+ T cells as the "helpers."In recent years, numerous studies support the notion that CD4+ T cells play an indispensable role in antitumor responses. Here, we summarize and discuss the current knowledge regarding the roles of CD4+ T cells in antitumor responses and immunotherapy, with a focus on the molecular and cellular mechanisms behind these observations. These new insights on CD4+ T cells may pave the way to further optimize cancer immunotherapy.

WTAP enhances the instability of SYTL1 mRNA caused by YTHDF2 in bladder cancer.

Bladder cancer (BCa) is the most frequent type of cancer in humans. The association between m6A modification and the anti-tumor effects of natural killer (NK) cells has been described in BCa. This study intended to investigate the implications of m6A regulators in modulating SYTL1 expression in BCa and the association with the anti-tumor effects of NK cells. The prognostic role of SYTL1 in BCa was investigated using bioinformatics analysis, and the correlation between SYTL1 expression and NK cells was analyzed. The effects of SYTL1 on the anti-tumor response of NK-92 cells were examined by RT-qPCR, cytotoxicity, western blot, and ELISA assays. The relationships among WTAP, YTHDF2, and SYTL1 were investigated by RT-qPCR, RIP-qPCR, ELISA, and actinomycin D treatment. Finally, the effects of WTAP and SYTL1 on BCa tumor growth and the anti-tumor response of NK cells were verified in vivo. SYTL1 was reduced in BCa tissues and had a prognostic significance, which was related to NK cell-mediated anti-tumor responses. NK-92 cells produced toxicity to BCa cells, which was further enhanced by SYTL1 overexpression in BCa cells through prompting LDH, NKG2D, NKp30, and NKp44 and IFN-γ levels. WTAP enhanced the degradation of the SYTL1 mRNA by YTHDF2. WTAP and YTHDF2 impaired the anti-tumor response of NK cells in BCa. SYTL1 inhibited the BCa progression in mice while enhancing the anti-tumor response of NK cells. WTAP inhibited the anti-tumor response of NK cells to BCa cells by promoting the degradation of SYTL1 mRNA through YTHDF2-mediated m6A methylation.

Abstract LB426: Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway

Abstract IDO1 is an immune checkpoint regulator and mediator of tumor immune evasion (1). The cellular and molecular mechanisms of IDO1-mediated immune suppression are not fully understood. We describe here that GPR35, the putative receptor for kynurenic acid (KYNA), a metabolite of IDO1-mediated tryptophan catabolism, couples to Hippo-YAP pathway. GPR35 activation, either by overexpression or by treatment with tryptophan metabolites resulted in Hippo inactivation and YAP-directed transcription via TEAD family of transcription factors. Recently, YAP is found to be essential for tumor immune escape. Yap1 deficiency in T-lineage-specific knockout mice, or by dosing a YAP inhibitor, impairs Treg formation and function, leading to tumor growth inhibition (2). YAP also plays an inhibitory role in CD8 T cell function, especially in activated CTL usually found in the TME [3,4]. Through medicinal chemistry design and a structure-vs-activity relationship study effort, we discovered a GPR35-selective inhibitor, here termed TMER1i. TMER1i potently blocks GPR35 in Hippo suppression and YAP activation. We showed that GPR35 is expressed in the T-cell lineage of the immune cells including naïve CD4 T cells, Treg and CD8 T cells. Treatment of purified human naïve CD4 T cells with anti-CD3/anti-CD28 under an IDO1+ TME-like culture condition in vitro induced a robust T cell activation and differentiation toward Treg phenotype, as shown previously using mouse naïve CD4 T cells by others (5). Importantly, TMER1i efficiently blocked the Treg differentiation under these conditions. When dosed in human-PBMC reconstituted NCG mice bearing HT29 tumors, TMER1i dose-dependently increased the infiltration of human CD8 T cells within tumors in a statistically significant manner, and a trending lower Treg cell infiltration, accompanied by a tumor growth inhibition. qPCR analysis of the HT29 tumors grown in the PBMC-NCG mice indicated that IDO1 was highly expressed in the tumors. These results indicated that GPR35 selective inhibitor TMER1i enhanced an anti-tumor immune activity in an IDO1+ tumor microenvironment. Taken together, these studies provided evidence for a role of GPR35 in mediating IDO1 immune suppression by regulating Hippo-YAP pathway in Treg and CTL cells in the immune system. Thus, GPR35 inhibitors have a potential to be novel immuno-therapeutic agents effective in treating patients with IDO1-positive tumors. Reference1.Munn, DH, and Mellor, AL. (2007) Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J. Clin. Invest. 117:1147-11542.Ni, X. et al. Yap is essential for Treg-mediated suppression of antitumor Immunity. Cancer Discovery 2018; 8:1026-10433.Lebid, A. et al. (2020) YAP attenuates CD8 T cell-mediated anti-tumor response. Frontiers in Immunology 11:1-84.Stampouloglou, E. et al. (2020) YAP suppresses T-cell function and infiltration in the tumor microenvironment. PLoS Biol 18(1): e30005915.Fallarino, F. et al. (2006) The combined effects of tryptophan starvation and tryptophan catabolites down-regulate T cell receptor æ-chain and induce a regulatory phenotype in naïve T cells. J Immunol 176:6752-6761 Citation Format: Hongmei He, Nam Hee Kim, Sangdon Han, Zhi Liang Chu. Evidence for a role of GPR35 in IDO1-mediated tumor immune escape by regulating Hippo-YAP pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB426.

Dual molecule targeting HDAC6 leads to intratumoral CD4+ cytotoxic lymphocytes recruitment through MHC-II upregulation on lung cancer cells

BackgroundDespite the current therapeutic treatments including surgery, chemotherapy, radiotherapy and more recently immunotherapy, the mortality rate of lung cancer stays high. Regarding lung cancer, epigenetic modifications altering cell cycle, angiogenesis...

Abstract 2664: Targeting PD-L1/CMTM6 in myeloid cells results in elevated release of CCL2 to improve CD8 T cell-mediated antitumor response

Abstract Background: Although targeting myeloid cells has become one of the next generation targets for cancer immunotherapy, there is a lack of therapeutic tools to specifically target these cells. Our group has developed a novel humanized PD-L1-targeting antibody, H1A, that induces PD-L1 degradation via blockade of PD-L1 and CMTM6, therefore limiting PD-L1 intrinsic signaling while also preventing interactions with PD-1. In the immune cell compartment, CMTM6 expression is restricted to myeloid cells, making these cells the primary target of H1A. Recently, we unexpectedly found that targeting intrinsic PD-L1 in myeloid cells via H1A resulted in increased release of CCL2 from myeloid cells. In this study, we examined the role of CCL2/CCR2 signaling in CD8 T cell function using human peripheral blood-derived immune cells and evaluated the therapeutic potential of H1A antibody using our humanized PD-L1/PD-1 mouse model. Methods: Human PBMCs derived from healthy donors were treated with H1A or Atezolizumab, a blocking PD-L1 antibody, followed by mass cytometry and flow cytometry to evaluate immune cell phenotype and functional states. PD-L1 expressing myeloid cells isolated from human PBMCs were treated with H1A or Atezolizumab and assessed for CCL2 secretion using multiplex immunoassays. CCL2 production and transcription were confirmed by flow cytometry and single-cell RNA-sequencing. CCR2 blockade was used on human PBMCs to evaluate the effects of CCL2/CCR2 signaling on T cell function alongside H1A treatment. Finally, in vivo antitumor activity of H1A and Atezolizumab were compared in two humanized PD-L1 tumor models. Results: H1A reduced the frequency of regulatory T cells and increased effector T cells, in whole PBMCs upon global T cell activation with anti-CD3/CD28. H1A enhanced the secretion and production of CCL2 from myeloid cells, which was confirmed at a transcript level. CCR2 blockade diminished CD8 T cells’ ability to differentiate into effector cells in H1A treated PBMCs. H1A demonstrated comparable therapeutic effects with Atezolizumab in treatment of immunogenic and ICI-responsive mouse tumor MC38, which is CD8 T cell-dependent, but showed superior therapeutic effects in treatment of ICI-insensitive mouse tumor E0771 compared to Atezolizumab. Both treatments elicited durable memory specific immune responses upon E0771 tumor rechallenge. Conclusion: H1A demonstrated a superior antitumor activity compared to Atezolizumab in our preclinical models via a new mechanism by which H1A caused degradation of intrinsic PD-L1 in myeloid cells. This causes more release of CCL2 to improve T cell function via CCR2 upon T cell activation. Thus, H1A is emerging as a new immunotherapy tool to target PD-L1/CMTM6 in myeloid cells for treatment of cancers that are refractory to current immune checkpoint inhibitor therapy. Citation Format: Michelle Alexandria Hsu, Xin Liu, Ying Li, Whitney Barham, Kevin Pavelko, Jake Hirdler, Fabrice Lucien, Haidong Dong. Targeting PD-L1/CMTM6 in myeloid cells results in elevated release of CCL2 to improve CD8 T cell-mediated antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2664.

Abstract 6629: Cancer-associated nerves regulate anti-tumor immunity in pancreatic cancer

Abstract Nerves serve an emerging component of the tumor microenvironment (TME), contributing to tumorigenesis and cancer progression. Neural invasion (NI) is a common feature of pancreatic ductal adenocarcinoma (PDAC), accounting for distant metastasis and local recurrence. However, the mechanism by which tumor associated nerves (TANs) regulate anti-tumor immunity remains largely elusive. Here, we investigated the association between TANs and immunosuppressive TME to develop more effective therapeutic strategies for PDAC. Using tumor samples of PDAC patients, we identified that the nerves invaded by cancer cells showed increased adrenergic activation accompanied with no parasympathetic or sensory innervation changes. Patients with severe NI were associated with increased catecholamine levels. Additionally, single-cell RNA-sequencing and spatial transcriptome analysis of tumors from patients with PDAC revealed that peri-neural niches of severe NI patients were characterized by decreased CD8+ T cells and increased activity of M2 polarization of tumor-associated macrophages (TAMs), and regulatory T (Treg) cells, which was validated by multiplexed immunofluorescence staining. We further identified that CD8+ T cells expressing β-adrenaline receptors (β-ARs) reduced proliferation and cytokine production when exposure to catecholamines in a dose-dependent manner or co-cultured with dorsal root ganglia pretreated with cancer cells. KrasLSL-G12D; p53LoxP; Pdx1-CreER (KPC) mice treated with catecholamines accelerated tumor progression by inhibiting T cell-mediated antitumor responses, which was abrogated by surgical sympathectomy of KPC mice. Especially, in an orthotopic PDAC model, nonselective β-blocker propranolol in combination with immune checkpoint blockade (ICB) significantly enhanced active CD8+ T cells with concurrent reduced tumor growth. We suggest a novel regulatory mechanism of inhibiting anti-tumor immunity that can promote cancer progression mediated by TANs invasion. These data establish a close connection between the nerves, cancer cells, and immune cells and provide the potential novel targets of therapeutic approaches in PDAC. Citation Format: Ruiyuan Xu, Jianlu Song, Yuan Chen, Xinpeng Yin, Chengcheng Wang, Yupei Zhao. Cancer-associated nerves regulate anti-tumor immunity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6629.

Abstract 4048: Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer

Abstract Despite recent advances in treatment, lung adenocarcinoma (LUAD), particularly harboring Kras mutations, is still the leading cause of cancer mortality. We and others have previously shown that Kras mutant LUAD development and progression is associated with intrinsic inflammatory characteristics driven by epithelial NF-κB activation, increased levels of IL-6 (which is transcriptionally regulated by NF-κB), and activation of the IL-6-responsive transcription factor STAT3. We further demonstrated that IL-6 blockade suppresses lung tumorigenesis by reducing tumor-associated immunosuppressive myeloid populations and induction of a T cell-mediated anti-tumor response. The recent discovery of a novel SOS1-targeting compound (BI-3406) that selectively inhibits the KRAS-SOS1 interaction and leads to anti-proliferative activity has opened up the possibility to more efficiently inhibit Kras mutant lung cancer. Accordingly, in this study we used a mouse model of KRAS mutant LUAD (CC-LR) to compare the therapeutic effects of BI-3406, IL-6 blockade, and MEK inhibitor (trametinib). We also tested whether inhibiting IL-6 driven pro-tumor immune response would provide a synergistic/additive response and increase the tumor inhibitory effect of either BI-3406 or Trametinib. Eight cohorts of 14-week-old female mice were treated with either anti-IL-6 antibody, trametinib, or BI-3406, or their combinations for 4 weeks, and their tumor burden was compared at the end of study. We observed a significant reduction in tumor multiplicity and tumor area in response to all monotherapies, with the highest effect in the trametinib treated cohort. Neither combination therapy led to decreased tumor burden over either of the monotherapy regimens in our model. However, we observed an increase in T helper 1 and CD8 T expressing IFNγ cell populations in the cohort treated with Trametinib and anti-IL-6 Ab combination. In addition, using qPCR analysis of RNA extracted from the whole lung we found a higher expression of IFNγ along with a reduced expression of IL-10 in the cohort treated with BI-3406 and anti-IL-6 Ab combination. This suggests that these combinations may have the potential to improve the anti-tumor immune responses. Hence, we conclude that further adjustment of the treatment regimen (doses, interval, duration) may allow to obtain improved anti-tumor efficacy with these combinations. Overall, this study provides a new research perspective for the improvement of currently available treatment modalities for patients with Kras mutant lung cancer that could be further explored preclinically. Citation Format: Katherine E. Larsen, Maria J. Arredondo Sancristobal, Melody Zarghooni, Avantika Krishna, Maria T. Grimaldo, Nastaran Karimi, Arnav Gaitonde, Michael J. Clowers, Seyed Javad Moghaddam. Comparative effects of SOS1 inhibitor with IL-6 blockade and their combination on Kras mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4048.