Intratumoral NK Cells Research Articles

A mechanistic study of radiotherapy on intratumoral NK cell infiltration augmentation by regulating the EZH2/CXCL10 pathway in hepatocellular carcinoma cells

Objective: To investigate the effect and associated mechanism of tumor tissue-infiltrating NK cells after receiving radiotherapy for hepatocellular carcinoma (HCC). Methods: A HCC tumor-bearing mouse model was constructed using human hepatocellular carcinoma cell line (SK-Hep-1) and divided into four groups: control, radiotherapy, NK cell clearance, and NK clearance combined with radiotherapy. Tumor growth condition was simultaneously recorded. The NK cell ratio in peripheral blood and the NK cell intratumoral infiltration condition were detected by flow cytometry and immunohistochemistry. Lentiviral-constructed SK-Hep-1 cells was used to detect the effect of radiotherapy on the regulation of CXCL10 and NK cell chemotaxis following EZH2 overexpression. SK-Hep-1 cells were irradiated in vitro and in vivo. The expression levels of EZH2 and CXCL10 mRNA and protein in the two groups of cell lines and mouse tumor tissues were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and immunohistochemistry. The chemotaxis and blocking experiments were used to validate the chemotaxis effect of CXCL10 on NK cells. The independent sample t-test was used to compare the groups. P<0.05 was considered statistically significant. Results: The HCC tumor-bearing mouse model experiment showed that HCC tumor growth was most remarkable in the NK clearance combined with the radiotherapy group compared to the radiotherapy group (P<0.05). Compared with the control group, the number of NK cells in the peripheral blood of nude mice in the radiotherapy group was significantly reduced, while the NK cell intratumoral infiltration was significantly increased (P<0.05). Flow cytometry and immunohistochemistry showed in vitro and in vivo expressional alterations. The average expression levels of EZH2 mRNA and protein in hepatocellular carcinoma cell lines and tumor tissues were decreased in the radiotherapy group than the control group and mouse tumor tissues (P<0.05), while the mRNA and protein expression levels of CXCL10 increased (P<0.05). The cell supernatant following radiotherapy enhanced NK cell chemotaxis but inhibited CXCL10 neutralization. EZH2 overexpression validated that radiotherapy up-regulated CXCL10 mRNA and down-regulated protein expression levels in in vitro and in vivo experiments (P<0.05). The chemotactic effect on NK cells was significantly weakened with EZH2 overexpression following radiotherapy. Conclusion: NK cells, as immune effector cells, are directly involved in radiotherapy- activated anti-HCC immunity. Importantly, radiotherapy inhibits EZH2 expression in hepatocellular carcinoma, thereby upregulating CXCL10 expression and enhancing intratumoral NK cell invasion.

H3K27M diffuse midline glioma is homologous recombination defective and sensitized to radiotherapy and NK cell-mediated antitumor immunity by PARP inhibition.

Radiotherapy (RT) is the primary treatment for diffuse midline glioma (DMG), a lethal pediatric malignancy defined by histone H3 lysine 27-to-methionine (H3K27M) mutation. Based on the loss of H3K27 trimethylation producing broad epigenomic alterations, we hypothesized that H3K27M causes a functional double-strand break (DSB) repair defect that could be leveraged therapeutically with PARP inhibitor and RT for selective radiosensitization and antitumor immune responses. H3K27M isogenic DMG cells and orthotopic brainstem DMG tumors in immune deficient and syngeneic, immune competent mice were used to evaluate the efficacy and mechanisms of PARP1/2 inhibition by olaparib or PARP1 inhibition by AZD9574 with concurrent RT. H3K27M mutation caused an HRR defect characterized by impaired RT-induced K63-linked polyubiquitination of histone H1 and inhibition of HRR protein recruitment. H3K27M DMG cells were selectively radiosensitized by olaparib in comparison to isogenic controls, and this effect translated to efficacy in H3K27M orthotopic brainstem tumors. Olaparib and RT induced an innate immune response and induction of NK cell (NKG2D) activating ligands leading to increased NK cell-mediated lysis of DMG tumor cells. In immunocompetent syngeneic orthotopic DMG tumors, either olaparib or AZD9574 in combination with RT enhanced intratumoral NK cell infiltration and activity in association with NK cell-mediated therapeutic responses and favorable activity of AZD9574. The HRR deficiency in H3K27M DMG can be therapeutically leveraged with PARP inhibitors to radiosensitize and induce an NK cell-mediated antitumor immune response selectively in H3K27M DMG, supporting the clinical investigation of best-in-class PARP inhibitors with RT in DMG patients. H3K27M DMG are HRR defective and selectively radiosensitized by PARP inhibitor.PARP inhibitor with RT enhances NKG2D ligand expression and NK cell-mediated lysis.NK cells are required for the therapeutic efficacy of PARP inhibitor and RT. Radiotherapy is the cornerstone of H3K27M-mutant diffuse midline glioma treatment, but almost all patients succumb to tumor recurrence with poor overall survival, underscoring the need for RT-based precision combination therapy. Here, we reveal HRR deficiency as an H3K27M-mediated vulnerability and identify a novel mechanism linking impaired RT-induced histone H1 polyubiquitination and the subsequent RNF168/BRCA1/RAD51 recruitment in H3K27M DMG. This model is supported by selective radiosensitization of H3K27M DMG by PARP inhibitor. Notably, the combination treatment results in NKG2D ligand expression that confers susceptibility to NK cell killing in H3K27M DMG. We also show that the novel brain penetrant, PARP1-selective inhibitor AZD9574 compares favorably to olaparib when combined with RT, prolonging survival in a syngeneic orthotopic model of H3K27M DMG. This study highlights the ability of PARP1 inhibition to radiosensitize and induce an NK cell-mediated antitumor immunity in H3K27M DMG and supports future clinical investigation.

Abstract 1340: Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™

Abstract Natural Killer (NK) cells are highly responsive cytotoxic immune cells of the innate immune system with well characterized anti-tumor properties. Their ability to directly kill malignant cells and elicit an adaptive immune response makes them a promising candidate for a precision guided immunotherapy in oncology. Bicycle® peptides are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle® platform technology to discover and evaluate a new class of fully synthetic molecules termed NK tumor immune cell agonists (NK-TICA™). The NK-TICA™ consists of chemically coupled Bicycle® peptides that bind specifically to the key activating receptor, NKp46, and to tumor antigens, that results in highly potent, antigen-dependent receptor activation and NK cell function. We demonstrate potent, selective binding of our Bicycle® peptides to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner. In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle® NK-TICA™ as a multifunctional immune cell engager will promote the modulation and anti-tumor activity of peripheral and intra-tumoral NK cells to solid tumors. Citation Format: Fay Dufort, Tucker Ezell, Alexandra Rezvaya, Kathleen Ho, Christopher Leitheiser, Philip Brandish, Kevin McDonnell, Nicholas Keen. Modulation of the natural killer cell immune response to tumor with a synthetic tumor -immune cell agonist, NK-TICA™ [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 1340.

Combined IL6 and CCR2 blockade potentiates antitumor activity of NK cells in HPV-negative head and neck cancer

BackgroundWhile T cell-activating immunotherapies against recurrent head and neck squamous cell carcinoma (HNSCC) have shown impressive results in clinical trials, they are often ineffective in the majority of patients. NK cells are potential targets for immunotherapeutic intervention; however, the setback in monalizumab-based therapy in HNSCC highlights the need for an alternative treatment to enhance their antitumor activity.MethodsSingle-cell RNA sequencing (scRNA-seq) and TCGA HNSCC datasets were used to identify key molecular alterations in NK cells. Representative HPV-positive ( +) and HPV-negative ( −) HNSCC cell lines and orthotopic mouse models were used to validate the bioinformatic findings. Changes in immune cells were examined by flow cytometry and immunofluorescence.ResultsThrough integration of scRNA-seq data with TCGA data, we found that the impact of IL6/IL6R and CCL2/CCR2 signaling pathways on evasion of immune attack by NK cells is more pronounced in the HPV − HNSCC cohort compared to the HPV + HNSCC cohort. In orthotopic mouse models, blocking IL6 with a neutralizing antibody suppressed HPV − but not HPV + tumors, which was accompanied by increased tumor infiltration and proliferation of CD161+ NK cells. Notably, combining the CCR2 chemokine receptor antagonist RS504393 with IL6 blockade resulted in a more pronounced antitumor effect that was associated with more activated intratumoral NK cells in HPV − HNSCC compared to either agent alone.ConclusionsThese findings demonstrate that dual blockade of IL6 and CCR2 pathways effectively enhances the antitumor activity of NK cells in HPV-negative HNSCC, providing a novel strategy for treating this type of cancer.

PD-L1-expressing natural killer cells predict favorable prognosis and response to PD-1/PD-L1 blockade in neuroblastoma

ABSTRACT T/NK cell-based immunotherapy has achieved remarkable success in adult cancers but has limited efficacy in pediatric malignancies including high-risk neuroblastoma (NB). Immune defects of NB tumor microenvironment are poorly understood compared with adults. Here, we described the unique characteristics of NB immune contexture and determined the phenotype signatures of PD-L1-expressing CD8+ T and NK cells in NB tumors by systemically analyzing the spatial distribution of T and NK cells and the distinct expression of programmed death 1 (PD-1) and its ligand (PD-L1) in patients with NB. We found that PD-L1-expressing CD8+ T and NK cells in NB tumors were highly activated and functionally competent and associated with better clinical outcomes. Intratumoral NK cells were a favorable prognostic biomarker independent of CD8+ T cells, PD-1/PD-L1 expression, tumor stage, MYCN amplification, and risk classification. NK cells combined with anti-PD-1/PD-L1 antibodies showed potent antitumor activity against both MYCN-amplified and non-amplified NBs in vitro and in vivo, and PD-L1-expressing NK cells associated with improved antitumor efficacy. Collectively, we raise novel insights into the role of PD-L1 expression on CD8+ T-cell and NK-cell activation. We highlight the great potential of intratumoral NK cells in better defining risk stratification, and predicting survival and response to anti-PD-1/PD-L1 therapy in NB. These findings explain why single anti-PD-1/PD-L1 therapy may not be successful in NB, suggesting its combination with NK cell-adoptive cellular therapy as a promising strategy for relapsing/refractory NB. This study provides a potential prospect that patients with PD-L1-expressing NK cells may respond to anti-PD-1/PD-L1 therapy.

Tumor infiltrating natural killer cells in soft tissue sarcoma are predominantly CD56 dim: Implications for outcomes

Abstract Natural killer (NK) cells have been shown to be important mediators of anti-tumor responses, including in soft tissue sarcomas (STS). NK cells show significant heterogeneity, depending on maturation, tissue residency, and inflammatory environment. As previous studies have suggested that tumor-infiltrating NK cells (TiNKs) acquire a less cytotoxic CD56 brighttissue resident phenotype, we sought to compare blood versus tumor NK cell phenotype in STS and evaluate any association with survival. Blood and tumor from 27 STS patients undergoing surgery from 2018–2022 were collected prospectively for flow cytometry and retrospectively analyzed. 52% were female, the mean age was 59, and 74% were AJCC stage 3. Increasing absolute number of NK cells in the blood correlated with longer survival (P&amp;lt;0.005, r=0.6). As expected, CD56 dimNK cells predominated in the blood (91.5±5.8% of CD3-CD56+ lymphocytes compared to 80.1±13% in tumor, P&amp;lt;0.005). In contrast, CD56 brightcells were enriched in tumor representing 18.5±13% compared to 8.4±5.8% in blood (P&amp;lt;0.005). Although CD56 brightNK cells were approximately 2.4±3-fold higher in tumor compared to blood, CD56 dimNK cells still represented the majority of TiNKs. Higher proportions of both overall NK cells and CD56 dimNK cells in STS tumors were associated with better metastasis-free survival on Kaplan-Meier analysis (P&amp;lt;0.05). CD56 dimTiNKs had higher NKp46 expression than CD56 brights. In conclusion, both blood and intra-tumoral NK cells appear prognostic in STS. Although the proportion of CD56 brightTiNKs in STS is higher than blood the majority of TiNKs are CD56 dimconsistent with a cytotoxic phenotype. Better characterization of NK subsets in STS is likely to have translational significance.

Development of a deep pathomics score for predicting hepatocellular carcinoma recurrence after liver transplantation

Background and purposeTumor recurrence after liver transplantation (LT) impedes the curative chance for hepatocellular carcinoma (HCC) patients. This study aimed to develop a deep pathomics score (DPS) for predicting tumor recurrence after liver transplantation using deep learning.Patients and methodsTwo datasets of 380 HCC patients who underwent LT were enrolled. Residual convolutional neural networks were used to identify six histological structures of HCC. The individual risk score of each structure and DPS were derived by a modified DeepSurv network. Cox regression analysis and Concordance index were used to evaluate the prognostic significance. The cellular exploration of prognostic immune biomarkers was performed by quantitative and spatial proximity analysis according to three panels of 7-color immunofluorescence.ResultsThe overall classification accuracy of HCC tissue was 97%. At the structural level, immune cells were the most significant tissue category for predicting post-LT recurrence (HR 1.907, 95% CI 1.490–2.440). The C-indices of DPS achieved 0.827 and 0.794 in the training and validation cohorts, respectively. Multivariate analysis for recurrence-free survival (RFS) showed that DPS (HR 4.795, 95% CI 3.017–7.619) was an independent risk factor. Patients in the high-risk subgroup had a shorter RFS, larger tumor diameter and a lower proportion of clear tumor borders. At the cellular level, a higher infiltration of intratumoral NK cells was negatively correlated with recurrence risk.ConclusionsThis study established an effective DPS. Immune cells were the most significant histological structure related to HCC recurrence. DPS performed well in post-LT recurrence prediction and the identification of clinicopathological features.

Abstract 2915: Intratumoral co-injections of NK cells and neutralizing antibodies against the NKG2A inhibitory pathway achieve efficacious anti-tumor responses in a CD8 T cell and cDC1-dependent manner

Abstract Local intratumoral NK cell activity is known to foster adaptive T cell-mediated immunity against cancer, that conceivably is inhibited by the interactions of the inhibitory receptor NKG2A with the MHC class I molecule HLA-E in humans or Qa-1b in mice. In this study, we found that intratumoral delivery of NK cells significantly attains therapeutic effects only if co-injected with anti-NKG2A and anti-Qa1b blocking monoclonal antibodies (mAb) in two solid mouse tumors models. Such therapeutic activity was contingent on endogenous CD8 T cells and on type-1 conventional dendritic cells (cDC1), which mediate cross-priming of tumor antigens. Moreover, the anti-tumor effects can be enhanced upon combination with anti-PD-1 mAb. This combination achieves at least partial abscopal efficacy against distant untreated tumors. In xenografted mice with HLA-E-expressing human cancer cells, intratumoral co-injection of allogeneic human NK cells and the clinical-grade anti-NKG2A mAb monalizumab synergistically achieved therapeutic effects and gave rise to stronger antibody-dependent cellular cytotoxicity (ADCC) in conjunction with cetuximab. In conclusion, these studies provide evidences for the therapeutic potential of on-site NK cell-based immunotherapies that may recruit endogenous T-cell responses. Citation Format: Maite Alvarez, Maria C. Ochoa, Carmen Molina, Alvaro Teijeira, Saray Garasa, Sandra Sanchez-Gregorio, Irene Olivera, Javier Glez-Vaz, Asunta Cirella, Gabriel Gomis, Jose Gonzalez-Gomariz, Pedro Berraondo, Ignacio Melero. Intratumoral co-injections of NK cells and neutralizing antibodies against the NKG2A inhibitory pathway achieve efficacious anti-tumor responses in a CD8 T cell and cDC1-dependent manner [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 2915.

KLRF1, a novel marker of CD56bright NK cells, predicts improved survival for patients with locally advanced bladder cancer.

Bladder tumor-infiltrating CD56bright NK cells are more tumor cytotoxic than their CD56dim counterparts. Identification of NK cell subsets is labor-intensive and has limited utility in the clinical setting. Here, we sought to identify a surrogate marker of bladder CD56bright NK cells and to test its prognostic significance. CD56bright and CD56dim NK cells were characterized with the multiparametric flow (n=20) and mass cytometry (n=21) in human bladder tumors. Transcriptome data from bladder tumors (n=351) profiled by The Cancer Genome Atlas (TCGA) were analyzed. The expression levels of individual markers in intratumoral CD56bright and CD56dim NK cells were visualized in tSNE plots. Expressions of activation markers were also compared between Killer Cell Lectin-Like Receptor Subfamily F Member 1 (KLRF1)+ and KLRF1- NK cells. Intratumoral CD56bright NK cells displayed a more activated phenotype compared to the CD56dim subset. Multiple intratumoral cell types expressed CD56, including bladder tumor cells and nonspecific intratumoral CD56 expression was associated with worse patient survival. Thus, an alternative to CD56 as a marker of CD56bright NK cells was sought. The activation receptor KLRF1 was significantly increased on CD56bright but not on CD56dim NK cells. Intratumoral KLRF1+ NK cells were more activated and expressed higher levels of activation molecules compared with KLRF1- NK cells, analogous to the distinct effector function of NK cells across CD56 expression. High intratumoral KLRF1 was associated with improved recurrence-free survival (hazard ratio [HR] 0.53, p=0.01), cancer-specific survival (HR 0.47, p=0.02), and overall survival (HR 0.54, p=0.02) on multivariable analyses that adjusted for clinical and pathologic variables. KLRF1 is a promising prognostic marker in bladder cancer and may guide treatment decisions upon validation.

The prognostic impact of the immune signature in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.

PD-1 expression on mouse intratumoral NK cells and its effects on NK cell phenotype

SummaryAlthough PD-1 was shown to be a hallmark of T cells exhaustion, controversial studies have been reported on the role of PD-1 on NK cells. Here, we found by flow cytometry and single cell RNA sequencing analysis that PD-1 can be expressed on MHC class I-deficient tumor-infiltrating NK cells in vivo. We also demonstrate distinct alterations in the phenotype of PD-1-deficient NK cells and a more mature phenotype which might reduce their capacity to migrate and kill in vivo. Tumor-infiltrating NK cells that express PD-1 were highly associated with the expression of CXCR6. Furthermore, our results demonstrate that PD-L1 molecules in membranes of PD-1-deficient NK cells migrate faster than in NK cells from wild-type mice, suggesting that PD-1 and PD-L1 form cis interactions with each other on NK cells. These data demonstrate that there may be a role for the PD-1/PD-L1 axis in tumor-infiltrating NK cells in vivo.

The Combination of Radiotherapy and Complement C3a Inhibition Potentiates Natural Killer cell Functions Against Pancreatic Cancer.

Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral NK cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiation therapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell-based immunotherapy against pancreatic cancer, its combination with radiation therapy hold greater therapeutic benefit.

Abstract 6046: Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma

Abstract Background: Osteosarcoma (OS) is the most common bone malignancy in childhood and adolescence. Though the prognosis of OS patients has been improved by multimodal therapies, patients with metastatic disease still have a very poor prognosis. We previously reported a novel TBL1/β-catenin inhibitor, Tegavivint, suppresses OS primary tumor and lung metastasis using patient-derived cell lines. Wnt/β-catenin signaling is reported to influence intratumoral T cell infiltration and using murine syngeneic OS models we investigated the effects of Tegavivint on improving immune checkpoint inhibition and the OS tumor immune microenvironment. Methods: We used murine OS cell lines F331 and F420 for in vitro and in vivo studies. We performed cytotoxicity studies using the CCK-8 assay and western blots to investigate downstream β-catenin signaling perturbations secondary to Tegavivint. Intratibial injections were performed into C57BL/6 mice using both murine cell lines. Tegavivint and anti-PD-1 antibody was administered as monotherapy or in combination. After treatment, we compared the tumor volume in the control, monotherapy (Tegavivint or PD-1 only) and combination therapy groups (Tegavivint and PD-1). We assessed T cell tumor infiltration using immunohistochemistry and flow cytometry. Results: In vitro, Tegavivint suppresses the growth of F331 and F420 cell lines with IC50s of 9.52nM and 49.4nM, respectively after 72hours of treatment. In vivo, anti-PD1 monotherapy did not show any significant anti-tumor activity, but Tegavivint monotherapy significantly suppressed the growth of murine tumors (p&amp;lt;0.05) and the combination therapy further improved overall anti-tumor activity. IHC staining showed an increase in CD8-positive T cells in Tegavivint monotherapy and combination therapy treated tumors compared with the control tumors. Furthermore, flow cytometry validated the increase in CD8-positive T cell infiltration and also showed an increase in intratumoral NK cells in Tegavivint treated tumors. Conclusions: These results indicate that TBL1 inhibition, via Tegavivint, inhibits nuclear β-catenin activity and alters the tumor immune microenvironment and enhances immune checkpoint blockade in OS. Future directions include more comprehensive profiling and molecular analysis of the tumor and immune microenvironment, and evaluation of additional immune modulatory approaches for the treatment of OS. Citation Format: Kengo Nakahata, Bikesh K. Nirala, Ryan L. Shuck, Lyazat Kurenbekova, Jason T. Yustein. Targeting TBL1 inhibits nuclear β-catenin activity and enhances immune checkpoint inhibition efficacy in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6046.

Abstract 2325: Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers

Abstract We discovered a crucial change common to all platinum resistant (CR) NSCLC cells, wherein a high level of reactive oxygen species (ROS) modulates tumor metabolism. CR cells activate the kynurenine (KYN) pathway (KP) during excessive oxidative stress to maintain high ROS for proliferation by catabolizing tryptophan (TRP) to KYN via indoleamine 2,3-dioxygenase-1 (IDO1); and by expressing (3-5x) higher levels of programmed death-ligand 1 (PD-L1) compared to platinum sensitive cells (p&amp;lt;0.02; n=8). We detected significant increases in IDO1 activity (3-4x), identified by the KYN/TRP ratio, in CR cells’ culture media (p&amp;lt;0.05; n=8). Extracellular KYN is known to program naïve T-cells to the immune suppressive regulatory T-cell (T-reg) phenotype. Using a human peripheral blood mononuclear cell (PBMC) co-culture system, we found a higher frequency of regulatory T-cells (Tregs; CD4+CD25+Foxp3+) and a lower frequency of effector T-cells (Teffs CD3+CD8+) and NK cell (CD3-CD314+CD335+) activity in co-culture with CR cells compared to cisplatin sensitive cancer cells. Importantly, inhibition of IDO1 and Tryptophan 2,3-Dioxygenase (TDO2), the enzymes involved in the rate limiting step in the KP, with the dual inhibitor AT-0174 at 20µM (48h) suppressed Tregs, but enhanced Teffs and NK cell populations in CR co-cultures. Next, we used a syngeneic mouse model of LLC (Lewis lung cancer) vs cisplatin resistant LLC-CR, to examine the effects of AT-0174 (170 mg/kg; P.O. once daily) in combination with anti-PD1 antibody (10 mg/ml; I.P. once every 3 days). Consistent with the in vitro model, analysis of mice with LLC-CR tumors found higher intratumoral T-reg and lower NK cell populations. Treatment with AT-0174 resulted in higher tumor infiltrating lymphocytes of Teffs and NK and lower Treg frequency in LLC-CR tumors than treatment with a selective IDO1 inhibitor (epacadostat). More importantly, the combination of AT-0174+antiPD1 reversed the immunosuppressive tumor microenvironments with further enhancement in Teffs and NK frequencies and diminished tumor growth in CR allografts with a significant reduction of total tumor weight compared with LLC mouse tumors (n=10, P&amp;lt;0.01). Our new findings support the potential therapeutic efficacy of the dual IDO/TDO inhibitor AT-0174 as an immuno-therapeutic treatment that enhances tumor immune surveillance and blocks tumor metabolism related survival mechanisms in platinum resistant NSCLC tumors. Support by the Dept. of Veterans Affairs. Citation Format: Sydney Spector, Chunjing Wu, Dan Nguyen, George Theodore, Ashley Garcia, Lynn Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting kynurenine pathway using novel IDO/TDO dual inhibitor (AT0174) to modulate tumor microenvironment in platinum resistant non-small cell lung cancer cancer: An immunometabolism compliment markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2325.

Abstract 3513: INBRX-121, a safe and efficacious molecular targeted cytokine that enhances NK cell-mediated tumor killing

Abstract IL-2 is a powerful cytokine that enhances the activity of and induces the expansion of tumoricidal effector cells including T and NK cells. Despite promising signs of activity, the clinical utility of IL-2 is limited due to severe treatment-associated toxicities including vascular leak syndrome and the concurrent expansion of immunosuppressive regulatory T cells, which blunts efficacy. We developed a molecular targeted cytokine (MTC) platform that combines an engineered IL-2 variant with reduced affinity for the IL-2 receptor and high affinity single-domain antibodies (sdAb) to restrict IL-2 receptor signaling to cells expressing the target antigen (termed cis-signaling). INBRX-121 is an MTC designed to deliver IL-2 to NK cells using an NKp46-specific sdAb as the targeting moiety. In vitro, INBRX-121 induces dose-dependent STAT5 phosphorylation and proliferation in NK cells with comparable potency to wild-type IL-2, while showing no NKp46-independent activity at concentrations of more than three magnitudes higher than that required for NK cell activity. INBRX-121 enhances NK cell activation and cytotoxic capacity, thereby reducing the NK-to-target cell ratio as well as the target receptor density needed for optimal tumor cell killing. Furthermore, INBRX-121 lowers the threshold for antibody-dependent cellular cytotoxicity (ADCC) and increases maximal target cell killing in combination with ADCC-competent antibodies to an extent that may lessen the need for Fc engineering to enhance binding to the low affinity CD16A variant. In vivo, INBRX-121 demonstrates potent anti-tumor activity with increased intra-tumoral NK cell accumulation and upregulation of NK cell activation markers. INBRX-121 monotherapy reduces primary tumor growth and the formation of metastasis, while combination with ADCC-competent antibodies further increases NK cell activity to convert tumor growth suppression into complete tumor elimination. INBRX-121 is well tolerated in rodents and non-human primates and no overt signs of toxicity have been observed in cynomolgus monkeys up to the highest administered dose of 30 mg/kg. Our MTC platform provides a novel path to overcoming the limitations of IL-2 therapy through the pinpointed delivery of IL-2 activity to target cells of interest. In the case of INBRX-121 this ensures potent enhancement of the naturally broad anti-tumor activity of NK cells, which have distinct advantages over T cell therapeutics. Unlike T cells, tumor recognition by NK cells is independent of MHC-presented antigens and can elicit immediate cytotoxic activity. Further, many of the toxicities associated with T cell therapeutics have not been observed with NK-centric treatments. INBRX-121 therefore provides a promising safe and efficacious treatment option for a broad array of cancer indications both as a monotherapy and in combination with ADCC-enabled antibodies. Citation Format: Heather Kinkead, Florian J. Sulzmaier, Anya Polovina, Nadja Kern, Angelica Sanabria, Jason Ho, Chelsie Macedo, Ryan Henderson, Abrahim Hussain, Rajay Pandit, William Crago, Emily Rowell, Brendan P. Eckelman. INBRX-121, a safe and efficacious molecular targeted cytokine that enhances NK cell-mediated tumor killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3513.

Abstract 2520: Ovarian tumors escape anti-tumor function of NK cells by inducing CISH expression and its inhibition prevents tumor progression

Abstract Background: Ovarian cancer (OVCA), a lethal malignancy of women, mainly disseminates locally in the peritoneal cavity and the local immune system plays important roles in inhibiting its progression. Although NK cells, a member of innate immunity, recognize and exert anti-tumor immune responses, tumors escape NK cell immunity and progress to late stages by inducing factors like inhibitory receptors and cytokines. IL-15, a cytokine, stimulates NK cell function and persistent exposure to IL-15 induces cytokine inducible SH2-containing protein (CISH), an inhibitory receptor and a marker of NK cell exhaustion. It is unknown if ovarian tumors use this phenomenon in their favor as the tumor secretes enhanced levels of IL-15. Furthermore, dietary supplementation with root powder of Ashwagandha (ASH), a natural herb and an anti-stress compound has shown to decrease the rates of OVCA metastasis and enhance the frequency of intratumoral NK cells in a preclinical model. However, it is unknown if ASH supplementation improves NK cell frequency by inhibiting CISH. The goal of this study was to examine if OVCA progresses by inducing CISH-expression in NK cells and if dietary ASH supplementation reduces NK cell exhaustion by decreasing CISH expression. Materials and methods: Two studies were conducted. Study 1: Exploratory study with clinical specimens including normal ovaries (n=5) and ovarian high grade serous carcinoma at early and late stages (n=5 each). Study 2: Prospective study using the laying hen model of OVCA supplemented with or without 2% dietary ASH root powder for 90 days (n=7 for each group; normal, early and late stage). Hen normal and tumor ovarian tissues were collected at the end of the study. Both the clinical and hen specimens were used for immunohistochemistry, Western blotting and gene expression assays to determine the frequency of CISH-expressing NK-cells and intensity of GRP78 expression (a marker of cellular stress). Data were collected and analyzed by ANOVA and unpaired t-test and considered significant when P&amp;lt;0.05. Results: CISH-expressing NK cells were localized in normal and tumor ovaries in women and hens. Compared with normal ovaries, frequency of CISH-expressing NK cells was significantly higher in tumors at early and late stages in patients and hens. Similarly, strong expression of CISH gene and protein (29kDa) was detected in tumor tissues. Enhanced expression of CISH positively correlated with expression of GRP78. Compared with untreated, ASH supplementation reduced tumor progression in 5 out of 7 hens and significantly decreased the frequency of CISH-expressing NK cells and GRP78 (P&amp;lt;0.001). Conclusion: The results of this study suggest that expression of CISH by NK cells and cellular stress were associated with OVCA progression and dietary ASH supplementation reduced NK cell exhaustion by decreasing CISH and GRP78. Support: Swim Across America and NIH CA187309 Citation Format: Jasmin C. Acosta, Amy K. Stasik, Janice M. Bahr, Animesh Barua. Ovarian tumors escape anti-tumor function of NK cells by inducing CISH expression and its inhibition prevents tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2520.

Fractalkine Elicits Chemotactic, Phenotypic, and Functional Effects on CX3CR1+CD27- NK Cells in Obesity-Associated Cancer.

Esophagogastric adenocarcinomas (EAC) are obesity-associated malignancies underpinned by severe immune dysregulation and inflammation. Our previous work indicates that NK cells migrate to EAC omentum, where they undergo phenotypic and functional alterations and apoptosis. In this study, we investigate whether such erroneous chemotaxis to omentum is paralleled by compromised NK cell infiltration of EAC patient tumor and examine the role of the inflammatory chemokine fractalkine in shaping the NK cell-mediated response. Our data show diminished NK cell frequencies in EAC tumor compared with those in the circulation and reveal that intratumoral NK cell frequencies decline as visceral obesity increases in EAC patients. Our in vitro findings demonstrate that antagonism of fractalkine receptor CX3CR1 significantly reduces NK cell migration to EAC patient-derived, omental adipose tissue-conditioned media, but not toward tumor-conditioned media. These data suggest fractalkine is a key driver of NK cell chemotaxis to omentum but has a lesser role in NK cell homing to tumor in EAC. We propose that this may offer a novel therapeutic strategy to limit NK cell depletion in the omentum of obese EAC patients, and our data suggest the optimal timing for CX3CR1 antagonism is after neoadjuvant chemoradiotherapy. Our functional studies demonstrate that fractalkine induces the conversion from CX3CR1+CD27- to CX3CR1-CD27+ NK cells and increases their IFN-γ and TNF-α production, indicative of its role in shaping the dominant NK cell phenotype in EAC omentum. This study uncovers crucial and potentially druggable pathways underpinning NK cell dysfunction in obesity-associated cancer and provides compelling insights into fractalkine's diverse biological functions.

Abstract 1440: Novel imipridone ONC206 suppresses ovarian cancer progression through modulating immune cell response

Abstract Advanced ovarian cancer (stages III and IV) is the most lethal gynecologic malignancy in the United States. Survival of this cancer has not improved to a major extent over the past decade. High-grade serous ovarian cancer (HGSC), in particular, is notable for its initial sensitivity to chemotherapy using platinum and taxane combination following debulking surgery. However, the vast majority of these cancers (&amp;gt;75-80%) recur within 12-24 months after diagnosis, and patients die of progressively chemotherapy-resistant disease. The identification of prognostic or predictive markers, for ovarian cancer is crucial to the development of therapeutic targets and thus improvement of survival. ONC201 is the first clinical bitopic antagonist of dopamine receptor D2 (DRD2), that is well tolerated and currently being investigated in several clinical trials for oncology. ONC206 is a chemical derivative of ONC201 with the same impridone core structure, which is also a DRD2 antagonist that exhibits distinct receptor pharmacology and nanomolar potency in various preclinical cancer models. However, the effects of ONC206 on HGSC progression and the mechanism of action have not been thoroughly explored. Preliminary data demonstrated that overexpression of DRD2 is associated with poor overall survival rates in patients with advanced stage HGSC. ONC206-treated HGSC cells demonstrated significantly lower growth rates and higher percentage of apoptotic cells than ONC201-treated cells did in vitro. Besides, intraperitoneal injection of ONC206 (100 mg/kg) twice a week into HGSC-bearing immunocompetent mice demonstrated decreased tumor volume in vivo. In addition, by using suspension mass cytometry with a panel of 28 antibodies, fresh tumor tissues from ONC206-treated mice showed markedly increase in intratumoral NK cells and activated CD8+ T cells densities. These data suggest that ONC206 is more potent than ONC201 in suppressing HGSC growth directly. In addition, ONC206 may also suppresses HGSC growth through activating immune cell response in tumor tissues. Further studies that define the molecular mechanism by which DRD2 modulates the tumor suppressive effect of ONC206 on HGSC cells by suppressing HGSC directly or enhancing anti-tumor immune cell activity will be perform to establish ONC206 as a novel therapeutic agent in treatment of HGSC patients. Citation Format: Chi Lam Au Yeung, Wen Hu, Sammy Ferri-Borgogno, Rohinton S. Tarapore, Joshua E. Allen, Karen H. Lu, Samuel C. Mok. Novel imipridone ONC206 suppresses ovarian cancer progression through modulating immune cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1440.

Gene expression analysis of sarcoma-infiltrating NK cells identifies signatures correlating with favorable prognosis and potential new targets for augmenting NK function

Abstract Success of current T-cell based immunotherapies in soft tissue sarcomas (STS) is limited while pre-clinical and clinical studies have shown evidence of NK activity in STS. To investigate the effects of the tumor microenvironment (TME) on tumor-infiltrating NK cell gene expression, we isolated circulating and tumor-infiltrating NK and CD3 T cells from sarcoma patients undergoing surgery (N=4) and performed RNA sequencing analysis. Comparing Differential Gene Expression (DGE) of flow-sorted intra-tumoral NK cells to circulating NK cells, we identified upregulation of genes involved in mitogen signaling inhibition (DUSP4) and metabolic function (SMPD3, SLC7A5), but not of genes typically associated with cytotoxic function (e.g. IFNG, GZMB). In contrast, intra-tumoral T cells showed significant upregulation of both activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. We then queried the TCGA STS dataset and stratified cases into “NK Activating TME” and “NK Suppressing TME” based on the CIBERSORT NK signature analysis program (based on high or low proportion of activated NK cells, respectively). Patients with NK Activating TME had significantly improved survival compared to NK Suppressive TME (log-rank p = 0.03). DGE identified unique tumor-associated gene ontology between NK activated versus suppressive subsets with upregulation of the retinoic acid pathway. Unlike T cells which demonstrate significant DGE between circulating and tumor-infiltrating subsets, NK cells appear to have similar gene expression between blood and tumor. Therefore, although NK cells appear to be prognostic in STS, their anti-tumor effects may be dictated by systemic rather than local factors.

Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

BackgroundNatural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor...