Checkpoint Signaling Pathways Research Articles

PD-1 inhibitor combined with Docetaxel exerts synergistic anti-prostate cancer effect in mice by down-regulating the expression of PI3K/AKT/NFKB-P65/PD-L1 signaling pathway.

Docetaxel is a yew compound antitumor agent with accurate antitumor efficacy, but its application is limited due to the high and serious adverse effects, and finding effective combination therapy options is a viable strategy. Immune checkpoint inhibitors have become hotspots in enhancing anti-tumor immunity by blocking immune checkpoint signaling pathways, but their response rate to monotherapy use is not high and the efficacy is minimal. To explore the anti-tumor effects and mechanisms of the combination of PD-1 inhibitors and Docetaxel through in vivo experiments and develop a feasible combination treatment for the therapy of prostate cancer. Tumor-bearing mice were subcutaneously injected with 0.1ml RM-1 cells. Treatment were taken when the tumor growed up to 3mm, after which the tumor and spleen were removed to test the antitumor effect with Flow cytometric (FACS) analysis, Immunohistochemistry, Western Blot. In this experiment, we found that PD-1 inhibitors combined with Docetaxel had a synergistic effect on mouse prostate cancer, inhibited the growth of prostate cancer, improved survival and reduced adverse reactions, increased spleen and tumor infiltrative CD4+ and CD8+ T cells, especially in group combination with low-dose Docetaxel, and were related to the PI3K/AKT/NFKB-P65/PD-L1 signaling pathway. Our study confirms that PD-1 inhibitors in combination with Docetaxel are a viable combination strategy and provide a safe and effective combination option for the clinical treatment of prostate cancer.

Tumor-associated macrophage enhances PD-L1-mediated immune escape of bladder cancer through PKM2 dimer-STAT3 complex nuclear translocation

Tumor-associated macrophages (TAMs) are important components of the tumor microenvironment (TME) and strongly associated with poor prognosis and drug resistance, including checkpoint blockade immunotherapy in solid tumor patients. However, the mechanism by which TAM affects immune metabolism reprogramming and immune checkpoint signalling pathway in the TME remains elusive. In this study we found that transforming growth factor-beta (TGF-β) secreted by M2-TAMs increased the level of glycolysis in bladder cancer (BLCA) and played important role in PD-L1-mediated immune evasion through pyruvate kinase isoenzymes M2 (PKM2). Mechanistically, TGF-β promoted high expression of PKM2 by promoting the nuclear translocation of PKM2 dimer in conjunction with phosphorylated signal transducer and activator of transcription (p-STAT3), which then exerted its kinase activity to promote PD-L1 expression in BLCA. Moreover, SB-431542 (TGF-β blocker) and shikonin (PKM2 inhibitor) significantly reduced PD-L1 expression and inhibited BLCA growth and organoids by enhancing anti-tumor immune responses. In conclusion, M2-TAM-derived TGF-β promotes PD-L1-mediated immune evasion in BLCA by increasing the PKM2 dimer-STAT3 complex nuclear translocation. Combined blockade of the TGF-β receptor and inhibition of PKM2 effectively prevent BLCA progression and immunosuppression, providing a potential targeted therapeutic strategy for BLCA.

Synthesis, growth inhibitory activity against tumor cells, and structure-activity relationship of CGK733 and its analogs.

CGK733 was reported as a compound that inhibited ATM/ATR kinase activities and blocked their checkpoint signaling pathways with great selectivity. However, this paper was subsequently retracted, and the truth about the activity of CGK733 remains unclear. We synthesized various analogs of CGK733 with a modification of the carboxylic acid moiety and/or the aniline derivative moiety to accumulate knowledge of the structure-activity relationship of this compound. Growth inhibitory activity of CGK733 and novel 35 analogs against HeLa S3 cells was evaluated, and the structure-activity relationship revealed that analogs with the 2-naphthyl or 4-fluorophenyl group instead of the benzhydryl group have activity comparable to CGK733 and that the 3-nitro group on the aniline moiety significantly affects the activity.

Endobronchial Ultrasound Guided Transbronchial Needle Aspiration and PD-L1 Yields

PurposeImmunotherapy is a leading approach for treating advanced non-small cell lung cancer (NSCLC) by targeting the PD-1/PD-L1 checkpoint signaling pathway, particularly in tumors expressing high levels of PD-L1 (Jug et al. in J Am Soc Cytopathol 9:485–493, 2020; Perrotta et al. in Chest 158: 1230–1239, 2020). Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive method to obtain tissue for molecular studies, including PD-L1 analysis, in unresectable tumors (Genova et al. in Front Immunol 12: 799455, 2021; Wang et al. in Ann Oncol 29: 1417–1422, 2018). This study aimed to assess the adequacy of PD-L1 assessment in EBUS-TBNA cytology specimens.MethodsData was collected retrospectively from patients who underwent EBUS-TBNA between 2017 and 2021 for suspected lung cancer biopsy. Samples positive for NSCLC were examined for PD-L1 expression. EBUS was performed by experienced practitioners, following institutional guidelines of a minimum of five aspirations from positively identified lesions. Sample adequacy for molecular testing was determined by the pathology department.ResultsThe analysis involved 387 NSCLC cases (149 squamous cell, 191 adenocarcinoma, 47 unspecified). Of the 263 EBUS-TBNA specimens tested for PD-L1, 237 (90.1%) were deemed adequate. While 84% adhered to the protocol, adherence did not yield better results. Significantly higher PD-L1 adequacy was observed in squamous cell carcinomas (93.2%) compared to adenocarcinoma (87.6%). The number of aspirations and sedation type did not correlate with PD-L1 adequacy in either cancer type, but lesion size and location had a significant impact in adenocarcinomas. Adenocarcinoma exhibited higher PD-L1 expression (68%) compared to squamous cell carcinoma (48%).ConclusionEBUS-TBNA offers high yields for assessing immunotherapy markers like PD-L1, with satisfactory adequacy regardless of NSCLC subtype, lesion size, or location.

Network pharmacology and experimental evaluation strategies to decipher the underlying pharmacological mechanism of Traditional Chinese Medicine CFF-1 against prostate cancer.

Prostate cancer (PCa) is a common malignancy in elderly men. We have applied Traditional Chinese Medicine CFF-1 in clinical treatments for PCa for several years. Here, we aimed to identify the underlying mechanism of CFF-1 on PCa using network pharmacology and experimental validation. Active ingredients, potential targets of CFF-1 were acquired from the public databases. Subsequently, protein-protein interaction (PPI) and the herbs-active ingredients-target network was constructed. A prognostic model for PCa was also constructed based on key targets. In vitro experiments using PCa cell lines CWR22Rv1 and PC-3 were carried out to validate the potential mechanism of CFF-1 on PCa. A total of 112 bioactive compounds and 359 key targets were screened from public databases. PPI and herbs-active ingredients-target network analysis determined 12 genes as the main targets of CFF-1 on PCa. Molecular docking studies indicated that the primary active ingredients of CFF-1 possess strong binding affinity to the top five hub targets. DNMT3B, RXRB and HPRT1 were found to be involved in immune regulation of PCa. In vitro, CFF-1 was found to inhibit PCa cell proliferation, migration, invasion and induce apoptosis via PI3K-Akt, HIF-1, TNF, EGFR-TKI resistance and PD-1 checkpoint signaling pathways. This study comprehensively elucidates the underlying molecular mechanism of CFF-1 against PCa, offering a strong rationale for clinical application of CFF-1 in PCa treatment.

An open-label, multicenter study investigating RP3 oncolytic immunotherapy in combination with first- or second-line systemic atezolizumab plus bevacizumab in patients with locally advanced unresectable or metastatic hepatocellular carcinoma.

TPS576 Background: Despite advances in treatment for unresectable hepatocellular carcinoma (HCC), long-term survival rates remain poor. The combination of atezolizumab (Atezo) plus bevacizumab (Bev) is approved frontline therapy for advanced HCC, but a minority of patients (pts) respond and secondary resistance usually occurs within months. HCC has an immune-suppressed tumor microenvironment (TME), mediated by activated immune checkpoint signaling and angiogenesis pathways, which may contribute to therapeutic resistance. RP3 is a genetically modified herpes simplex virus type 1 (HSV-1) that expresses the fusogenic gibbon ape leukemia virus glycoprotein with the R sequence deleted (GALV-GP-R–), an anti–CTLA-4 antibody-like molecule, CD40 ligand, and 4-1BB ligand. The direct oncolytic effect coupled with immune stimulation by RP3 in the TME is intended to provide systemic antitumor activity and synergize with anti–PD-1/PD-L1 agents, such as Atezo. Preclinical data have demonstrated improved distribution of oncolytic HSV within tumors in combination with Bev, supporting the clinical combination of RP3 with Bev. This study will evaluate the safety and efficacy of RP3 combined with Atezo plus Bev as first- (1L) and second-line (2L) systemic therapies for unresectable and advanced HCC (NCT05733598). Methods: The 1L and 2L cohorts will each enroll up to 30 pts. Pts in the 1L cohort may not have received prior systemic treatment; pts in the 2L cohort must have progressed on or following 1 prior line of systemic therapy, which must have included a PD-1/PD-L1–directed agent. Key inclusion criteria include advanced unresectable HCC with ≥1 measurable tumor of ≥1 cm in longest diameter, Child-Pugh class A, and an Eastern Cooperative Oncology Group performance status of 0 to 1. Key exclusion criteria include untreated esophageal and/or gastric varices with bleeding or at high risk for bleeding and macroscopic invasion of the tumor into any major blood vessel(s) and/or main bile ducts. Pts with a history of medically refractory hepatic encephalopathy and/or hepato-renal syndrome are also excluded. Pts in the 1L cohort will receive Atezo 1200 mg and Bev 15 mg/kg every 3 weeks (Q3W) together with RP3 intratumorally Q3W for a total of up to 8 doses. Pts in the 2L cohort will receive RP3 every 2 weeks for 4 doses with Bev Q3W starting on cycle (C)1 day (D)1, then RP3 and Bev Q3W for up to 4 more doses with Atezo Q3W being added on C4D1. The primary endpoint is the overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary endpoints are safety, ORR using HCC modified RECIST, duration of response, complete response rate, and progression-free survival. Clinical trial information: NCT05733598 .

Abstract B052: Small molecule RPA inhibitors abrogate the ATR kinase signaling pathway

Abstract Replication Protein A (RPA) is the major single-stranded DNA (ssDNA) binding protein in the cell and plays a role in replication, recombination, and the DNA damage response (DDR) and repair. RPA functions to protect ssDNA from degradation and also serves as an interaction hub to recruit replication and DDR machinery. Under normal cellular conditions, RPA protein abundance is in excess to that of the ssDNA generated during S phase replication so as to protect from replication fork collapse and replication catastrophe in the event of elevated levels of replication stress. RPA inhibition is thus a promising cancer therapeutic strategy that targets oncogenic cells with increased replication stress due to rapid cell proliferation, genotoxic stressors, DDR inhibition, etc, and therefore an increased ssDNA burden. We have previously developed the RPA inhibitor (RPAi) NERx329 that disrupts RPA-ssDNA binding and exhibits single agent anti-cancer activity in vitro and in vivo presumably due to chemical RPA exhaustion. When a cell experiences extensive replication stress, elevated levels of RPA-bound ssDNA are recognized by the kinase ataxia telangiectasia and Rad2-related (ATR) through the ATR interacting protein (ATRIP). ATR is then activated through interactions with DNA topoisomerase 2-binding protein 1 (TopBP1) and phosphorylates downstream targets largely to direct the G2/M checkpoint signaling pathway through checkpoint kinase 1 (CHK1). In this work, we biochemically reconstitute this signaling pathway in order to evaluate an RPAi mechanism of action. We utilize a ssDNA substrate that mimics a stalled replication fork and demonstrate the RPA-dependence of ATR activation by monitoring phosphorylation of multiple ATR substrates: RPA, TopBP1, and p53. We then show that RPAi disrupt ATR kinase activity. Collectively, this works describes a novel mechanism of action of RPAi whereby RPA inhibition results in replication catastrophe by blocking the ATR activity and the G2/M checkpoint pathway. Citation Format: Matthew R Jordan, Diana Ainembabazi, Pamela S VanderVere-Carozza, Navnath S Gavande, Katherine S Pawelczak, John J Turchi. Small molecule RPA inhibitors abrogate the ATR kinase signaling pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B052.

Single-cell transcriptomics reveals ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia exerts antitumor effects by alleviating macrophage suppression in breast cancer

The potential of Ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia (FVIO-MHT) in solid tumor therapy has been demonstrated. However, the impact of FVIO-MHT on the tumor microenvironment (TME) remains unclear. This study utilized single-cell transcriptome sequencing to examine the alterations in the TME in response to FVIO-MHT in breast cancer. The results revealed the cellular composition within the tumor microenvironment (TME) was primarily modified due to a decrease in tumor cells and an increased infiltration of myeloid cells. Subsequently, an enhancement in active oxygen (ROS) metabolism was observed, indicating oxidative damage to tumor cells. Interestingly, FVIO-MHT reprogrammed the macrophages’ phenotypes, as evidenced by alterations in the transcriptome characteristics associated with both classic and alternative activated phenotypes. And an elevated level of ROS generation and oxidative phosphorylation suggested that activated phagocytosis and inflammation occurred in macrophages. Additionally, cell–cell communication analysis revealed that FVIO-MHT attenuated the suppression between tumor cells and macrophages by inhibiting phagocytic checkpoint and macrophage migration inhibitory factor signaling pathways. Inhibition of B2m, an anti-phagocytosis checkpoint, could promote macrophage-mediated phagocytosis and significantly inhibit tumor growth. These data emphasize FVIO-MHT may promote the antitumor capabilities of macrophages by alleviating the suppression between tumor cells and macrophages.

Immune Checkpoint Pathway Expression in Lymphocyte Subpopulations in Patients with Common Variable Immunodeficiency and Chronic Lymphocytic Leukemia.

This study aims to gain a deeper understanding of chronic lymphocytic leukemia (CLL) and common variable immunodeficiency (CVID) by studying immune cells and specific immune checkpoint signaling pathways. The analysis of the percentage of selected immune points and their ligands (PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200) on peripheral blood lymphocyte subpopulations was performed using flow cytometry, and additional analyses determining the serum concentration of the above-mentioned molecules were performed using enzyme immunoassay tests. The obtained results indicate several significant changes in the percentage of almost all tested molecules on selected subpopulations of T and B lymphocytes in both CVID and CLL patients in relation to healthy volunteers and between the disease subunits themselves. The results obtained were also supported by the analysis of the serum concentration of soluble molecules tested. By uncovering valuable insights, we hope to enhance our comprehension and management of these conditions, considering both immunodeficiencies and hematological malignancies. Understanding the role of these signaling pathways in disease development and progression may lead to the development of modern, personalized diagnostic and therapeutic strategies. Ultimately, this knowledge may enable the monitoring of the immune system in patients with CVID and CLL, paving the way for improved patient care in the future.

Mechanism of herb pair containing Astragali Radix and Spatholobi Caulis in the treatment of myelosuppression based on network pharmacology and experimental investigation

Ethnopharmacological relevanceThe Astragali Radix and Spatholobi Caulis herb pair (ARSC) is one of the most commonly used herbal combinations for bone marrow suppression. According to traditional Chinese medicine, Astragali Radix strengthens the spleen and replenishes qi, while Spatholobi Caulis is a hematinic agent that promotes blood circulation and enrichment. The compatibility of the two helps the body to tonify the spleen and kidneys and compensate for visceral deficiencies. However, the multi-target mechanism of ARSC in bone marrow suppression has remained largely unknown. Aim of the studyThe aim of this study is to explore the key targets and signaling pathways of the traditional Chinese herbal pair ARSC for the treatment of bone marrow suppression. Materials and methodsThe active components of ARSC and targets for myelosuppression were screened using network databases. Cytoscape 3.8.0 was used to construct compound-target, compound-disease-target and protein-protein interaction (PPI) networks. Go-function and pathway enrichment analyses were performed to explore the potential mechanism. In vivo animal experiments were conducted to verify the molecular mechanisms. ResultsThe 36 active compounds were identified from the ARSC, and a total of 108 genes involved in myelosuppression were screened. VEGFA, IL6, TNF, JUN, STAT3, PTGS2, CASP3 and MMP9 genes were identified as potential drug targets in the PPI network analyzed by CytoHubba. Enrichment analysis indicated that ARSC may treat myelosuppression through various biological processes, such as apoptosis, TNF-α signaling pathway via NF-κB, PI3K/AKT/mTOR signaling pathway, IL6/JAK/STAT3 signaling pathway, P53 signaling pathway and G2/M checkpoint signaling pathway. The results of the experiment showed that the aqueous extract of ARSC significantly alleviated myelosuppression, reduced the apoptosis rate of bone marrow cells, upregulated the mRNA expression levels of TNF-α, IL-6 and VEGF, and promoted NF-κB phosphorylation in myelosuppressed mice. ConclusionsThis study identified the active components and relevant mechanisms of ARSC in the treatment of myelosuppression. Our findings predicted that ARSC could treat bone marrow suppression through multiple components, multiple targets and multiple pathways. Pharmacological experiments showed that ARSC alleviated fluorouracil-induced myelosuppression by reducing the apoptosis rate of bone marrow cells and regulating the TNF-α/NF-κB signaling pathway.

ERCC6L facilitates the onset of mammary neoplasia and promotes the high malignance of breast cancer by accelerating the cell cycle

BackgroundBreast cancer (BC) is the leading cause of morbidity and the second leading cause of death among female malignant tumors. Although available drugs have been approved for the corresponding breast cancer subtypes (ER-positive, HER2+) currently, there are still no effective targeted drugs or treatment strategies for metastatic breast cancer or triple-negative breast cancer that lack targets. Therefore, it is urgent to discover new potential targets. ERCC6L is an essential protein involved in chromosome separation during cell mitosis. However, the effect of ERCC6L on the tumorigenesis and progression of breast cancer is unclear.Methods and resultsHere, we found that ERCC6L was highly expressed in breast cancer, especially in TNBC, which was closely related to poor outcomes of patients. An ERCC6L conditional knockout mouse model was first established in this study, and the results confirmed that ERCC6L was required for the development of the mammary gland and the tumorigenesis and progression of mammary gland cancers. In in vitro cell culture, ERCC6L acted as a tumor promoter in the malignant progression of breast cancer cells. Overexpression of ERCC6L promoted cell proliferation, migration and invasion, while knockdown of ERCC6L caused the opposite results. Mechanistically, ERCC6L accelerated the cell cycle by regulating the G2/M checkpoint signalling pathway. Additionally, we demonstrated that there is an interaction between ERCC6L and KIF4A, both of which are closely related factors in mitosis and are involved in the malignant progression of breast cancer.ConclusionsWe first demonstrated that ERCC6L deficiency can significantly inhibit the occurrence and development of mammary gland tumors. ERCC6L was found to accelerate the cell cycle by regulating the p53/p21/CDK1/Cyclin B and PLK/CDC25C/CDK1/Cyclin B signalling pathways, thereby promoting the malignant progression of breast cancer cell lines. There was a direct interaction between KIF4A and ERCC6L, and both are closely associated with mitosis and contribute to growth and metastasis of breast tumor. To sum up, our results suggest that ERCC6L may be used as a promising target for the treatment of BC.

Safety and efficacy of immune checkpoint inhibitors after allogeneic hematopoietic cell transplantation.

The immune system plays a major role in preventing infections and cancers. Impairment in immunity may facilitate the development of neoplasia owing to defective immune surveillance, among other mechanisms. Immune evasion plays a significant role in relapse after allogeneic hematopoietic cell transplantation (alloHCT); one purported mechanism is through immune checkpoint signaling pathways. Checkpoint inhibitors (CPIs) are FDA approved for relapsed classical Hodgkin's Lymphoma (cHL), primary mediastinal large B cell Lymphoma (PMBCL) and other solid tumors. Retrospective studies evaluating the outcomes of alloHCT after prior exposure to CPIs showed favorable survival outcomes but high rates of graft-versus-host disease (GVHD); the risk appears to be lower when using post-transplant cyclophosphamide as GVHD prophylaxis. CPIs have increasingly been used to prevent or treat post-alloHCT relapse. Available data, albeit limited, supports the clinical activity of CPIs in post-alloHCT relapse; however, serious and even fatal cases of GVHD have been reported. The optimal timing, schedule, dosing, and patients likely to benefit from this strategy are yet to be identified. In this review, we highlight the immune system's role in cancer surveillance and relapse prevention and discuss the current clinical evidence of CPIs use in post-alloHCT relapse.

Glycolysis induces Th2 cell infiltration and significantly affects prognosis and immunotherapy response to lung adenocarcinoma.

Glycolysis has a major role in cancer progression and can affect the tumor immune microenvironment, while its specific role in lung adenocarcinoma (LUAD) remains poorly studied. We obtained publicly available data from The Cancer Genome Atlas and Gene Expression Omnibus databases and used R software to analyze the specific role of glycolysis in LUAD. The Single Sample Gene Set Enrichment Analysis (ssGSEA) indicated a correlation between glycolysis and unfavorable clinical outcome, as well as a repression effect on the immunotherapy response of LUAD patients. Pathway enrichment analysis revealed a significant enrichment of MYC targets, epithelial-mesenchymal transition (EMT), hypoxia, G2M checkpoint, and mTORC1 signaling pathways in patients with higher activity of glycolysis. Immune infiltration analysis showed a higher infiltration of M0 and M1 macrophages in patients with elevated activity of glycolysis. Moreover, we developed a prognosis model based on six glycolysis-related genes, including DLGAP5, TOP2A, KIF20A, OIP5, HJURP, and ANLN. Both the training and validation cohorts demonstrated the high efficiency of prognostic prediction in this model, which identified that patients with high risk may have a poorer prognosis and lower sensitivity to immunotherapy. Additionally, we also found that Th2 cell infiltration may predict poorer survival and resistance to immunotherapy. The study indicated that glycolysis is significantly associated with poor prognosis in patients with LUAD and immunotherapy resistance, which might be partly dependent on the Th2 cell infiltration. Additionally, the signature comprised of six genes related to glycolysis showed promising predictive value for LUAD prognosis.

An open-label, multicenter study investigating RP3 oncolytic immunotherapy in combination with first- or second-line systemic atezolizumab and bevacizumab therapy in patients with locally advanced unresectable or metastatic hepatocellular carcinoma.

TPS4178 Background: Despite advances in treatment for unresectable hepatocellular carcinoma (HCC), long-term survival rates remain poor. The combination of bevacizumab (Bev) and atezolizumab (Atezo) is the preferred frontline therapy for advanced HCC, but a minority of patients (pts) respond, and secondary resistance usually occurs within months. HCC has an immune-suppressed tumor microenvironment, mediated by activated immune checkpoint signaling and angiogenesis pathways, which may contribute to therapeutic resistance. RP3 is a genetically modified herpes simplex virus type 1 (HSV-1) that expresses the fusogenic gibbon ape leukemia virus glycoprotein with the R sequence deleted (GALV-GP-R–), an anti–CTLA-4 antibody-like molecule, CD40 ligand, and 4-1BB ligand. The direct oncolytic effect coupled with immune stimulation by RP3 in the tumor microenvironment is intended to provide systemic antitumor activity and enhance therapeutic response to anti–PD-1/PD-L1 agents, such as Atezo. Preclinical data have demonstrated improved distribution of oncolytic HSV within tumors in combination with Bev, supporting the clinical combination of RP3 with Bev. This study will evaluate the safety and efficacy of RP3 combined with Atezo and Bev as first- (1L) and second-line (2L) systemic therapies for unresectable and advanced HCC. Methods: The 1L and 2L cohorts will each enroll up to 30 pts. Pts in the 1L cohort may not have received prior systemic treatment; pts in the 2L cohort must have progressed on or following one prior line of systemic therapy, which must have included a PD-1/PD-L1−directed agent. Key inclusion criteria include advanced, unresectable HCC with ≥1 measurable tumor of ≥1 cm in longest diameter, Child-Pugh Class A, and Eastern Cooperative Oncology Group performance status of 0−1. Key exclusion criteria include untreated esophageal and/or gastric varices with bleeding or at high risk for bleeding and macroscopic invasion of tumor into any major blood vessel(s) and/or main bile ducts. Pts with a history of medically refractory hepatic encephalopathy and/or hepato-renal syndrome are also excluded. Pts in the 1L cohort will receive 1200 mg Atezo and 15 mg/kg Bev every 3 weeks (Q3W) together with RP3 intratumorally Q3W for a total of up to 8 doses. Pts in the 2L cohort will receive RP3 every 2 weeks for 4 doses with Bev Q3W starting on cycle (C) 1 day (D) 1, then RP3 and Bev Q3W for up to 4 more doses with Atezo Q3W being added on C4D1. The primary endpoint is overall response rate (ORR) by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1. Secondary endpoints are safety, ORR using HCC modified RECIST, duration of response, complete response rate, and progression-free survival. Clinical trial information: NCT05733598 .

Relationship between ICOS and ICOS ligand RNA expression patterns with tumor type, tumor mutational burden, and clinically relevant immune checkpoints.

e14669 Background: ICOS (inducible T-cell co-stimulator) has a unique ligand (ICOSL). Together they contribute to both antitumor responses/immune stimulation and potentiating immunosuppression. The interaction of ICOS and ICOSL can stimulate CD4 and CD8 ICOS-positive T-cell populations, but also can increase immunosuppressive regulator T-cells. ICOS agonists and antagonists are both under development as novel therapies. We aimed to quantify ICOS and ICOSL expression relationships with tumor type and other clinically relevant checkpoint inhibitors along with evaluating relationships between ICOS and ICOSL expression patterns. Methods: Data from 514 patients with advanced solid tumors at the University of California San Diego were used in the analysis. RNA expression levels of ICOS, ICOS ligand, PD-1, PD-L1, LAG-3, and CTLA-4 from tumor specimens were analyzed at OmniSeq. Tumor mutational burden (TMB) was also evaluated. Univariate analyses (Fischer’s exact tests) and multivariate analysis (logistic regression) were conducted in SAS v 9.4 to determine relationships between high (>=75 percentile RNA rank) and low (0-24 percentile RNA rank) ICOS and ICOSL expression with age, gender, cancer type, high LAG-3, high TMB, high CTLA4, high PD-1, and high PD-L1. The relationship between ICOS and ICOSL high or low expression were also assessed. Results: Seventy patients had high ICOS (14%) and 192 had high ICOSL (37%) while low ICOS was seen in 226 (44%) and low ICOSL was seen in 76 (15%). High ICOS RNA levels were independently associated with patients not having colorectal cancer (p=0.0009), tumors with high PD-1 expression (p=0.025), tumors with high PD-L1 expression (p<0.0001), and tumors with high CTLA-4 expression (p<0.0001). Low ICOS RNA levels were independently associated with patients whose tumors did not have high PD-1 (p=0.0006), tumors that did not have high PD-L1 (p=0.01), tumors that did not have high CTLA-4 expression (p<0.0001), and those with low ICOSL expression (p=0.0007). High ICOSL was independently associated with a diagnosis of colorectal cancer (p=0.0015), tumors that did not have high PD-L1 expression (p=0.003), and tumors with high CTLA4 (p=0.0001). Low ICOSL was independently associated with patients who did not have colorectal cancer (p=0.0001) and tumors with low ICOS expression (p=0.0032). Conclusions: ICOS and ICOSL RNA expression patterns can provide insight into the complicated ICOS/ICOSL immune checkpoint signaling pathway and help support rational immunotherapy combinations as part of a precision medicine approach. Both stimulators and inhibitors of the ICOS/ICOSL axis are being developed, and their proper application may require interrogation of the individual patient’s tumor/environment.

Anthracycline Therapy Modifies Immune Checkpoint Signaling in the Heart.

Cancer survival rates have increased significantly because of improvements in therapy regimes and novel immunomodulatory drugs. Recently, combination therapies of anthracyclines and immune checkpoint inhibitors (ICIs) have been proposed to maximize neoplastic cell removal. However, it has been speculated that a priori anthracycline exposure may prone the heart vulnerable to increased toxicity from subsequent ICI therapy, such as an anti-programmed cell death protein 1 (PD1) inhibitor. Here, we used a high-dose anthracycline mouse model to characterize the role of the PD1 immune checkpoint signaling pathway in cardiac tissue using flow cytometry and immunostaining. Anthracycline treatment led to decreased heart function, increased concentration of markers of cell death after six days and a change in heart cell population composition with fewer cardiomyocytes. At the same time point, the number of PD1 ligand (PDL1)-positive immune cells and endothelial cells in the heart decreased significantly. The results suggest that PD1/PDL1 signaling is affected after anthracycline treatment, which may contribute to an increased susceptibility to immune-related adverse events of subsequent anti-PD1/PDL1 cancer therapy.

Integrated analysis of genomic and transcriptomic profiles to examine CD73/NT5E as a driver of drug-resistance and immunosuppression in biliary cancer (BTC).

606 Background: Despite the improved outcome achieved by the addition of the anti-PD-L1 durvalumab to standard chemotherapy, the vast majority of BTC do not benefit from chemo-immunotherapy. Immunosuppressive microenvironment is a dominant feature of BTC, involved in tumour progression and drug resistance. Here, we investigated the biological role and treatment implications of the adenosine-producing enzyme CD73 in a clinically-annotated cohort of BTC. CD73, an ecto-5’-nucleotidase (NT5E), creates an immunosuppressive tumour-promoting microenvironment by converting ATP to adenosine. Methods: Immunohistochemistry for CD73, CD4/CD8 and FOXP3 as well as whole-exome and transcriptomic sequencing were performed on resected specimens of 80 BTC (Illumina Platform). Spatial Transcriptomics was performed by using Visium Spatial Gene Expression-10x Genomics). Silencing of CD73 was obtained by transient transfection od CD73-siRNA, while genetic Knock-out by using CRISPR-Cas system technology in two BTC cell lines. Tumor growth was assessed in 2D and in 3D cell culture by using MTS assay and spheroid growth analysis before and after treatment with selected drugs. Statistical and Survival, correlation analyses were performed. Results: High CD73 expression (CD73high) was associated with older age (>70 years, p=0.01), gallbladder subsite (p=0.03), and nodal involvement (p=0.04). CD73high tumours were significantly enriched in infiltrating FOXP3+ T lymphocytes (p<0.001). CD73high status was independent predictor of poorer prognosis at the multivariate analysis (p=0,03) together with ECOG PS ≥2 (p=0,001) and the pathological stage (p=0,025) and was associated with a remarkably shorter RFS in patients treated with adjuvant chemotherapy (p=0,011). At the transcriptomic level, CD73high tumours were significantly enriched in upregulated EMT, TNF-alfa/NFKB, hypoxia and G2/M checkpoint signaling pathways and p53, BMI1, MEL18, EGFR and K-RAS genes. In in vitro models, siRNA-mediated depletion and CRISPR-CAS9 gene KO of CD73 sensitized both BTC 2D and 3D culture to cisplatin/gemcitabine treatment. The pharmacological inhibition of CD73 by AMCP improved the sensitivity of BTC cell lines to cisplatin/gemcitabine treatment. Finally, Spatially resolved transcriptomics of CD73high revealed a critical role of CD73 in tumor immunity and therapeutic response. Conclusions: We showed that CD73high BTC display aggressive biological features, poorer prognosis and resistance to standard chemotherapy. The therapeutic targeting of this adenosinergic ectonucleotidase by clinically-available compounds has the potential to enhance the efficacy of conventional treatment in BTC.

An open-label, multicenter study investigating RP3 oncolytic immunotherapy in combination with first- or second-line systemic atezolizumab and bevacizumab therapy in patients with locally advanced unresectable or metastatic hepatocellular carcinoma.

TPS623 Background: Despite advances in treatment for unresectable hepatocellular carcinoma (HCC), long-term survival rates remain poor. The combination of bevacizumab (Bev) and atezolizumab (Atezo) is the preferred frontline therapy for advanced HCC, but a minority of patients (pts) respond, and secondary resistance usually occurs within months. HCC has an immune-suppressed tumor microenvironment, mediated by activated immune checkpoint signaling and angiogenesis pathways, which may contribute to therapeutic resistance. RP3 is a genetically modified herpes simplex virus 1 (HSV-1) that expresses the fusogenic GALV-GP R- protein, an anti–CTLA-4 antibody-like molecule, CD40 ligand, and 4-1BB ligand. The direct oncolytic effect coupled with immune stimulation by RP3 in the tumor microenvironment is intended to provide systemic anti-tumor activity and reverse therapeutic resistance to anti-PD-1/PD-L1 agents. Pre-clinical data has demonstrated improved distribution of oncolytic HSV within tumors in combination with Bev supporting the clinical combination of RP3 with Bev. RP3 combined with anti-PD1 therapy has demonstrated clinical activity and safety in pts with various malignant solid tumors. This study will evaluate the safety and efficacy of RP3 combined with Atezo and Bev as 1st (1L) and 2nd (2L) line systemic therapies for inoperable and advanced HCC. Methods: The 1L and 2L cohorts will each enroll up to 30 pts with advanced, unresectable HCC. Pts in the 1L cohort may not have received prior systemic treatment; pts in the 2L cohort must have progressed on or following one prior line of systemic therapy, which must have included a PD-(L)1 directed agent. Key inclusion criteria include advanced unresectable HCC with at least 1 measurable tumor of ≥1 cm in longest diameter, Childs-Pugh Class A, and ECOG Score of 0 or 1. Important exclusion criteria include untreated esophageal and/or gastric varices with bleeding or at high risk for bleeding, and macroscopic invasion of tumor into major blood vessels and/or main bile ducts. Pts with a history of medically refractory hepatic encephalopathy and/or hepato-renal syndrome are also excluded. Pts in 1L cohort will receive 1200 mg Atezo and 15mg/kg Bev Q3W together with RP3 intratumorally (IT) Q3W for a total of up to 8 doses. In 2L pts will receive RP3 Q2W for 4 doses with Bev Q3W starting on C1D1, then RP3 and Bev Q3W for up to 4 more doses with Atezo Q3W being added on C4D1. The primary endpoint includes overall response rate (ORR) by RECIST v1.1. Secondary endpoints are safety, ORR using HCC mRECIST, duration of response, complete response rate, and progression-free survival.

Diversin upregulates the proliferative ability of colorectal cancer by inducing cell cycle proteins

Colorectal cancer (CRC) is a common gastrointestinal tumour with increasing incidence worldwide. However, the underlying molecular mechanism of CRC proliferation is not completely clear. Diversin，as an ankyrin repeat-containing protein, is upregulated in various solid tumours and accelerates cancer progression by promoting cell proliferation and increasing S phase fraction of cells. In this study, 71 CRC samples and corresponding adjacent tissue samples were included. The expression of diversin in tissues was verified via immunohistochemical analysis. The MTS assay and flow cytometry (FCM) was used to measure cell proliferation and cell cycle. Results of immunohistochemical analysis revealed that diversin was highly expressed in human CRC tissues and was significantly associated with tumour differentiation, clinical stage and lymph node metastasis. The analysis based on the CRC data from The Cancer Genome Atlas (TCGA) database showed that a high expression of diversin correlated with the poor prognosis of CRC. Results of the MTS assay indicated that the overexpression of diversin promoted the proliferation of CRC cells, while its downregulation had an inhibitory effect on CRC cell proliferation. FCM analysises presented that diversin increased the flux of the CRC cell cycle from G1 to S and regulated cycle-related proteins, namely, P21, P27, cyclin E, CDK2, cyclin D and CDK4. The results suggest that diversin contributes to CRC proliferation that involves the distribution of the cell cycle. In CRC tissues, the expression of diversin has closely related to the prognosis. The higher the expression levels of diversin, the worse the prognosis. In vitro, diversin could increase the proliferative ability of CRC cells through the G1–S checkpoint and JNK signalling pathway, confirming that diversin contributes to CRC development.

Checkpoint Nano-PROTACs for Activatable Cancer Photo-Immunotherapy.

Checkpoint immunotherapy holds great potential to treat malignancies via blocking the immunosuppressive signaling pathways, which however suffers from inefficiency and off-target adverse effects. Herein, checkpoint nano-proteolysis targeting chimeras (nano-PROTACs) in combination with photodynamic tumor regression and immunosuppressive protein degradation to block checkpoint signaling pathways for activatable cancer photo-immunotherapy are reported. These nano-PROTACs are composed of a photosensitizer (protoporphyrin IX, PpIX) and an Src homology 2 domain-containing phosphatase 2 (SHP2)-targeting PROTAC peptide (aPRO) via a caspase 3-cleavable segment. aPRO is activated by the increased expression of caspase 3 in tumor cells after phototherapeutic treatment and induces targeted degradation of SHP2 via the ubiquitin-proteasome system. The persistent depletion of SHP2 blocks the immunosuppressive checkpoint signaling pathways (CD47/SIRPα and PD-1/PD-L1), thus reinvigorating antitumor macrophages and T cells. Such a checkpoint PROTAC strategy synergizes immunogenic phototherapy to boost antitumor immune response. Thus, this study represents a generalized PROTAC platform to modulate immune-related signaling pathways for improved anticancer therapy.