Checkpoint Targets Research Articles

Differences in genomic, transcriptomic, and immune landscape of prostate cancer (PCa) based on site of metastasis (mets).

21 Background: In patients with metastatic PCa, specific sites of mets are associated with differential overall survival, with lymph node (LN) only disease having the best survival, followed by bone, lung, and liver, with each showing progressively worse survival. However, the biology of tropism to specific sites and differential outcomes has not been fully explored. Herein, utilizing a large, multi-institutional, real-world dataset, we hypothesized that differences in molecular and immune landscape of PCa based on site of mets may explain different clinical outcomes. Methods: DNA (592-gene panel or whole exome) and RNA (whole transcriptome) sequencing was performed for primary PCa (PPCa) and unpaired PCa mets sites (non-visceral: NV and visceral: V) and sequenced at Caris Life Sciences (Phoenix, AZ). Mismatch repair deficiency/microsatellite instability-high (dMMR/MSI-H) was tested by IHC/NGS, respectively. Tumor mutational burden (TMB) (high >10 mut/Mb) was assessed. Androgen receptor (AR) signaling score and Neuroendocrine Prostate Cancer (NEPC) score were calculated using previously defined RNA-based signatures. Pathway enrichment was determined by Gene Set Enrichment Analysis (GSEA). Immune cell fractions calculated by deconvolution of WTS using quanTIseq. Statistical significance was determined by chi-square and Mann-Whitney U tests. All analyses done with CODEai. Results: 6074 PCa samples, including 3411 PPCa, 1634 NV (bone = 784, LN = 850) and 1029 V (liver: 468, lung: 177, bladder: 241, other: 143) were evaluated. Age at sample collection ranged 30 to 70+ years. Compared to PPCa, significantly higher mutation rates were observed for multiple genes in V (RB1 8% vs 3%, TP53 43% vs 31%, PTEN 12 % vs 8%, CTNNB1 8% vs 3%, AR 13% vs 1%, APC 15% vs 5%, AKT1 4% vs 2%; p<0.05 for all) and NV mets (AR 9% vs 1%, SPOP 7% vs 10%, TP53 38% vs 31%, CTNNB1 6% vs 3%, and KMT2C 8 % vs 5%; p<0.05 for all). Alterations of RB1, APC and AR were more frequent, and SPOP less common in liver mets compared to PPCa, while SPOP, KRAS, PIK3CA, APC and BAP1 mutations were more common in lung mets (p < 0.05 for all). Compared to PPCa, V and NV mets had higher AR signaling and interferon-γ scores, while V mets had higher NEPC scores and were more frequently PD-L1+ (SP142) and TMB-high (p<0.05 for all). No differences in dMMR/MSI-H was observed between PPCa, V and NV mets. AR-V7 variant prevalence was significantly higher in V (36%) and NV (33%) mets compared to PPCa (7%, p-value < 0.05). GSEA identified E2F, G2M checkpoint and MYC target pathways as being more upregulated in both V and NV mets compared to PPCa (q<0.05). Compared to PPCa, V and NV mets were significantly less enriched in macrophage M2, NK cells and Tregs, while V mets were also less enriched in B cells and neutrophils (p<0.05). Conclusions: We elucidate molecular and immunologic mechanisms of metastatic tropism in advanced PCa. These data may facilitate future drug development.

NPM3 as an Unfavorable Prognostic Biomarker Involved in Oncogenic Pathways of Lung Adenocarcinoma via MYC Translational Activation.

The nucleoplasmin/nucleophosmin (NPM) family was previously regarded as a critical regulator during disease development, and its mediation in carcinogenesis has achieved intensive attention recently. However, the clinical importance and functional mechanism of NPM3 in lung adenocarcinoma (LUAD) have not been reported yet. This study aimed to investigate the role and clinical significance of NPM3 in the development and progression of LUAD, including the underlying mechanisms. The expression of NPM3 in pan-cancer was analyzed via GEPIA. The effect of NPM3 on prognosis was analyzed by the Kaplan-Meier plotter and the PrognoScan database. In vitro, cell transfection, RT-qPCR, CCK-8 assay, and wound healing assay were employed to examine the role of NPM3 in A549 and H1299 cells. Gene set enrichment analysis (GSEA) was performed using the R software package to analyze the tumor hallmark pathway and KEGG pathway of NPM3. The transcription factors of NPM3 were predicted based on the ChIP-Atlas database. Dual-luciferase reporter assay was applied to verify the transcriptional regulatory factor of the NPM3 promoter region. The NPM3 expression was found to be markedly higher in the LUAD tumor group than the normal group and to be positively correlated with poor prognosis, tumor stages, and radiation therapy. In vitro, the knockdown of NPM3 greatly inhibited the proliferation and migration of A549 and H1299 cells. Mechanistically, GSEA predicted that NPM3 activated the oncogenic pathways. Further, the NPM3 expression was found to be positively correlated with cell cycle, DNA replication, G2M checkpoint, HYPOXIA, MTORC1 signaling, glycolysis, and MYC targets. Besides, MYC targeted the promoter region of NPM3 and contributed to the enhanced expression of NPM3 in LUAD. The overexpression of NPM3 is an unfavorable prognostic biomarker participating in oncogenic pathways of LUAD via MYC translational activation and it contributes to tumor progression. Thus, NPM3 could be a novel target for LUAD therapy.

Upregulation of vesicle-associated membrane protein 7 in breast cancer tissues.

Vesicle-associated membrane protein 7 (VAMP7) plays oncogenic roles in cancers. However, its clinical significance in breast cancer (BC) tissues remains unknown. To elucidate the clinical implications of VAMP7, as well as its involvement in the tumor microenvironment and molecular pathways of breast cancer. BC (n=100) and non-cancerous breast tissues (n= 100) were collected for an immunohistochemical experiment (1:200). The protein expression level of VAMP7 was determined by using a semi-quantitative scoring method. High-throughput RNA-sequencing data of BC tissues were analyzed to confirm the mRNA expression trend of VAMP7. Additionally, the largest BC prognosis cohort data were collected to mine the potential impact VAMP7 has on BC progression. The association between VAMP7 and the microenvironment of BC was evaluated by using a CIBERSORT algorithm. Moreover, we explored the co-expressed molecular mechanisms of VAMP7 in BC by calculating Pearson correlation coefficients and overexpressed genes. Finally, the biological mechanism underlying the relationship between VAMP7 and the key pathways was also explored using gene set enrichment analysis (GSEA). Potential therapeutic strategies were predicted targeting VAMP7. VAMP7 protein was significantly over-expressed in BC tissue than that in controls (p< 0.001). Compared with 459 normal breast tissues and 113 non-cancerous breast tissues, the expression level of VAMP7 mRNA was significantly increased in 1111 BC tissues. CD4+T cells, macrophages, and naïve B cells had a higher infiltration rate in BC tissues with high VAMP7 expression, while regulatory T cells and CD8+T cells had a lower infiltration rate. Over-expressed VAMP7 was associated with macrophages activation and transition from M1 to M2 polarization. Upregulated VAMP7 could predicted poorer OS, DMFS, PPS, and RFS outcomes. Upregulated VAMP7 co-expressed genes were significantly enriched in the cell cycle checkpoints. GSEA confirmed that over-expressed VAMP7 are markedly associated with functional enrichment in cell cycle related categories, including mitotic spindle, G2M checkpoint, and E2F targets. KU-55933 was predicted as a putative therapeutic drug for BC targeting VAMP7. VAMP7 was upregulated in BC tissue and correlated with poor prognosis of BC patients. VAMP7 may promote BC progression by targeting the cell cycle pathway.

T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain as a promising immune checkpoint target for the treatment of SLE.

Immune checkpoints (ICs) play a pivotal role in orchestrating immune regulation, crucial for the maintenance of immune tolerance and prevention of autoimmune diseases. One noteworthy example among these immune regulators is T cell immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT). The TIGIT pathway's inhibition or the absence of TIGIT has been linked to the hyperactivation and excessive proliferation of T cells, rendering individuals more susceptible to autoimmune diseases and exacerbating inflammatory responses. Conversely, the activation of TIGIT has exhibited promising outcomes in ameliorating autoimmune disorders, as observed in murine models of systemic lupus erythematosus (SLE). Consequently, a judicious exploration of the co-inhibitory axis appears warranted for the effective management of pathogenic immune responses in SLE. In light of compelling evidence, this review undertakes a comprehensive examination of TIGIT's characteristics within the context of autoimmunity, offering insights into its potential as a therapeutic target for SLE.

Overexpression of tousled-like kinase 2 predicts poor prognosis in HBV-related hepatocellular carcinoma patients after radical resection.

Background: Tousled-like kinase 2 (TLK2) is integral to DNA repair, replication, and cell cycle regulation, crucial for maintaining genome stability and integrity. However, the expression and prognostic value of TLK2 in hepatitis B viral (HBV) -related hepatocellular carcinoma (HCC) remains unclear. Methods: We examined TLK2 expression and prognostic implications in pan-cancer by using diverse databases. Subsequently, TLK2 expression in HBV-related HCC tissues and adjacent tissues was assessed using quantitative real-time PCR and immunohistochemistry. The prognostic value of TLK2 was assessed through ROC curves, time-dependent ROC curves, Cox regression, Kaplan-Meier curve, and decision curve analysis. Additionally, analyses of immune infiltration, protein-protein interactions, key molecules of tumor-related signaling pathways, molecular subtypes, and TLK2-associated differentially expressed genes (DEGs) were conducted, along with GO/KEGG and GSEA enrichment analyses. Results: TLK2 expression was significantly higher in HCC tissues compared to adjacent tissues and correlated with gender, AFP levels, albumin-bilirubin (ALBI) grade, microvascular invasion (MVI), maximum tumor diameter, tumor number, and TNM stage. TLK2 overexpression emerged as an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in HBV-related HCC patients. An integrated OS nomogram model, incorporating TLK2, age, ALBI grade, MVI, and tumor number, displayed enhanced prognostic capability (C-index: 0.765, 95% CI: 0.732-0.798) in predicting OS and has a higher net benefit than the TNM stage. Moreover, TLK2 expression correlated closely with immune cell infiltration and key molecules of signaling pathways. Functional enrichment analyses highlighted significant associations with DNA duplex unwinding, double-strand break repair, DNA replication, cell cycle, E2F targets, G2M checkpoint, and MYC targets V1. Conclusion: TLK2 is notably overexpressed in HBV-related HCC and emerges as a promising prognostic biomarker, necessitating further validation.

Combination strategy exploration for prior treated recurrent or metastatic nasopharyngeal carcinoma in the era of immunotherapy

To assess the efficacy and safety of the combination of immune checkpoint inhibitors (ICIs) and target therapy (anti-angiogenesis or EGFR inhibitors) as a second-line or subsequent treatment for recurrent or metastatic nasopharyngeal carcinoma (R/M NPC), we conducted a retrospective study. In this study, previously treated R/M NPC patients were administered one of the following treatment: ICIs combined with target therapy and chemotherapy (ITC), ICIs combined with target therapy alone (IT), ICIs combined with chemotherapy (IC), or chemotherapy alone (C). The primary endpoint under consideration was progression-free survival (PFS), while secondary endpoints included overall survival (OS), objective response rate (ORR), disease control rate (DCR), and safety measures. A total of 226 patients participated in this study, with 70 receiving the ITC regimen, 48 receiving IT, 48 treated with IC, and 60 undergoing C alone. The median PFS for the four cohorts was 20.67, 13.63, 12.47, and 7.93 months respectively. Notably, ITC regimen yielded the most favorable PFS among these cohorts. The ITC cohort exhibited a comparable tumor response and safety profile to the IT and IC cohorts (p > 0.05), but superior tumor response compared to the C cohort (p < 0.05). The ITC regimen also conferred a significant improvement in OS when comparing to C alone (HR 0.336, 95%CI 0.123–0.915, p = 0.033). The IT and IC regimens achieved a nearly identical PFS (HR 0.955, 95%CI 0.515–1.77, p = 0.884), although the IT regimen was associated with a lower occurrence of SAEs in contrast to the IC regimen (p < 0.05). In addition, the IT regimen demonstrated superior PFS (HR 0.583, 95%CI 0.345–0.985, p = 0.044) and fewer SAEs when compared to C alone (p < 0.05). These findings collectively support the notion that the combination of ICIs, target and chemotherapy exhibits robust antitumor activity in previously treated R/M NPC patients, without a significant increase in adverse events.

A transcriptomic approach to explore the immune landscape of patient with pancreatic ductal adenocarcinoma with prognostic impact.

681 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by its immunologically cold tumor microenvironment (TME) with scarce T cell infiltration and few molecular signatures of immune activation. To date, the immunotherapies including the immune checkpoint blockade of PD-1, and CTLA-4 either single or in combination have shown modest results in PDAC tumors. Recently, the Lymphocyte Activation Gene (LAG3) has emerged as a promising checkpoint target as indicated by preclinical and clinical data. The aim of this study is to explore the immune profile of a series of PDAC and to evaluate its prognostic impact. Methods: A 5-mm thick section of Formalin-Fixed Paraffin-Embedded (FFPE) tissue from a retrospective cohort of 28 PDAC cases were analyzed using the Precision Immunooncology panel (PIP, HTG Molecular Diagnostics) on a NextSeq 550 sequencer (Illumina). The RNA expression of 1392 immune-related genes were analyzed. Principal Components (PC) and Manhattan distance were studied for data visualization and clustering. Maxstat algorithm (maxstat v0.7-25) was used to establish optimal cut-offs for variable categorization. Log-rank and Cox regression were used for both univariate and multivariate for survival. All tests were two-tailed. The statistical analysis was performed using R studio (R version 4.0.3). Lastly, immunohistochemistry (IHC) for CD4, CD8, CD20, PD1, PD-L1, and LAG-3 proteins was performed on tissue microarrays (TMAs) to assess their correlation with RNA expression. Results: Unsupervised analysis of the gene expression identified two clusters of patients with differentially prognostic information. These two groups were defined with a logistic regression model of 14 genes ( BTLA, CXCR5, DLX6, KLHDC9, KRT13, LAG3, LGSN, MICB, SIGLEC5, SPINK5, SPN, TBX21, UPK2, ZIC5) showing for cluster1 and cluster 2 a median survival of 13 (0-97.583) and 57 (0.67-256.08) months, respectively (p-value = 0.0088). This predictive model was independently validated in the PDAC dataset of the Cancer Genome Atlas (TCGA) (p&lt;0.0001). Interestingly, cluster 1, was characterized by the significant overexpression of LAG3 which has recently been proposed as the next immune checkpoint (IC) receptor target. Moreover, Cluster 1, exhibited the overexpression of other ICs such as PD1, PD-L1, IDO1, LIF and CTLA4, defining cluster 1 as an immunologically hot tumor. IHC for LAG3 and other ICs showed a moderate correlation with HTG results. Conclusions: In this study, a prognostic transcriptomic-based signature of 14 genes has been defined and validated for PDAC. This signature clearly identifies two prognostic groups that could constitute the basis for tailored immunotherapy with specific IC inhibitors. LAG-3 is a promising target for immunotherapy in PDAC patients.

Immune Checkpoint Inhibitor-Induced Inflammatory Arthritis: Overview of Therapies and a Personalized Approach to Optimized Combined Therapy.

Immune checkpoint inhibitors (ICIs), including anti-cytotoxic T lymphocyte antigen 4, anti-programmed cell death 1, and anti-programmed cell death ligand 1 (PD-L1) antibodies, are currently widely used in oncology clinical practice, achieving considerable success in improving disease outcomes. New checkpoint targets are being discovered and investigated through basic science research and clinical trials. ICI remove negative regulatory immune signals on T cells, leading to immune activation and induction of antitumor immunity. Patients who receive ICI, however, are at risk for developing immune-related adverse events (irAEs), which are attributed to increased T cell activity against antigens in both tumors and in healthy tissues, to increased inflammatory cytokine levels, to increased levels of preexisting autoantibodies, and to enhanced complement-mediated inflammation. Arthritis is one of the most common irAEs. ICI-induced rheumatic irAEs are categorized by levels of severity which guide the choice of treatment options. Management of ICI-induced rheumatic irAEs includes the use of glucocorticoids, disease-modifying antirheumatic drugs (mainly methotrexate), and biological agents (e.g., tumor necrosis factor, interleukin-6 receptor, and CD20 inhibitors). This review aims to summarize the current ICI subtypes, their role in rheumatic irAEs development, and therapies currently used in clinical practice to manage irAEs. In addition, we propose to use an ex vivo personalized diagnostic assay for the selection of the most effective ICI with antirheumatic drugs combinations that will inhibit the advancement of ICI-induced adverse events.

Profiling of VEGF Receptors and Immune Checkpoints in Recurrent Respiratory Papillomatosis.

Recurrent respiratory papillomatosis (RRP) is caused by human papilloma virus (HPV) infection of the aerodigestive tract that significantly impacts quality-of-life including the ability to communicate and breathe. Treatment was traditionally limited to serial ablative procedures in the O.R. with possible local adjuvant therapy, but new systemic therapies, such as Vascular endothelial growth factor (VEGF) inhibitors, are showing significant promise. This study aims to determine whether rationale exists for combination therapeutic approaches using VEGF inhibitors and/or immune checkpoint blockade. Using fresh specimens from the O.R., we performed flow cytometry on papilloma, normal adjacent tissue, and blood. Papilloma and surrounding tissue were examined for expression of PD-L1, PD-L2, Galectin-9, VEGFR2, and VEGFR3. CD8+ and CD4+ T cells were assayed for expression of PD-1, TIGIT, LAG3, and TIM3. Our data shows that papilloma tissue exhibits significantly higher levels of PD-L1 and PD-L2 compared to adjacent tissue. Elevated levels of the VEGF receptor VEGFR3 were also observed in papilloma tissue. When examining T cells within the papilloma, elevated PD-1 and TIGIT expression was observed on CD8+ T cells, while levels of PD-1, TIGIT, and TIM3 were elevated on CD4+ T cells compared to PBMCs. Heterogenous marker expression was observed between individuals. Our analysis shows that RRP tissue shows elevated levels of multiple immune check point targets and VEGFR3, with varied patterns unique to each papilloma patient. Some of these immune checkpoint markers already have novel immunotherapies available or in development, providing molecular rationale to offer these systemic treatments to selected patients affected by RRP alongside VEGF inhibitors. Laryngoscope, 134:2819-2825, 2024.

TNFSF12 is associated with breast cancer prognosis and immune cell infiltration.

Breast cancer (BRCA) is one of the most common cancers in women and is the leading cause of cancer-related deaths in women. TNFSF12, originally a member of the TNF superfamily, is considered a key molecule that is associated with poor prognosis of many cancers. However, its role in progression of BRCA remains unclear. In this study, the expression profile and clinical information of TNFSF12 across various cancers were obtained from The Cancer Genome Atlas (TCGA) database. Differences in TNFSF12 expression levels between carcinoma and paraneoplastic cancers were compared, and its association with prognosis was examined. Functional enrichment analysis was conducted to explore the potential signaling pathways and biological functions linked with TNFSF12. Moreover, the correlation between TNFSF12 and immune cell infiltration, response to immune checkpoint inhibitors (ICIs), and response to chemotherapy were evaluated. TNFSF12 level in BRCA and normal serum was detected by ELISA. TNFSF12 was lowly expressed in BRCA and is significantly associated with PAM50. TNFSF12 low expression correlates with poor overall survival, particularly among HER2-positive patients. Patients with high level of TNFSF12 expression are usually accompanied with elevated levels of various immune cells, including CD8 T cells, cytotoxic cells, DCs, eosinophils, iDCs, mast cells, neutrophils, NK CD56bright cells, NK cells, pDC, T cells, Tem, and TFH Th17 cells, and exhibit sensitivity to immune checkpoint inhibitors. Functional enrichment analysis indicates significant activation of KRAS signaling, TNFA signaling via NFKB, and epithelial-mesenchymal transition (EMT) in the high TNFSF12 expression group, while MTORC1 signaling, MYC, G2M checkpoint, and E2F targets are inhibited. Furthermore, patients in the low expression group demonstrate higher sensitivity to paclitaxel and rapamycin, whereas those in the high expression group show increased sensitivity to erlotinib and foretinib. ELISA analysis also confirmed a significant decrease of TNFSF12 protein levels in BRCA patients. This study presents a comprehensive analysis of the close correlation between TNFSF12 and prognosis, immune response, as well as the effectiveness of chemotherapeutic agents in BRCA patients.

Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release.

Background: Cancer cell evasion of the immune response is critical to cancer development and metastases. Clinicians' ability to kickstart the immune system to target these rogue cells is an ever-growing area of research and medicine. This study delved into the relationship between lipid metabolism, High Mobility Group Box 1 protein (HMGB1)-a pro-inflammatory damage-associated molecular pattern protein-and immune regulation within non-small cell lung adenocarcinoma (NSCLC). Method: To address this question, we used a combination of proteomics, molecular biology, and bioinformatic techniques to investigate the relationship between fatty acids and immune signals within NSCLC. Results: We found that the expression of stearoyl CoA desaturase 1 (SCD1) was decreased in NSCLC tumors compared to normal tissues. This emphasized the critical role of lipid metabolism in tumor progression. Interestingly, monounsaturated fatty acid (MUFA) availability affected the expression of programmed death ligand-1 (PD-L1), a pivotal immune checkpoint target in lung cancer cells and immune cells, as well as HMGB1, suggesting a novel approach to modulating the immune response. This study uncovered a complex interplay between SCD1, PD-L1, and HMGB1, influencing the immunological sensitivity of tumors. Conclusion: Our work underscores the critical importance of understanding the intricate relationships between lipid metabolism and immune modulation to develop more effective NSCLC treatments and personalized therapies. As we continue to explore these connections, we hope to contribute significantly to the ever-evolving field of cancer research, improving patient outcomes and advancing precision medicine in NSCLC.

T cell-Intrinsic Peripheral Tolerance: A Checkpoint Target to Treat Autoimmunity

T cell-Intrinsic Peripheral Tolerance: A Checkpoint Target to Treat Autoimmunity

Cisplatin and Procaterol Combination in Gastric Cancer? Targeting Checkpoint Kinase 1 for Cancer Drug Discovery and Repurposing by an Integrated Computational and Experimental Approach.

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer.

Clinical Application of ImmunoPET Targeting Checkpoint Inhibitors.

In the last decade, monoclonal antibodies (mAbs) targeting CTLA-4, PD-1, or PD-L1 have been developed and immune checkpoint inhibitors (ICIs) have become the main approach in cancer immunotherapy. However, not all patients benefit from ICI therapy and some are at risk of developing treatment-induced side-effects. These aspects, in parallel with the imaging challenges related to response assessments during immunotherapy, have driven scientific research to the discovery of new predictive biomarkers to individualize patients who could benefit from ICIs. In this context, molecular imaging using PET (positron emission tomography), which allows for whole-body tumor visualization, may be a promising non-invasive method for the determination of patients' sensitivity to antibody drugs. Several PET tracers, diverse from 2-[18F]FDG (or 2-Deoxy-2-[18F]fluoroglucose), have been developed to image immune checkpoints (ICs) or key elements of the immune system, although most of them are still in preclinical phases. Herein, we present the current state of the ImmunoPET-targeting of IC proteins with mAbs and antibody fragments, with a main focus on the latest developments in clinical molecular imaging studies of solid tumors. Moreover, given the relevance of the immune system and of tumor-infiltrating lymphocytes in particular in the prediction of the benefit of ICIs, we dedicate a portion of this review to ImmunoPET-targeting T cells.

MYC Pathway Upregulation Identified in Non-Responders to Hypomethylating Agents and Presents a Therapeutic Target in Acute Myeloid Leukemia

Background: In the current era of acute myeloid leukemia (AML) management, hypomethylating agents (HMAs) remain as the backbone of combination regimens for front-line treatment of elderly or unfit patients (pts) and/or as salvage therapy in relapsed/refractory pts. Data on biological predictors of HMA response are limited. Our previous work explored cytidine deaminase (CDA), known to inactivate HMAs, and nucleophosmin 1 (NPM1), determined by core pathway analysis to indirectly influence CDA expression. No clear correlation was identified in pt samples between CDA protein expression and NPM1 status or response, and pharmacogenomic analysis showed no CDA single nucleotide polymorphisms were predictive of response to HMAs. We aimed to expand prior findings using RNA sequencing (RNA seq) and gene set enrichment analysis (GSEA) to identify gene signatures predictive of HMA response and propose potential therapeutic targets. Methods: AML pts with banked samples who received frontline, bridging, or salvage HMA-based therapy between January 2014 to December 2018 were reviewed. Responses following at least 2 cycles of HMA were categorized as complete response (CR), CRi (CR with incomplete hematologic recovery), morphologic leukemia-free state (MLFS), complete hematologic response (CHR), or refractory. Pts were categorized as responders (CR, CRi, MLFS, CHR) or non-responders (refractory). Tumor cells were purified using immunomagnetic selection from bone marrow aspirates collected at diagnosis (dx). RNA seq was performed on 20 available pt tumor samples. Unsupervised clustering was performed and GSEA was completed to compare identified clusters using a false discovery rate (FDR) cut-off of less than 25%. GSEA-derived gene pathway scores were assessed by Wilcoxon rank-sum test. To evaluate AML cell lines to explore the non-responder phenotype, viability of AML cell lines was assessed following treatment with HMA azacitidine (Aza) and MS177 (a proteolysis targeting chimeric EZH2 degrader, also known to degrade MYC). Immunoblotting was used to assay proteolysis targeting chimeric (PROTAC)-induced MYC degradation. Results: Unsupervised clustering of RNA seq from 20 tumor samples at dx, labeled for response and NPM1 status, revealed 3 distinct groups: a non-responder group, a responder group, and a mixed response group ( Figure 1A). GSEA comparing the non-responder group to the responder group revealed several enriched genes in the non-responder group, including MYC targets (FDR &amp;lt;0.001), E2F targets (FDR &amp;lt;0.001), and G2M checkpoint (FDR=0.244). Upregulation of MYC targets was confirmed by enrichment profiling. Application of enrichment pathway scores revealed significant differences between non-responders and responders for E2F targets (p=0.026), G2M checkpoint (p=0.026), and MYC targets (p=0.0022). To explore MYC as a therapeutic target, we assayed MYC degradation in leukemia cells treated with two PROTAC degraders. The PROTAC MS177 induced marked MYC degradation in HL-60 cells ( Figure 1B). Cytotoxicity assays showed that in Kasumi-1 cells, the 50% inhibitory concentrations (IC50) for Aza and MS177 were 2.9 µM (responder phenotype) and 1.1 µM, respectively ( Figure 1C); in HL-60 cells the IC50 for Aza and MS177 were 10.9 µM (non-responder phenotype) and 0.06 µM, respectively ( Figure 1D). MS177 also showed efficacy in cytotoxicity assays using pt derived tumor samples. Conclusions: Using tumor samples from AML pts receiving HMAs, we identified a non-responder gene phenotype enriched with MYC upregulation. GSEA-derived pathway scoring differentiated non-responders from responders in our dataset. To further evaluate whether the enrichment pathway score is predictive for response across other AML cohorts, we are applying enrichment scores to publicly available AML datasets. With MYC upregulation identified as a potential driver of HMA failure, MYC degradation or inhibitory targets are recognized as potential therapeutic targets for pts with a non-responder gene signature. We established the HL-60 AML cell line, found to be less sensitive to Aza than the Kasumi-1 cell line, demonstrated greater sensitivity to MYC degradation compared to Kasumi-1 cells and may reflect the non-responder phenotype. Further in vitro efforts are ongoing to evaluate MYC targets for the HMA non-responder phenotype.

Deconvoluting Clonal and Cellular Architecture in IDH-Mutant Acute Myeloid Leukemia

Introduction Mutations in Isocitrate dehydrogenase 1/2 ( IDH) occur in 20% of acute myeloid leukemias (AML) and frequently co-occur with DNMT3A, NPM1, SRSF2, and NRAS. In clonal hematopoiesis (CH), IDH mutations confer high risk of AML transformation. Recently developed methods linking single cell genotype to single cell transcriptomic profiles and cell lineage offer the opportunity to define the transcriptomic consequences of each mutation across leukemic cell lineages and clonal phylogenies. Here, we develop an integrative single cell laboratory and analytical framework to study the genetic, clonal and cellular heterogeneity of IDH mutated CH and AML, across disease stages (diagnosis and relapse) and deliver a high-resolution map of IDH-mutant AML. Methods Bone marrow or peripheral blood mononuclear samples from 15 AML patients, 2 CH individuals, and 12 unaffected age-matched donors were profiled by single cell RNA-sequencing. For the AML cases, a median of 3 samples per patient were profiled under treatment with IDH inhibitor (IDHi) monotherapy (n=5 patients), IDHi in combination with intensive chemotherapy (NCT02632708, n=6) or in combination with hypomethylating agent (n=2). This dataset comprised 259,610 cells across AML (n=190,133), CH (n=16,705) and normal controls (n=52,772). Genotyping of Transcriptomes (Nam et al., 2019) (for IDH1/2, NPM1, SRSF2, and NRAS mutations) across 6 AML patients enabled genotyping 23% of cells at diagnosis for at least one clone-defining mutation. Results Through meta-analysis of 3,653 diagnostic AML samples profiled by a targeted DNA panel (Papaemmanuil et al., 2016; Tazi et al., 2022), we identified DNMT3A, SRSF2 and NPM1 as critical co-operative mutations in IDH-mutant AML. Amongst the 15 AML patients, 6 had IDH1 mutation, 8 had IDH2 mutations, and 1 had both IDH1 and IDH2. The co-occurring mutations in DNMT3A (n=11 patients), NPM1 (n=5), SRSF2 (n=5) reflected the genomic landscape of IDH-mutant AML. At diagnosis, IDH-mutant AML was enriched in immature hematopoietic stem and progenitor-like blasts. Compared to normal hematopoiesis, IDH-mutant AML blasts upregulated inflammatory response markers including TNFɑ/NFκB-signaling and downregulated proliferation-associated pathways including G2M Checkpoint, and MYC and E2F Targets, and DNA damage response. To investigate how co-mutations modify the IDH-mutant phenotype, we established an integrative single-cell profiling approach to assign single cells to distinct phylogenetic subclones and extract clone-specific transcriptional programs. SRSF2 and NPM1 co-mutations modulated the AML blast phenotype, towards erythroid ( SRSF2) or granulomonocytic lineage ( NPM1). NRAS- or SRSF2-mutant subclones defined distinct gene expression-derived clusters and upregulated proliferation and metabolic pathways. Furthermore, NPM1 co-mutated blasts upregulated KMT2A-target genes. Next, we tracked clonal responses to combination IDHi and chemotherapy using GoT and targeted DNA sequencing. In 4 cases, the ancestral clone, defined by IDH and DNMT3A mutations, retained clonal dominance in both mature and immature myeloid compartments at complete response (median 4 weeks). Conversely, subclones defined by mutations in NPM1, NRAS, or SRSF2, were either reduced or not detectable (limit of detection 2%). In two cases, mutations were not detected after therapy. In patients who relapsed after IDHi monotherapy treatment, blasts upregulated stemness, fatty acid metabolism, OxPHOS, and inflammation at relapse compared to diagnosis. Furthermore, MHC Class II was downregulated at relapse, pointing to immune evasion of AML as a potential relapse mechanism. Finally, we investigated the role of IDH2 mutations at CH. IDH-mutant CH-derived monocytes upregulated inflammation-associated pathways, mirroring the phenotypes observed in IDH-mutant AML at diagnosis. Conclusion We defined how IDH mutations and their frequent co-mutations dysregulated hematopoiesis. We leveraged state-of-the-art single cell techniques to assign leukemic cells to phylogenetic subclones and delineated the role of serially acquired co-mutations in shaping lineage bias and transcriptional programs. Furthermore, we defined genetic, clonal and transcriptional changes at three stages of the AML disease process: diagnosis, remission, and relapse under IDH inhibitor therapy.

A single-cell atlas of immune checkpoint molecules in atherosclerosis in ApoE-/- mice

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

The Novel Checkpoint Target Lymphocyte-Activation Gene 3 Is Highly Expressed in Cutaneous Squamous Cell Carcinoma.

Lymphocyte activation-gene 3 (LAG-3) is an emerging next-generation immune checkpoint molecule. We aim to define the expression pattern of LAG-3 in cutaneous squamous cell carcinoma (cSCC) as a first step to understand the role of LAG-3 in cSCC prognosis and therapy. To define the expression pattern of LAG-3 in cSCC as a first step to understand the role of LAG-3 in cSCC prognosis and therapy. To test whether LAG-3 is expressed on cSCC infiltrating lymphocytes, we isolated CD8 + T lymphocytes from three SCC tumors using flow cytometry and performed single-cell RNA sequencing for LAG-3 and programmed cell death protein -1 (PD-1). In addition, we evaluated LAG-3 mRNA expression in formalin-fixed, paraffin-embedded tissue using NanoString technology. Single-cell RNA sequencing showed that LAG-3 is expressed more than PD-1 in CD8 + tumor infiltrating lymphocytes (50.8% vs 35.2%, respectively). Quantifying LAG-3 mRNA expression showed that compared with normal skin, LAG-3 mRNA is approximately 8 fold higher in immunocompetent associated SCC tumors and approximately 2 fold higher in transplant associated SCC tumors ( p -values <.05). In addition, LAG-3 mRNA was expressed 7.2 fold higher in T2a SCC tumors compared with normal skin ( p -value <.05). Lymphocyte activation-gene 3 is expressed on SCC infiltrating T lymphocytes at a higher percentage than PD-1. In addition, LAG-3 mRNA expression is significantly higher in SCC tumors. Ongoing studies will be performed to define its role as an immune-related biomarker and as a therapeutic target.

Evidence-based management of primary and secondary CNS lymphoma

Central nervous system (CNS) lymphoma has traditionally had very poor outcomes however advances in management have resulted in dramatic improvements and long-term survival of patients. We describe the evidence for treatment strategies for these aggressive disorders. In primary CNS lymphoma there are randomized trial data to inform treatment decisions but these are lacking to guide management in secondary CNS lymphoma. Dynamic assessment of patient fitness and frailty is key throughout treatment, alongside delivery of CNS-bioavailable therapy and enrolment in clinical trials, at each stage of the disease. Intensive high-dose methotrexate-containing induction followed by consolidation with autologous stem cell transplantation with thiotepa-based conditioning is recommended for patients who are fit. Less intensive chemoimmunotherapy, novel agents (including Bruton tyrosine kinase inhibitors, cereblon targeting immunomodulatory agents, and checkpoint inhibitors in the context of clinical trials), and whole brain radiotherapy may be reserved for less fit patients or disease which is chemoresistant. Data regarding the efficacy of chimeric antigen receptor T-cells therapy is emerging, and concerns regarding greater toxicity have not been realized. Future areas of prospective studies include the identification of those at high risk of developing CNS lymphoma, management in elderly or frail patients as well as incorporating novel agents into regimens, particularly for those with chemoresistant disease.

B7H3 targeting gold nanocage pH-sensitive conjugates for precise and synergistic chemo-photothermal therapy against NSCLC

BackgroundThe combination of drug delivery with immune checkpoint targeting has been extensively studied in cancer therapy. However, the clinical benefit for patients from this strategy is still limited. B7 homolog 3 protein (B7-H3), also known as CD276 (B7-H3/CD276), is a promising therapeutic target for anti-cancer treatment. It is widely overexpressed on the surface of malignant cells and tumor vasculature, and its overexpression is associated with poor prognosis. Herein, we report B7H3 targeting doxorubicin (Dox)-conjugated gold nanocages (B7H3/Dox@GNCs) with pH-responsive drug release as a selective, precise, and synergistic chemotherapy-photothermal therapy agent against non-small-cell lung cancer (NSCLC).ResultsIn vitro, B7H3/Dox@GNCs exhibited a responsive release of Dox in the tumor acidic microenvironment. We also demonstrated enhanced intracellular uptake, induced cell cycle arrest, and increased apoptosis in B7H3 overexpressing NSCLC cells. In xenograft tumor models, B7H3/Dox@GNCs exhibited tumor tissue targeting and sustained drug release in response to the acidic environment. Wherein they synchronously destroyed B7H3 positive tumor cells, tumor-associated vasculature, and stromal fibroblasts.ConclusionThis study presents a dual-compartment targeted B7H3 multifunctional gold conjugate system that can precisely control Dox exposure in a spatio-temporal manner without evident toxicity and suggests a general strategy for synergistic therapy against NSCLC.