Immunosuppressive Protein Research Articles

Granzyme B (GZMB)-Positive Tumor-Infiltrating Lymphocytes in Lung Adenocarcinoma: Significance as a Prognostic Factor and Association with Immunosuppressive Proteins.

Granzyme B (GZMB) is a serine protease produced by cytotoxic lymphocytes that reflects the activity of anti-tumor immune responses in tumor-infiltrating lymphocytes (TILs); however, the prognostic significance of GZMB+ TILs in lung adenocarcinoma is poorly understood. We analyzed 273 patients with pathological stage (pStage) I-IIIA lung adenocarcinoma who underwent surgery at Kyushu University from 2003 to 2012. We evaluated GZMB+ TIL counts by immunohistochemistry. We set the cut-off values at 12cells/0.04mm2 for GZMB+ TILs and divided the patients into GZMB-High (n=171) and GZMB-Low (n=102) groups. Then, we compared the clinicopathological characteristics of the two groups and clinical outcomes. Programmed cell death ligand-1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) expression in tumor cells was also evaluated, and combined prognostic analyses of GZMB+ TILs with PD-L1 or IDO1 were performed. GZMB-Low was significantly associated with pStage II-III, PD-L1 positivity, and IDO1 positivity. Disease-free survival (DFS) and overall survival (OS) in the GZMB-Low group were significantly worse than in the GZMB-High group. In multivariable analysis, GZMB-Low was an independent prognostic factor for both DFS and OS. Furthermore, combined prognostic analyses of GZMB+ TILs with PD-L1 or IDO1 showed that GZMB-Low with high expression of these immunosuppressive proteins had the worst prognosis. We analyzed GZMB+ TIL counts in lung adenocarcinoma and elucidated its prognostic significance and association with PD-L1 and IDO1. GZMB+ TIL counts might reflect the patient's immunity against cancer cells and could be a useful prognostic marker of lung adenocarcinoma.

Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.

NAV-003, a bispecific antibody targeting a unique mesothelin epitope and CD3ε with improved cytotoxicity against humoral immunosuppressed tumors.

Mesothelin (MSLN) is a cell surface protein overexpressed in a number of cancer types. Several antibody- and cellular-based MSLN targeting agents have been tested in clinical trials where their therapeutic efficacy has been moderate at best. Previous studies using antibody and Chimeric Antigen Receptor-T cells (CAR-T) strategies have shown the importance of particular MSLN epitopes for optimal therapeutic response, while other studies have found that certain MSLN-positive tumors can produce proteins that can bind to subsets of IgG1-type antibodies and suppress their immune effector activities. In an attempt to develop an improved anti-MSLN targeting agent, we engineered a humanized divalent anti-MSLN/anti-CD3ε bispecific antibody that avoids suppressive factors, can target a MSLN epitope proximal to the tumor cell surface, and is capable of effectively binding, activating, and redirecting T cells to the surface of MSLN-positive tumor cells. NAV-003 has shown significantly improved tumor cell killing against lines producing immunosuppressive proteins in vitro and in vivo. Moreover, NAV-003 demonstrated good tolerability in mice and efficacy against patient-derived mesothelioma xenografts co-engrafted with human peripheral blood mononuclear cells. Together these data support the potential for NAV-003 clinical development and human proof-of-concept studies in patients with MSLN-expressing cancers.

MDB-45. SELECTIVE B7-H3 INHIBITORS NI1 AND NI4 REDUCE AGGRESSIVENESS OF GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a primary childhood malignancy of the central nervous system (CNS). Clinically heterogenous MB are sub-grouped into four molecular subgroups, i.e. Wingless (WNT), Sonic Hedgehog (SHH), Group 3, and Group 4. Among all, Group 3 MB (G3MB) is known to be the most aggressive form, with less than 50% five-year survival. G3MB are associated with large-cell/anaplastic (LCA) histology, distinctive methylation profile, amplification of MYC, and isochromosome 17q (i17q). Targeting these high-risk molecular features unique to G3MB has been challenging, necessitating alternative approaches to therapy for G3-MB. One innovative approach is immunotherapy, which is largely impeded by inhibitory immune checkpoints. B7 homolog 3 (B7-H3/CD276), an immunosuppressive immune checkpoint protein and member of B7 superfamily, is significantly enriched in pediatric malignancies of CNS including G3MB. The enhanced expression of B7-H3/CD276 has been associated with aggressive and metastatic potential. B7-H3/CD276 is also a primary target of miR-1253, a novel tumor suppressor gene silenced in G3MB. Using a structure-based drug discovery pipeline, we screened the ChemBridge library of compounds to discover selective B7-H3 small-molecule inhibitors. Selective B7-H3 inhibitors Ni1 and Ni4 exhibited strong binding scores, CNS penetrance, and oral bioavailability. In vitro, both compounds had appreciably low IC50 values of 3µM (Ni1) and 0.75µM (Ni4). Further, both compounds inhibited wound healing and colony formation. Ni1 shifted the thermostability of B7-H3, suggesting in vitro biding. Ni1 has also undergone PK/PD and MTD studies in vivo, showing stability in serum, good blood-brain barrier permeability, and a half-life of 8 hours. We are now elucidating B7-H3-activated downstream signaling through JAK2/STAT3 signaling and testing the in vivo efficacy for Ni1 against G3MB tumors in a murine model.

Conjugating 4β-NH-(5-Aminoindazole)-Podophyllotoxin and Galectin-1-Targeted Aptamer for Synergistic Chemo-Immunotherapy of Hepatocellular Carcinoma.

By conjugating a chemotherapeutic candidate drug 4β-NH-(5-aminoindazole)-podophyllotoxin (βIZP) and an immunosuppressive protein galectin-1 targeted aptamer AP74, a chemo-immunotherapy molecule (AP74-βIZP) was developed to against liver cancer. AP74-βIZP could target galectin-1 and enrich in tumor microenvironment to improve the tumor inhibition ratio by 6.3% higher than that of βIZP in HepG2 xenograft model. In safety evaluation, βIZP could not be released from AP74-βIZP in normal tissues with low glutathione level. Therefore, the degrees of organs injury and myelosuppression after the treatment with AP74-βIZP were lower than those with βIZP. After 21 days treatment at the drug dose of 5mg kg-1 , AP74-βIZP did not exhibit weight loss of mice, while the weight was significantly reduced by 24% and 14% in oxaliplatin and βIZP, respectively. In immune synergy, AP74-IZP enhanced CD4/CD8 cell infiltration to promote the expression of cell factor (i.e., IL-2, TNF-α, and IFN-γ), which further improved the antitumor activity. The tumor inhibition ratio of AP74-βIZP was 70.2%, which was higher than that of AP74 (35.2%) and βIZP (48.8%). Because of the dual effects of chemotherapy and immunotherapy, AP74-βIZP exhibited superior activity and lower toxicity. The approach developed in this work should be applicable to the other chemotherapy drugs. This article is protected by copyright. All rights reserved.

Comprehensive analysis of the oncogenic and immunological role of FAP and identification of the ceRNA network in human cancers.

Fibroblast activation protein-alpha (FAP) is a transmembrane serine protease involving in tissue remodeling. Previous studies report that FAP is highly expressed in certain tumors and participated in oncogenesis. However, there is still lack of systematic and in-depth analysis of FAP based on clinical big data. Here, we comprehensively map the FAP expression profile, prognostic outcome, genetic alteration, immune infiltration across over 30 types of human cancers through multiple datasets including TCGA, CPTAC, and cBioPortal. We find that FAP is up-regulated in most cancer types, and increased FAP expression is associated with advanced pathological stages or poor prognosis in several cancers. Furthermore, FAP is significantly correlated with the infiltration of cancer-associated fibroblasts, macrophages, myeloid dendritic cells, as well as endothelia cells. Immunosuppressive checkpoint proteins or cytokines expression, microsatellite instability and tumor mutational burden analysis also indicate the regulation role of FAP in tumor progression. Gene enrichment analysis demonstrates that ECM-receptor interaction as well as extracellular matrix and structure process are linked to the potential mechanism of FAP in tumor pathogenesis. The ceRNA network is also constructed and identified the involvement of LINC00707/hsa-miR-30e-5p/FAP, LINC02535/hsa-miR-30e-5p/FAP, LINC02535/hsa-miR-30d-5p/FAP, as well as AC026356.1/hsa-miR-30d-5p/FAP axis in tumor progression. In conclusion, our study offers new insights into the oncogenic and immunological role of FAP from a pan-cancer perspective, providing new clues for developing novel targeted anti-tumor strategies.

Cytokine-targeted treatment in alopecia areata - new possibilities?

Alopecia areata is a form of non-scarring hair loss characterised by a recurrent and difficult-to-treat course. The aetiology of this disease is still incompletely understood. However, multiple immunological pathways and an abnormal cytokine profile in patients with alopecia areata have been described. There are many therapeutic options for patients with alopecia areata, including topical, intralesional or systemic corticosteroids, contact immunotherapy, phototherapy and non-corticosteroid immunosuppressive drugs such as cyclosporine, methotrexate or azathioprine. However, these treatments have limited efficacy and may be associated with side effects. In addition, Janus kinase inhibitors have been shown to cause metabolic disorders. Therefore, their use in patients with alopecia areata may be limited. Other cytokine-targeted therapies have been shown to be effective in alopecia areata, such as apremilast (a phosphodiesterase 4 inhibitor), ustekinumab (a human immunoglobulin (Ig) G1 kappa monoclonal antibody directed against interleukin 12 and 23), abatacept (a soluble fusion protein which links the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 to the modified Fc portion of human immunoglobulin G1), secukinumab (an IL-17A antagonist) and dupilumab (a monoclonal antibody that blocks interleukin 4 and interleukin 13). TNF inhibitors (such as infliximab, adalimumab and etanercept) have been described to be ineffective in alopecia areata. In addition, disease exacerbation after TNF therapy has been reported. Alefacept (an immunosuppressive dimeric fusion protein that consists of the extracellular CD2-binding part of human leukocyte function antigen-3 fused to the Fc part of human IgG1) and efalizumab (anti-CD11a monoclonal antibody) have not shown efficacy in alopecia areata. There are also isolated reports of alopecia areata after therapy with omalizumab (recombinant humanised monoclonal antibody anti-IgE), ixekizumab (inhibitor of IL-17A) and brodalumab (inhibitor of IL-17R).

Evaluation of 89Zr-Labeled Anti-PD-L1 Monoclonal Antibodies Using DFO and Novel HOPO Analogues as Chelating Agents for Immuno-PET.

Programmed death ligand 1 (PD-L1) is a type 1 transmembrane immunosuppressive protein that is expressed on a wide range of cell types, including cancer cells. Anti-PD-L1 antibodies have revolutionized cancer therapy and have led to improved outcomes for subsets of cancer patients, including triple-negative breast cancer (TNBC) patients. As a result, PET imaging of PD-L1 protein expression in cancer patients has been explored for noninvasive detection of PD-L1 expressing tumors as well as monitoring response to anti-PD-L1 immune checkpoint therapy. Previous studies have indicated that the in vivo stability and in vivo target detection of antibody-based radio-conjugates can be dramatically affected by the chelator used. These reports demonstrated that the chelator HOPO diminishes 89Zr de-chelation compared to DFO. Herein, we report an improved HOPO synthesis and evaluated a series of novel analogues for thermal stability, serum stability, PD-L1-specific binding using the BT-549 TNBC cell line, PET imaging in vivo, as well as biodistribution of 89Zr-labeled anti-PD-L1 antibodies in BT-549 xenograft murine models. A new chelator, C5HOPO, demonstrated high stability in vitro and afforded effective PD-L1 targeting in vivovia immuno-PET. These results demonstrated that an improved HOPO chelator is an effective chelating agent that can be utilized to image therapeutically relevant targets in vivo.

Argon gas plasma-treated physiological solutions stimulate immunogenic cell death and eradicates immunosuppressive CD47 protein in lung carcinoma.

Cold atmospheric plasma-treated liquids (PTLs) exhibit selective toxicity toward tumor cells and are provoked by a cocktail of reactive oxygen and nitrogen species in such liquids. Compared to the gaseous phase, these reactive species are more persistent in the aqueous phase. This indirect plasma treatment method has gradually gathered interest in the discipline of plasma medicine to treat cancer. PTL's motivated effect on immunosuppressive proteins and immunogenic cell death (ICD) in solid cancer cells is still not explored. In this study, we aimed to induce immunomodulation by plasma-treated Ringer's lactate (PT-RL) and phosphate-buffered saline (PT-PBS) solutions for cancer treatment. PTLs induced minimum cytotoxicity in normal lung cells and inhibited cancer cell growth. ICD is confirmed by the enhanced expression of damage-associated molecular patterns (DAMPs). We evidenced that PTLs induce intracellular nitrogen oxide species accumulation and elevate immunogenicity in cancer cells owing to the production of pro-inflammatory cytokines, DAMPs, and reduced immunosuppressive protein CD47 expression. In addition, PTLs influenced A549cells to elevate the organelles (mitochondria and lysosomes) in macrophages. Taken together, we have developed a therapeutic approach to potentially facilitate the selection of a suitable candidate for direct clinical applications.

Down-regulation of DNA key protein-FEN1 inhibits OSCC growth by affecting immunosuppressive phenotypes via IFN-γ/JAK/STAT-1

Oral squamous cell carcinoma (OSCC) escape from the immune system is mediated through several immunosuppressive phenotypes that are critical to the initiation and progression of tumors. As a hallmark of cancer, DNA damage repair is closely related to changes in the immunophenotypes of tumor cells. Although flap endonuclease-1 (FEN1), a pivotal DNA-related enzyme is involved in DNA base excision repair to maintain the stability of the cell genome, the correlation between FEN1 and tumor immunity has been unexplored. In the current study, by analyzing the clinicopathological characteristics of FEN1, we demonstrated that FEN1 overexpressed and that an inhibitory immune microenvironment was established in OSCC. In addition, we found that downregulating FEN1 inhibited the growth of OSCC tumors. In vitro studies provided evidence that FEN1 knockdown inhibited the biological behaviors of OSCC and caused DNA damage. Performing multiplex immunohistochemistry (mIHC), we directly observed that the acquisition of critical immunosuppressive phenotypes was correlated with the expression of FEN1. More importantly, FEN1 directly or indirectly regulated two typical immunosuppressive phenotype-related proteins human leukocyte antigen (HLA-DR) and programmed death receptor ligand 1 (PD-L1), through the interferon-gamma (IFN-γ)/janus kinase (JAK)/signal transducer and activator transcription 1 (STAT1) pathway. Our study highlights a new perspective on FEN1 action for the first time, providing theoretical evidence that it may be a potential immunotherapy target for OSCC.

Abstract 2899: Small molecule inhibitor of ubiquitin ligase CBL-B enhanced anti-tumor response of CAR-T and CAR-NK cell therapies

Abstract Introduction: Chimeric antigen receptor (CAR) T-cell therapy is an emerging therapeutic option for cancer treatment. However, its efficacy is limited, especially in solid tumors. This is partly because the CAR T cells become dysfunctional and exhausted in the tumor microenvironment. However, the key pathways responsible for impaired function of exhausted T cells remain unclear. Immunosuppressive cytokines or checkpoint proteins like TGF-β and PD-L1, are overproduced, which may lead to the downregulation of CD8+ T cell action and the promotion of T-reg maturation. E3 ligase Casitas B-Lineage Lymphoma Proto-Oncogene B (CBL-B) is a key negative modulator of T/NK-cell receptor and co-stimulatory regulation. CBL-B inhibition lowers the threshold of antigen-specific T/NK cell activation, even in absence of co-stimulatory signaling or the presence of an immune suppressive environment. Genetic ablation of CBL-B or functional inactivation of its E3 ligase activity in mice or primary human T cells enhances immune-mediated tumor growth control. Therefore, CBL-B inhibition may address the suboptimal response to current immunotherapies due to low inflammation, no/low co-stimulation signal or a high immune suppressive environment. Several groups demonstrated that deficiency of CBL-B overcomes endogenous CD8+ T-cell exhaustion, and deletion of CBL-B in CAR-T cells renders them resistant to exhaustion and deletion of CBL-B in CAR-NK cells provides greater resistance to TGF-β. Methods: We tested the CAR expression, T/NK cell activation and in vitro cytotoxicity when ex vivo culture of CAR-T or CAR-NK cells with CBL-B inhibitors. Results: We demonstrated that the combination of CBL-B inhibitor and CAR-NK cells elicited remarkable persistence in multiple runs of repeated cancer cell clearance assay, as compared to CAR-NK cells alone. Moreover, the combination of CAR-NK cells with both anti-PDL1-IL15 and CBL-B inhibitor showed further increased persistence in repeated cancer cell clearance assay. Conclusions: preclinical data provided evidence of CBL-B inhibitors to enhance key parameters associated with T/NK cell activation and reduce susceptibility to immune suppression. Our results suggest the combination strategy of cell therapy and immune regulating small/large molecule therapy might achieve better anti-tumor efficacy in both hematological and solid tumors. Citation Format: Cuiqing Yang, Yifang Wang, Tingting Liu, Fuwei Jiang, Chao Wang, Qingyang Wang, Qin Wang, Zhengtao Li, Wansun Mai, Gang Ye, Renhong Tang, Zhuoxiao Cao. Small molecule inhibitor of ubiquitin ligase CBL-B enhanced anti-tumor response of CAR-T and CAR-NK cell therapies [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 2899.

Abstract 5666: NAV-003, a full-length IgG1 bispecific antibody targeting a unique mesothelin epitope and CD3 to improve cytotoxicity against humoral immunosuppressed tumors

Abstract Mesothelin (MSLN) is a cell surface protein over-expressed in a number of cancer types. Several antibody- and cellular-based MSLN targeting agents have been tested in clinical trials where their therapeutic efficacy has been moderate at best. Previous studies using antibody and CAR-T based agents have shown the importance of particular MSLN epitopes for optimal therapeutic response, while other studies have found certain MSLN-positive tumors can produce proteins that can bind to subsets of IgG1-type antibodies and suppress their immune-effector activities. In an attempt to develop a more optimized anti-MSLN targeting agent, we engineered a humanized divalent anti-MSLN/anti-CD3ε bispecific antibody called NAV-003 that targets a MSLN epitope proximal to the tumor cell surface and an anti-CD3ε single-chain antibody capable of effectively binding, activating and directing T-cells to the surface of MSLN-bound tumor cells. NAV-003 has shown significantly improved tumor cell killing against lines producing immunosuppressive proteins in vitro and in vivo. Moreover, NAV-003 demonstrated good tolerability and efficacy against patient-derived mesothelioma xenograft models co-engrafted with human peripheral blood mononuclear cells. Together these data support the potential for NAV-003 clinical development and human proof-of-concept studies in patients with MSLN-expressing cancers. Citation Format: Luigi Grasso, Qun Jiang, Raffit Hassan, Nicholas C. Nicolaides, J. Bradford Kline. NAV-003, a full-length IgG1 bispecific antibody targeting a unique mesothelin epitope and CD3 to improve cytotoxicity against humoral immunosuppressed tumors. [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 5666.

Abstract 3065: Increased longevity and quality of life with treatment with a progesterone receptor modulator in a patient with treatment resistant stage IV small cell lung cancer

Abstract The progesterone receptor modulator, mifepristone, has been found to provide extension of life by years and marked palliative benefits in several cases of non-small cell lung cancer (NSCLC) despite progression with platinum-based chemotherapy, check-point inhibitors, and third-generation tyrosine inhibitors. Presented here is a case of small cell lung cancer (SCLC) treated with mifepristone. This patient was diagnosed at age 58 with stage IV NSCLC with EGFR deletion 19 mutation. He was treated with single agent osimertinib 80mg once/day. Two months later his serial CT scan showed improvement. He was treated with stereotactic ablative radiotherapy for one persisting lesion. He was able to conduct a very active life. However, clinical symptoms started to return after 8 months, and CT of the chest showed slightly increased size of a the previously treated right upper lobe mass with increased small right effusion, increased pleural nodularity, and new interlobular septal thickening. Biopsy of the pleural mass showed transformation to SCLC. He failed a clinical trial. He began mifepristone orally 200mg/day. Within 1 month he was able to resume normal activity including strenuous sports. CT scans 2 months later showed no longer any pleural effusions, pleural nodularity, or growth of the persisting nodule that had been previously radiated. His absence of clinical symptoms continued for 10 more months but CT now showed slight growth of the persisting lesion. He assumed that with the growth that mifepristone was losing its effectiveness, and after consultation with another oncologist, elected to be treated in another clinical study for a new promising drug for SCLC that precluded him from continuing mifepristone. He died 5 months later. The main benefit of mifepristone is believed to be by blocking membrane rather than nuclear progesterone receptors (PRs) that are responsible for producing an immunosuppressive protein called the progesterone induced blocking factor which allows various cancers to spread. A previous case of stage IV NSCLC without any tumor markers treated with mifepristone had no growth and good health x 2 ½ years when 1 lesion begins to grow slowly. His oncologist suggested stopping mifepristone and try a check-point inhibitor. Instead, he continued mifepristone. He died 3 years later from pneumonia still feeling quite well while the one lesion continued to grow slowly. Another case of rapidly progressing lung cancer diagnosed by CT scan corrected her severe hypoxia (PO2 70 mmHg) and hyponatremia (serum Na+ of 118 mmol/L) after 1 month of taking mifepristone. Her lesions mostly disappeared and 5 years later she died of a myocardial infarction. Though clinically suspicious of SCLC, it was not biopsy proven. Hopefully this case will inspire a clinical trial of PR antagonists for SCLC. Citation Format: Jerome H. Check, Carrie Wilson, Diane Check, Trina Poretta, Maya Srivastava. Increased longevity and quality of life with treatment with a progesterone receptor modulator in a patient with treatment resistant stage IV small cell lung cancer [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 3065.

Galectin-1: A Traditionally Immunosuppressive Protein Displays Context-Dependent Capacities

Galectin-Carbohydrate interactions are indispensable to pathogen recognition and immune response. Galectin-1, a ubiquitously expressed 14-kDa protein with an evolutionarily conserved β-galactoside binding site, translates glycoconjugate recognition into function. That galectin-1 is demonstrated to induce T cell apoptosis has led to substantial attention to the immunosuppressive properties of this protein, such as inducing naive immune cells to suppressive phenotypes, promoting recruitment of immunosuppressing cells as well as impairing functions of cytotoxic leukocytes. However, only in recent years have studies shown that galectin-1 appears to perform a pro-inflammatory role in certain diseases. In this review, we describe the anti-inflammatory function of galectin-1 and its possible mechanisms and summarize the existing therapies and preclinical efficacy relating to these agents. In the meantime, we also discuss the potential causal factors by which galectin-1 promotes the progression of inflammation.

PD-1 Independent Role of PD-L1 in Triple-Negative Breast Cancer Progression

Triple-negative breast cancer (TNBC) is a type of breast malignancy characterized by a high proliferative rate and metastatic potential leading to treatment failure, relapse, and poor prognosis. Therefore, efforts are continuously being devoted to understanding its biology and identifying new potential targets. Programmed death-ligand 1 (PD-L1) is an immunosuppressive protein that inactivates T cells by binding to the inhibitory receptor programmed death-1 (PD-1). PD-L1 overexpression in cancer cells contributes to immune evasion and, subsequently, poor survival and prognosis in several cancers, including breast cancer. Apart from its inhibitory impact on T cells, this ligand is believed to have an intrinsic role in cancer cells. This study was performed to clarify the PD-1 independent role of PD-L1 in TNBC MDA-MB-231 cells by knocking out the PD-L1 using three designs of CRISPR-Cas9 lentiviral particles. Our study revealed that PD-L1 knockout significantly inhibited MDA-MB-231 cell proliferation and colony formation in vitro and tumor growth in the chick embryo chorioallantoic membrane (CAM) model in vivo. PD-L1 knockout also decreased the migration and invasion of MDA-MB-231 cells in vitro. We have shown that PD-L1 knockout MDA-MB-231 cells have low levels of p-Akt and p-ERK in addition to some of their downstream proteins, c-Fos, c-Myc, p21, survivin, and COX-2. Furthermore, PD-L1 knockout significantly decreased the expression of Snail and RhoA. This study shows the intrinsic role of PD-L1 in TNBC independently of its binding to PD-1 receptors on T cells. It may pave the way for developing novel therapeutic strategies using PD-L1 inhibitors alone and in combination to treat TNBC more effectively.

Molluscum Contagiosum Virus Protein MC008 Targets NF-κB Activation by Inhibiting Ubiquitination of NEMO.

Molluscum contagiosum virus (MCV) is a human-adapted poxvirus that causes a common and persistent yet mild infection characterized by distinct, contagious, papular skin lesions. These lesions are notable for having little or no inflammation associated with them and can persist for long periods without an effective clearance response from the host. Like all poxviruses, MCV encodes potent immunosuppressive proteins that perturb innate immune pathways involved in virus sensing, the interferon response, and inflammation, which collectively orchestrate antiviral immunity and clearance, with several of these pathways converging at common signaling nodes. One such node is the regulator of canonical nuclear factor kappa B (NF-κB) activation, NF-κB essential modulator (NEMO). Here, we report that the MCV protein MC008 specifically inhibits NF-κB through its interaction with NEMO, disrupting its early ubiquitin-mediated activation and subsequent downstream signaling. MC008 is the third NEMO-targeting inhibitor to be described in MCV to date, with each inhibiting NEMO activation in distinct ways, highlighting strong selective pressure to evolve multiple ways of disabling this key signaling protein. IMPORTANCE Inflammation lies at the heart of most human diseases. Understanding the pathways that drive this response is the key to new anti-inflammatory therapies. Viruses evolve to target inflammation; thus, understanding how they do this reveals how inflammation is controlled and, potentially, how to disable it when it drives disease. Molluscum contagiosum virus (MCV) has specifically evolved to infect humans and displays an unprecedented ability to suppress inflammation in our tissue. We have identified a novel inhibitor of human innate signaling from MCV, MC008, which targets NEMO, a core regulator of proinflammatory signaling. Furthermore, MC008 appears to inhibit early ubiquitination, thus interrupting later events in NEMO activation, thereby validating current models of IκB kinase (IKK) complex regulation.

Lung Cancer - Standard Therapy and the Use of a Novel, Highly Effective, Well Tolerated, Treatment With Progesterone Receptor Modulators.

The most recent successful advances in lung cancer therapy have directly and increasingly focused on personalized tumor genetic/epigenetic/immunologic profiling, and the identification and development of novel pharmacologic agents aimed at those mutations [e.g., epidermal growth factor receptor (EGFR), Kristen rat sarcoma viral oncogene homolog (KRAS), anaplastic lymphoma kinase (ALK) and immunotherapy against programmed cell death protein 1 (PD-1) and its ligands] which have extended life and provided palliation for lung cancer-patients positive for these mutations. The objective of this study is to provide a review of the large number of drugs and their efficacy as of 2022, for lung cancer, but also introduce a novel treatment that has the potential, based on one controlled murine lung cancer study and 5 anecdotal human cases, that showed marked palliative and longevity benefits in very advanced lung cancer with no other treatment options, i.e., progesterone receptor (PR) antagonists targeting the immunosuppressive protein, the progesterone induced blocking factor (PIBF). Credibility, however, will only be provided when the efficacy can be demonstrated in a large series of lung cancer cases ideally with certain controls. Thus, the ultimate objective of the review is to interest oncologists with a large population of lung cancer patients to perform a well powered study to corroborate or refute the limited experience to date with PR antagonist therapy.

African Swine Fever Virus L83L Negatively Regulates the cGAS-STING-Mediated IFN-I Pathway by Recruiting Tollip To Promote STING Autophagic Degradation.

African swine fever (ASF) is a devastating infectious disease of pigs caused by the African swine fever virus (ASFV), which poses a great danger to the global pig industry. Many viral proteins can suppress with interferon signaling to evade the host's innate immune responses. Therefore, the development of an effective vaccine against ASFV has been dampened. Recent studies have suggested that the L83L gene may be integrated into the host genome, weakening the host immune system, but the underlying mechanism is unknown. Our study found that L83L negatively regulates the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. Overexpression of L83L inhibited IFN-β promoter and ISRE activity, and knockdown of L83L induced higher transcriptional levels of interferon-stimulated genes (ISGs) and phosphorylation levels of IRF3 in primary porcine alveolar macrophages. Mechanistically, L83L interacted with cGAS and STING to promote autophagy-lysosomal degradation of STING by recruiting Tollip, thereby blocking the phosphorylation of the downstream signaling molecules TBK1, IRF3, and IκBα and reducing IFN-I production. Altogether, our study reveals a negative regulatory mechanism involving the L83L-cGAS-STING-IFN-I axis and provides insights into an evasion strategy involving autophagy and innate signaling pathways employed by ASFV. IMPORTANCE African swine fever virus (ASFV) is a large double-stranded DNA virus that primarily infects porcine macrophages. The ASFV genome encodes a large number of immunosuppressive proteins. Current options for the prevention and control of this pathogen remain pretty limited. Our study showed that overexpression of L83L inhibited the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. In contrast, the knockdown of L83L during ASFV infection enhanced IFN-I production in porcine alveolar macrophages. Additional analysis revealed that L83L protein downregulated IFN-I signaling by recruiting Tollip to promote STING autophagic degradation. Although L83L deletion has been reported to have little effect on viral replication, its immune evade mechanism has not been elucidated. The present study extends our understanding of the functions of ASFV-encoded pL83L and its immune evasion strategy, which may provide a new basis for developing a live attenuated vaccine for ASF.

Optimization of CD200 checkpoint immunotherapy for treating glioblastoma.

Glioblastoma Multiforme (GBM) is a lethal brain cancer with a median survival between 10-14 months. The standard of care for GBM is a combination of resection, followed by chemotherapy, and radiotherapy. Recent advances in cancer immunotherapy have significantly improved the survival of patients in selected solid tumors. However, in the case of GBM, current immunotherapy has failed, potentially, due to obstacles including immunosuppressive molecules in the tumor microenvironment. CD200 is an immunosuppressive protein that is overexpressed on tumors suppressing the immune system. The University of Minnesota developed a novel CD200 checkpoint immunotherapy, currently in Phase I clinical trial (NCT04642937), targeting the activation receptors on immune cells with a peptide ligand to overcome its immunosuppressive effect on recurrent GBM. While the treatment shows promising results in patient outcomes, the CD200 mechanism in the dendritic cells is still unclear. In this study, we postulate a detailed pathway and develop an Ordinary Differential Equation model, based on the law of mass action, to describe its behavior. Using Global Sensitivity analysis techniques, we explore the parameter space and identify potential targets on the pathway to optimize the CD200 checkpoint therapy and improve its clinical impact.

Multi-omics profiling and digital image analysis reveal the potential prognostic and immunotherapeutic properties of CD93 in stomach adenocarcinoma.

Recent evidence highlights the fact that immunotherapy has significantly improved patient outcomes. CD93, as a type I transmembrane glycoprotein, was correlated with tumor-associated angiogenesis; however, how CD93 correlates with immunotherapy in stomach adenocarcinoma (STAD) remains unclear. TCGA, GTEx, GEO, TIMER2.0, HPA, TISIDB, TCIA, cBioPortal, LinkedOmics, and ImmuCellAI public databases were used to elucidate CD93 in STAD. Visualization and statistical analysis of data were performed by R (Version 4.1.3), GraphPad (Version 8.0.1), and QuPath (Version 0.3.2). CD93 was highly expressed in STAD compared with adjacent normal tissues. The overexpression of CD93 was significantly correlated with a poor prognosis in STAD. There was a negative correlation between CD93 expression levels with CD93 mutation and methylation in STAD. Our results revealed that CD93 expression was positively associated with most immunosuppressive genes (including PD-1, PD-L1, CTLA-4, and LAG3), immunostimulatory genes, HLA, chemokine, and chemokine receptor proteins in STAD. Furthermore, in STAD, CD93 was noticeably associated with the abundance of multiple immune cell infiltration levels. Functional HALLMARK and KEGG term enhancement analysis of CD93 through Gene Set Enrichment Analysis was correlated with the process of the angiogenesis pathway. Subsequently, digital image analysis results by QuPath revealed that the properties of CD93+ cells were statistically significant in different regions of stomach cancer and normal stomach tissue. Finally, we utilized external databases, including GEO, TISIDB, ImmuCellAI, and TCIA, to validate that CD93 plays a key role in the immunotherapy of STAD. Our study reveals that CD93 is a potential oncogene and is an indicative biomarker of a worse prognosis and exerts its immunomodulatory properties and potential possibilities for immunotherapy in STAD.