Off-target toxicity prediction in cellular cancer immunotherapies

Cytotherapy

2021 May 01

Murcia Pienkowski, V;Mazzocco, G;Niemiec, I;Sanecka-Duin, A;Krol, P;Myronov, O;Skoczylas, P;Kaczmarczyk, J;Blum, A;
| DOI: 10.1016/S1465324921004229

Background & Aim: One of the major bottlenecks of cancer cellular therapy development is off-target toxicity. It is caused by activated T-cells that unexpectedly recognize epitopes presented on healthy tissues instead of interacting only with the intended target on cancer cells. This mechanism can lead to severe immuno-toxicity resulting in organ dysfunction or even death. Unfortunately, experimental identification of epitopes that may trigger off-target toxicity is both costly and time consuming. In an attempt to accelerate this process, we created ArdImmune Tox, a computational tool assessing epitopes for their potential toxicity. Methods, Results & Conclusion: ArdImmune Tox builds up on the recent advances in computational immunology and Artificial Intelligence (AI). In the first step ArdImmune Tox mimics an experimental approach (X-scan) by simultaneously modifying multiple amino acids in the target peptide creating many possible off-target epitopes (OTE). Next, the peptide collection is mapped against a curated reference dataset encompassing proteomes from more than 1000 patients. In this step we retain peptides found in proteins expressed in healthy human tissues and keep track of their frequency. In order to establish which peptides are presented by the HLAs on the cell surface we use a dedicated machine-learning model developed in-house, which was trained on mass spectroscopy data of peptide-HLA (pHLA) presentation. These peptides are then scored for similarity to the target peptide based on selected physico-chemical properties. Importantly, only amino acids in positions identified by our approach as interacting with TCRs are considered. We present results of ArdImmune Tox on three cancer immunotherapy cases, where the OTEs were identified experimentally. In all of them ArdImmune Tox correctly identified the OTEs, ranking them among the 3 highest scoring potential off-target peptides, whereas BLAST algorithm-based approaches either positioned OTEs much lower in the ranking or even failed to identify them altogether. In conclusion, we introduce ArdImmuneTox - a novel, effective, in silico approach for the identification of peptide- based off-target toxicity and T-cell cross-reactivity in immunotherapies. Our approach shows superior performance to currently used sequence comparison-based methods. Our results indicate that ArdImmune-Tox can aid in the rapid and cost-effective selection of safe epitope targets for cancer immunotherapies.

Senescent alveolar macrophages promote early-stage lung tumorigenesis

Cancer cell

2023 May 24

Prieto, LI;Sturmlechner, I;Graves, SI;Zhang, C;Goplen, NP;Yi, ES;Sun, J;Li, H;Baker, DJ;
PMID: 37267954 | DOI: 10.1016/j.ccell.2023.05.006

Senescent cells play relevant but context-dependent roles during tumorigenesis. Here, in an oncogenic Kras-driven lung cancer mouse model, we found that senescent cells, specifically alveolar macrophages, accumulate early in neoplasia. These macrophages have upregulated expression of p16INK4a and Cxcr1, are distinct from previously defined subsets and are sensitive to senolytic interventions, and suppress cytotoxic T cell responses. Their removal attenuates adenoma development and progression in mice, indicating their tumorigenesis-promoting role. Importantly, we found that alveolar macrophages with these properties increase with normal aging in mouse lung and in human lung adenocarcinoma in situ. Collectively, our study indicates that a subset of tissue-resident macrophages can support neoplastic transformation through altering their local microenvironment, suggesting that therapeutic interventions targeting senescent macrophages may attenuate lung cancer progression during early stages of disease.

TNF-Related Apoptosis-Inducing Ligand (TRAIL) Loss in Canine Mammary Carcinoma

Veterinary and comparative oncology

2021 Aug 23

Kim, SH;Seung, BJ;Bae, MK;Lim, HY;Cho, SH;Sur, JH;
PMID: 34423555 | DOI: 10.1111/vco.12767

Escaping apoptosis is a hallmark of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a central molecule that regulates the extrinsic apoptotic pathway, has been widely investigated in human oncology; however, investigations focusing on the endogenous expression of TRAIL in canine tumors are lacking. Therefore, we aimed to examine the expression of endogenous TRAIL in canine mammary tumors and analyzed its correlation to downstream molecules Fas-associated death domain protein (FADD) and caspase-3, and to the apoptotic index. A total of 147 samples, classified as normal mammary gland (n = 9), mammary adenoma (n = 30), low-grade carcinoma (n = 42), and high-grade carcinoma (n = 66) were included in the immunohistochemical analyses, and 43 samples with sufficient levels of RNA were analyzed via RNA in situ hybridization and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. In immunohistochemistry, TRAIL protein expression was significantly decreased in high-grade carcinoma compared to those in normal mammary gland and adenoma, with similar downregulation of TRAIL mRNA expression. Also, FADD and caspase-3 expression positively correlated with TRAIL expression. However, the apoptotic index was paradoxically elevated in high-grade tumors. Overall, these results suggest that loss of TRAIL accompanied by dysregulation of TRAIL-induced extrinsic apoptotic pathway molecules could affect malignant progression of canine mammary tumors.This article is protected by

Preclinical pharmacology modeling of chimeric antigen receptor T therapies

Current opinion in pharmacology

2021 Oct 04

Kumari, R;Ouyang, X;Wang, J;Xu, X;Zheng, M;An, X;Li, QX;
PMID: 34619442 | DOI: 10.1016/j.coph.2021.08.008

Chimeric antigen receptor (CAR) T cells have largely been successful in treating hematological malignancies in the clinic but have not been as effective in treating solid tumors, in part, owing to poor access and the immunosuppressive tumor microenvironment. In addition, CAR-T therapy can cause potentially life-threatening side effects, including cytokine release syndrome and neurotoxicity. Current preclinical testing of CAR-T therapy efficacy is typically performed in mouse tumor models, which often fails to predict toxicity. Recent developments in humanized models and transgenic mice as well as in vitro three-dimensional organoids in early development and nonhuman primate models are being adopted for CAR-T cell efficacy and toxicity assessment. However, because no single model perfectly recapitulates the human immune system and tumor microenvironment, careful model selection based on their respective pros and cons is crucial for adequate evaluation of different CAR-T treatments, so that their clinical development can be better supported.

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation

Cell stem cell

2021 Jun 11

Sun, T;Annunziato, S;Bergling, S;Sheng, C;Orsini, V;Forcella, P;Pikiolek, M;Kancherla, V;Holwerda, S;Imanci, D;Wu, F;Meylan, LC;Puehringer, LF;Waldt, A;Oertli, M;Schuierer, S;Terracciano, LM;Reinker, S;Ruffner, H;Bouwmeester, T;Sailer, AW;George, E;Roma, G;de Weck, A;Piscuoglio, S;Lohmann, F;Naumann, U;Liberali, P;Cong, F;Tchorz, JS;
PMID: 34129813 | DOI: 10.1016/j.stem.2021.05.013

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.

PD-1/PD-L1 Pathway: A Therapeutic Target in CD30+ Large Cell Lymphomas

Biomedicines

2022 Jul 04

Xie, W;Medeiros, LJ;Li, S;Tang, G;Fan, G;Xu, J;
PMID: 35884893 | DOI: 10.3390/biomedicines10071587

The programmed death-ligands, PD-L1 and PD-L2, reside on tumor cells and can bind with programmed death-1 protein (PD-1) on T-cells, resulting in tumor immune escape. PD-1 ligands are highly expressed in some CD30+ large cell lymphomas, including classic Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma (PMBL), Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (EBV+ DLBCL), and anaplastic large cell lymphoma (ALCL). The genetic alteration of the chromosome 9p24.1 locus, the location of PD-L1, PD-L2, and JAK2 are the main mechanisms leading to PD-L1 and PD-L2 overexpression and are frequently observed in these CD30+ large cell lymphomas. The JAK/STAT pathway is also commonly constitutively activated in these lymphomas, further contributing to the upregulated expression of PD-L1 and PD-L2. Other mechanisms underlying the overexpression of PD-L1 and PD-L2 in some cases include EBV infection and the activation of the mitogen-activated protein kinase (MAPK) pathway. These cellular and molecular mechanisms provide a scientific rationale for PD-1/PD-L1 blockade in treating patients with relapsed/refractory (R/R) disease and, possibly, in newly diagnosed patients. Given the high efficacy of PD-1 inhibitors in patients with R/R CHL and PMBL, these agents have become a standard treatment in these patient subgroups. Preliminary studies of PD-1 inhibitors in patients with R/R EBV+ DLBCL and R/R ALCL have also shown promising results. Future directions for these patients will likely include PD-1/PD-L1 blockade in combination with other therapeutic agents, such as brentuximab or traditional chemotherapy regimens.

Combination immunotherapy including OncoVEXmGMCSF creates a favorable tumor immune micro-environment in transgenic BRAF murine melanoma

Cancer immunology, immunotherapy : CII

2022 Jan 09

Gartrell, RD;Blake, Z;Rizk, EM;Perez-Lorenzo, R;Weisberg, SP;Simoes, I;Esancy, C;Fu, Y;Davari, DR;Barker, L;Finkel, G;Mondal, M;Minns, HE;Wang, SW;Fullerton, BT;Lozano, F;Chiuzan, C;Horst, B;Saenger, YM;
PMID: 34999916 | DOI: 10.1007/s00262-021-03088-y

Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma.

Precision Cancer Medicine

Springer

2022 Jan 04

Jasani, B;Huss, R;Taylor, C;
| DOI: 10.1007/978-3-030-84087-7

Bharat Jasani, BSc, PhD, MBChB, FRCPath. Qualifying with honours in Biochemistry (University of Glasgow,1965-1969), PhD in Experimental Pathology and MBChB (honours distinction in Pathology) (University of Birmingham, 1970-1973), Dr Jasani initiated his clinical and research training in Histopathology, at Wales National School of Medicine in 1977, achieving MRCPath (1989), FRCPath (1997), and a Specialist Consultant’s status in Histopathology (Immunohistochemistry) (1993); and Personal Chair in Oncological Pathology (2003). Following his retirement as the Head of Histopathology and Academic Pathology (2003-2011), he continued as Professor of Pathology, Institute of Cancer & Genetics, Cardiff University & Associate Postgraduate Dean for Academic Medicine in Wales (2011-2015). He was then appointed as the Chair of Biomedical Sciences, Nazarbayev University School of Medicine. Astana, Kazakhstan, with Adjunct Professorship in Pathology, University of Pittsburgh School of Medicine, Pittsburg, USA (2015-2016). IN 2016 HE TOOK UP HIS CURRENT POSITION AS THE DIRECTOR OF PATHOLOGY, TARGOS MOLECULAR PATHOLOGY GMBH, KASSEL, GERMANY, WHICH WAS ACQUIRED BY DISCOVERY LIFE SCIENCES IN 2021. Over the past 40 years Dr Jasani has devoted himself to advancing cancer pathology based on the use of cancer biomarkers. As the Head of Molecular Diagnostic Unit, University Hospital of Wales (1982-2003), he led the development and establishment of diagnostic immunocytochemistry & molecular pathology services in Cardiff and Wales. He also promoted quality assurance and standardization of cancer biomarker analyses as Breast Cancer Module Leader of UKNEQAS of IHC/ISH, and UK’s lead representative on International Working Group on Standardization of Breast Cancer Biomarking, and the United States Sub-Committee on Quality Assurance for Immunocytochemistry. As Principal Investigator and Co-investigator of several translational research projects on development of predictive biomarkers for colorectal and breast cancer, he has also been invited to act as a Consultant and Key Opinion Leader to several leading biotech and pharmaceutical companies. He is an author of a book, over 50 chapters and reviews and more than 200 peer reviewed research publications.

The role of single-cell sequencing in studying tumour evolution

Faculty reviews

2021 May 26

Mossner, M;Baker, AC;Graham, TA;
PMID: 34131659 | DOI: 10.12703/r/10-49

Tumour evolution is a complex interplay between the acquisition of somatic (epi)genomic changes in tumour cells and the phenotypic consequences they cause, all in the context of a changing microenvironment. Single-cell sequencing offers a window into this dynamic process at the ultimate resolution of individual cells. In this review, we discuss the transformative insight offered by single-cell sequencing technologies for understanding tumour evolution.

Scientific reports

2022 Dec 27

Polverino, F;Mirra, D;Yang, CX;Esposito, R;Spaziano, G;Rojas-Quintero, J;Sgambato, M;Piegari, E;Cozzolino, A;Cione, E;Gallelli, L;Capuozzo, A;Santoriello, C;Berrino, L;de-Torres, JP;Hackett, TL;Polverino, M;D'Agostino, B;
PMID: 36575294 | DOI: 10.1038/s41598-022-26650-9

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1-2) COPD patients compared to severe-very severe (GOLD 3-4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1-2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma

Molecular cancer research : MCR

2023 Jun 06

Wang, X;Kutschat, AP;Aggrey-Fynn, J;Hamdan, FH;Graham, RP;Wixom, AQ;Souto, Y;Ladigan-Badura, S;Yonkus, JA;Abdelrahman, AM;Alva-Ruiz, R;Gaedcke, J;Strobel, P;Kosinsky, RL;Wegwitz, F;Hermann, P;Truty, MJ;Siveke, JT;Hahn, SA;Hessmann, E;Johnsen, SA;Najafova, Z;
PMID: 37279184 | DOI: 10.1158/1541-7786.MCR-22-0916

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft (PDX) models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a basal-like A subtype-specific transcribed enhancer program (B-STEP) in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histological approach for PDAC patient stratification by performing RNA in situ hybridization analyses for subtype-specific eRNAs on pathological tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single cell level in complex, heterogeneous, primary tumor material. Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single cell level in patient material can be used as a potential tool for treatment stratification.

Allogeneic CAR T Cells Targeting DLL3 Are Efficacious and Safe in Preclinical Models of Small Cell Lung Cancer

Clinical cancer research : an official journal of the American Association for Cancer Research

2023 Jan 23

Zhang, Y;Tacheva-Grigorova, SK;Sutton, J;Melton, Z;Mak, YSL;Lay, C;Smith, BA;Sai, T;Van Blarcom, T;Sasu, BJ;Panowski, SH;
PMID: 36692420 | DOI: 10.1158/1078-0432.CCR-22-2293

Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Delta-like ligand 3 (DLL3) is highly expressed on SCLC and several other types of neuroendocrine cancers, with limited normal tissue RNA expression in brain, pituitary, and testis, making it a promising CAR T-cell target for SCLC and other solid tumor indications.A large panel of anti-DLL3 scFv-based CARs were characterized for both in vitro and in vivo activity. To understand the potential for pituitary and brain toxicity, subcutaneous or intracranial tumors expressing DLL3 were implanted in mice and treated with mouse cross-reactive DLL3 CAR T cells.A subset of CARs demonstrated high sensitivity for targets with low DLL3 density and long-term killing potential in vitro. Infusion of DLL3 CAR T cells led to robust antitumor efficacy, including complete responses, in subcutaneous and systemic SCLC in vivo models. CAR T-cell infiltration into intermediate and posterior pituitary was detected, but no tissue damage in brain or pituitary was observed, and the hormone-secretion function of the pituitary was not ablated.In summary, the preclinical efficacy and safety data presented here support further evaluation of DLL3 CAR T cells as potential clinical candidates for the treatment of SCLC.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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