In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ~5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12–14 Gy) and proximal colon (13–14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10–12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6–180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

We have developed a method to assess target expression changes following exposure of plucked hairs to chemotherapeutic agents and other potential therapeutics. Successful ex vivo responses provide proof of concept data prior to clinical studies. Plucked hair is a valuable surrogate biomarker tissue to monitor pharmacodynamic (PD) responses in a clinical trial. The collection of hairs is minimally invasive, simple and is amenable to frequent sampling. Since hairs are epithelial appendages, many signaling pathways active in other epithelial tissues, including cancers, are also present in hairs. Highly proliferative plucked human scalp hair can be utilized as a surrogate tissue to measure proliferation, phosphorylation and DNA damage responses after treatment. Furthermore, hairs are highly vascularized, suggesting that administered Test Articles may be delivered efficiently to the hair in a similar manner to other highly vascularized tissues, including many tumor types. Human plucked hair (either from the scalp or beard) was placed in maintenance media in the presence of conventional or targeted chemotherapeutic agents, known for their different mechanisms of action (e.g. Gemcitabine, Carboplatin, Tarceva), for a range of time points. Hairs were then fixed post-exposure and longitudinal sections immunohistochemically labelled for various markers including p-ERK1/2, p-AKT, p-Chk1, g-H2AX, Ki67 and androgen receptor. Quantitative image analysis to measure the level of labelling was performed using an Aperio ScanScope. The relative number of positively labelled cell nuclei, or relative tissue area (depending on the labelling pattern of the target), of the hair outer root sheath labelled was analyzed, along with label intensity. In subsequent trial patients, formaldehyde fixed plucked hairs were returned to the lab, similarly sectioned, labelled and quantified. In addition, where antibody labelling is not possible or more detailed information on the modulation of a pathway may be useful, drug treated plucked hairs can be subjected to Next Generation Sequencing (NGS) gene expression analysis. The expected changes in labelling were observed. For example, Gemcitabine, which is linked to DNA polymerase inhibition, increased p-Chk1 labelling 4-fold after 4-8 hours and strongly induced p53 by 24 hours. Tarceva, an inhibitor of the EGF pathway, decreased levels of both p-ERK1/2 and p-AKT 3-fold after only 10 minutes. The alkylating agent carboplatin increased g-H2AX labelling up to 12-fold after 24 hours. We have demonstrated that plucked hair is a valuable PD biomarker tissue for various chemotherapy agents. Proof of concept studies performed ex vivo can inform the design of clinical validation studies by indicating optimum markers and timepoints. Further, each hair is an independent unit thereby allowing independent replicate tissues (hairs) to be sampled. Citation Format: Greg Tudor, Frida Ponthan, Adam Boanas, Aude-Marine Bonavita, Catherine Booth. Pharmacodynamic biomarkers: Evaluation of oncology drug target engagement in human plucked scalp and beard hair. [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 4350.

The early identification of GI toxicity caused by novel therapeutics is crucial for drug development pipelines. GI toxicity is one of the most frequent adverse effects amongst chemotherapeutics. Impaired barrier function and epithelial ulceration can lead to symptoms including diarrhoea, dehydration and susceptibility to systemic infection. We have identified a gene signature that can be utilised in an in vitro organoid model to predict relevant GI toxicity. Methods: Both mice and small intestinal organoids derived from human and mouse were exposed to chemotherapeutic agents known to cause GI toxicity. For in vivo assays mice were treated with 50 or 100mg/kg CPT11 for 4 or 8 hours or 100, 200 or 400mg/kg Iressa for 8 or 24 hours before small intestinal (SI) tissue was removed for RNA-Seq analysis. SI tissue was also analysed by histological staining and damage measured to confirm induction of GI-toxicity. Human and mouse organoids were treated with CPT11 and Iressa for 8 hours at a range of concentrations. The organoids were lysed, RNA extracted and an mRNA-Seq library was generated. Samples were sequenced using 2 × 75bp PE reads on a Next-Seq550. Sequences were aligned to the relevant genomes using BWA and normalised using DESeq2. Normalised gene counts were analysed using Partek Genomics Suite and Ingenuity Pathway Analysis (IPA) was used for gene set enrichment (GSEA) and pathway analysis of differentially expressed genes. Results: For all treatments and time points the mechanism of action (MOA) of each agent was evident from the GSEA and pathway analysis. CPT11 induced DNA damage and cell cycle arrest pathways, whereas Iressa demonstrated downregulation of cell cycle and chromosomal replication and mitotic genes. Analysis of toxicity pathways in IPA identified a number of differentially expressed genes that were consistent with increased toxicity. A gene signature was identified and a multivariate scoring system used to demonstrate signature engagement. This signature was activated in all SI tissues treated with CPT11 or Iressa, irrespective of the MOA of both agents. Conclusions: Deployment of this signature on mouse and human organoids treated with chemotherapeutic agents demonstrated activation of the signature, which could be an alternative method for GI toxicity testing. Further analysis of this signature in gene expression studies from GI toxicity inducing agents submitted to the NCBI Gene expression omnibus (GEO) demonstrated that the signature was activated with a variety of agents with different MOA’s such as NSAIDs, gamma-secretase inhibitors, CDK8/19 inhibitors and CAR-T-cells. Citation Format: Gary S. Beale, Aude Marine-Bonavita, Valentina Ubertini, Greg Tudor, Francesca Philips, Catherine Booth. Identification of a gastrointestinal (GI) toxicity gene signature that predicts GI-toxicity induction in small intestinal organoids [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 153.

The dose response relationship and corresponding values for mid-lethal dose and slope are used to define the dose- and time-dependent parameters of the hematopoietic acute radiation syndrome. The characteristic time course of mortality, morbidity, and secondary endpoints are well defined. The concomitant comorbidities, potential mortality, and other multi-organ injuries that are similarly dose- and time-dependent are less defined. Determination of the natural history or pathophysiology associated with the lethal hematopoietic acute radiation syndrome is a significant gap in knowledge, especially when considered in the context of a nuclear weapon scenario. In this regard, the exposure is likely ill-defined, heterogenous, and nonuniform. These conditions forecast sparing of bone marrow and increased survival from the acute radiation syndrome consequent to threshold doses for the delayed effects of acute radiation exposure due to marrow sparing, medical management, and use of approved medical countermeasures. The intent herein is to provide a composite natural history of the pathophysiology concomitant with the evolution of the potentially lethal hematopoietic acute radiation syndrome derived from studies that focused on total body irradiation and partial body irradiation with bone marrow sparing. The marked differential in estimated LD50/60 from 7.5 Gy to 10.88 Gy for the total body irradiation and partial body irradiation with 5% bone marrow sparing models, respectively, provided a clear distinction between the attendant multiple organ injury and natural history of the two models that included medical management. Total body irradiation was focused on equivalent LD50/60 exposures. The 10 Gy and 11 Gy partial body with 5% bone marrow sparing exposures bracketed the LD50/60 (10.88 Gy). The incidence, progression, and duration of multiple organ injury was described for each exposure protocol within the hematopoietic acute radiation syndrome. The higher threshold doses for the partial body irradiation with bone marrow sparing protocol induced a marked degree of multiple organ injury to include lethal gastrointestinal acute radiation syndrome, prolonged crypt loss and mucosal damage, immune suppression, acute kidney injury, body weight loss, and added clinical comorbidities that defined a complex timeline of organ injury through the acute hematopoietic acute radiation syndrome. The natural history of the acute radiation syndrome presents a 60-d time segment of multi-organ sequelae that is concomitant with the latent period or time to onset of the evolving multi-organ injury of the delayed effects of acute radiation exposure.