Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Assessing cerebrovascular reactivity (CVR) in rhesus macaques (Macaca mulatta) using a hypercapnic challenge and pseudo-continuous arterial spin labeling (pCASL).

Cerebrovascular reactivity (CVR) is a measure of cerebral small vessels' ability to respond to changes in metabolic demand and can be quantified using magnetic resonance imaging (MRI) coupled with a vasoactive stimulus. Reduced CVR occurs with neurodegeneration and is associated with cognitive decline. While commonly measured in humans, few studies have evaluated CVR in animal models. Herein, we describe methods to induce hypercapnia in rhesus macaques (Macaca mulatta) under gas anesthesia to measure cerebral blood flow (CBF) and CVR using pseudo-continuous arterial spin labeling (pCASL). Fifteen (13 M, 2 F) adult rhesus macaques underwent pCASL imaging that included a baseline segment (100% O2) followed by a hypercapnic challenge (isoflurane anesthesia with 5% CO2, 95% O2 mixed gas). Relative hypercapnia was defined as an end-tidal CO2 (ETCO2) ≥5 mmHg above baseline ETCO2. The mean ETCO2 during the baseline segment of the pCASL sequence was 34 mmHg (range: 23-48 mmHg). During this segment, mean whole-brain CBF was 51.48 ml/100g/min (range: 21.47-77.23 ml/100g/min). Significant increases (p<0.0001) in ETCO2 were seen upon inspiration of the mixed gas (5% CO2, 95% O2). The mean increase in ETCO2 was 8.5 mmHg and corresponded with a mean increase in CBF of 37.1% (p<0.0001). The mean CVR measured was 4.3%/mmHg. No anesthetic complications occurred as a result of the CO2 challenge. Our methods were effective at inducing a state of relative hypercapnia that corresponds with a detectable increase in whole brain CBF using pCASL MRI. Using these methods, a CO2 challenge can be performed in conjunction with pCASL imaging to evaluate CBF and CVR in rhesus macaques. The measured CVR in rhesus macaques is comparable to human CVR highlighting the translational utility of rhesus macaques in neuroscience research. These methods present a feasible means to measure CVR in comparative models of neurodegeneration and cerebrovascular dysfunction.

Total-Body Irradiation Alters White Matter Volume and Microstructural Integrity in Rhesus Macaques.

PURPOSE: Long-term survivors of brain irradiation can experience irreversible injury and cognitive impairment. T1-weighted and diffusion tensor magnetic resonance imaging (MRI) are used to evaluate brain volume and white matter (WM) microstructure in neurodevelopmental and neurodegenerative conditions. The goal of this study was to evaluate the long-term effects of single-dose total-body irradiation (TBI) or TBI with 5% partial-body sparing on brain volumetrics and WM integrity in macaques. METHODS AND MATERIALS: We used MRI scans from a cohort of male rhesus macaques (age range, 3.6-22.8 years) to compare global and regional brain volumes and WM diffusion in survivors of TBI (T1-weighted, n = 137; diffusion tensor imaging, n = 121; dose range, 3.5-10 Gy) with unirradiated controls (T1-weighted, n = 48; diffusion tensor imaging, n = 38). RESULTS: In all regions of interest, radiation affected age-related changes in fractional anisotropy, which tended to increase across age in both groups but to a lesser extent in the irradiated group (interaction P < .01). Depending on the region of interest, mean diffusivity decreased or remained the same across age in unirradiated animals, whereas it increased or did not change in irradiated animals. The increases in mean diffusivity were driven by changes in radial diffusivity, which followed similar trends across age. Axial diffusivity did not differ by irradiation status. Age-related changes in relative volumes in controls reflected normal trends in humans, with increasing WM and decreasing gray matter until middle age. Cerebrospinal fluid (CSF) volume did not differ across age in controls. WM volume was lower and CSF volume was higher in young irradiated macaques. WM volume was similar between groups, and CSF volume lower in older irradiated macaques. Gray matter volume was unaffected by radiation. CONCLUSIONS: TBI results in delayed WM expansion and long-term disruption of WM integrity. Diffusion changes suggest that myelin injury in WM is a hallmark of late-delayed radiation-induced brain injury.

Total-Body Irradiation Is Associated With Increased Incidence of Mesenchymal Neoplasia in a Radiation Late Effects Cohort of Rhesus Macaques (Macaca mulatta).

PURPOSE: Cancer is a severe delayed effect of acute radiation exposure. Total-body irradiation has been associated with an increased risk of solid cancer and leukemia in Japanese atomic bomb survivors, and secondary malignancies, such as sarcoma, are a serious consequence of cancer radiation therapy. The radiation late effects cohort (RLEC) of rhesus macaques (Macaca mulatta) is a unique resource of more than 200 animals for studying the long-term consequences of total-body irradiation in an animal model that closely resembles humans at the genetic and physiologic levels. METHODS AND MATERIALS: Using clinical records, clinical imaging, histopathology, and immunohistochemistry, this retrospective study characterized the incidence of neoplasia in the RLEC. RESULTS: Since 2007, 61 neoplasms in 44 of 239 irradiated animals were documented (18.4% of the irradiated population). Only 1 neoplasm was diagnosed among the 51 nonirradiated controls of the RLEC (2.0%). The most common malignancies in the RLEC were sarcomas (38.3% of diagnoses), which are rare neoplasms in nonirradiated macaques. The most common sarcomas included malignant nerve sheath tumors and malignant glomus tumors. Carcinomas were less common (19.7% of diagnoses), and consisted primarily of renal cell and hepatocellular carcinomas. Neoplasia occurred in most major body systems, with the skin and subcutis being the most common site (40%). RNA analysis showed similarities in transcriptional profiles between RLEC and human malignant nerve sheath tumors. CONCLUSIONS: This study indicates that total-body irradiation is associated with an increased incidence of neoplasia years following irradiation, at more than double the incidence described in aging, nonirradiated animals, and promotes tumor histotypes that are rarely observed in nonirradiated, aging rhesus macaques.

Pharmacokinetics and safety of TCMCB07, a melanocortin-4 antagonist peptide in dogs.

The melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 was developed for the treatment of cachexia. The objectives of this study were to examine pharmacokinetics and safety of TCMCB07 administered subcutaneously to healthy dogs. Dogs were treated with high- (2.25 mg kg-1 ) (n = 5) and low-dose TCMCB07 (0.75 mg kg-1 ) (n = 5) once daily for 28 days with a 14-day washout period between groups. Histamine levels, complete blood count, chemistry panel, blood pressure, 24-hour Holter recording, and pharmacokinetic parameters were monitored in the high-dose group. Physical examination changes were limited to weight gain and darkening of the coat color. There was no elevation of plasma histamine within 24 hours of injection but there was a significant elevation of plasma histamine across time. An approximately doubled eosinophil count and an approximately 25% increase, and then 25% decrease back to pre-treatment plasma phosphorous were also found, although both remained within the reference interval. Serial blood pressure and 24-hour Holter monitors revealed no clinically relevant changes. A difference was found in the AUC between dosing groups and a significant effect of dose, time, and interaction was noted for Vd . Low-dose TCMCB07 had a Cmax of 2.1 ug ml-1 at day 28, compared to high-dose TCMCB07 which had a Cmax 3.6 ug ml-1 at day 28. Once-daily subcutaneous administration of TCMCB07 was well-tolerated for up to 28 days in dogs when administered at doses one and three times (0.75 mg kg-1 and 2.25 mg kg-1 ) the predicted therapeutic dose and pharmacokinetic parameters are described. SIGNIFICANCE STATEMENT: Melanocortin-4 receptor (MC4R) antagonistic peptide TCMCB07 is safe at both low and high doses in dogs. Therapy was tolerated well as determined by physical examination, clinical pathology, and cardiovascular parameters; darkening of the coat was noted with treatment and resolved with discontinuation. Pharmacokinetics are described and further study in the naturally occurring canine model is warranted.

Cortical Thinning and Structural Bone Changes in Non-Human Primates after Single-Fraction Whole-Chest Irradiation.

Stereotactic body radiation therapy (SBRT) is associated with an increased risk of vertebral compression fracture. While bone is typically considered radiation resistant, fractures frequently occur within the first year of SBRT. The goal of this work was to determine if rapid deterioration of bone occurs in vertebrae after irradiation. Sixteen male rhesus macaque non-human primates (NHPs) were analyzed after whole-chest irradiation to a midplane dose of 10 Gy. Ages at the time of exposure varied from 45-134 months. Computed tomography (CT) scans were taken 2 months prior to irradiation and 2, 4, 6 and 8 months postirradiation for all animals. Bone mineral density (BMD) and cortical thickness were calculated longitudinally for thoracic (T) 9, lumbar (L) 2 and L4 vertebral bodies; gross morphology and histopathology were assessed per vertebra. Greater mortality (related to pulmonary toxicity) was noted in NHPs <50 months at time of exposure versus NHPs >50 months ( P = 0.03). Animals older than 50 months at time of exposure lost cortical thickness in T9 by 2 months postirradiation ( P = 0.0009), which persisted to 8 months. In contrast, no loss of cortical thickness was observed in vertebrae out-of-field (L2 and L4). Loss of BMD was observed by 4 months postirradiation for T9, and 6 months postirradiation for L2 and L4 ( P < 0.01). For NHPs younger than 50 months at time of exposure, both cortical thickness and BMD decreased in T9, L2 and L4 by 2 months postirradiation ( P < 0.05). Regions that exhibited the greatest degree of cortical thinning as determined from CT scans also exhibited increased porosity histologically. Rapid loss of cortical thickness was observed after high-dose chest irradiation in NHPs. Younger age at time of exposure was associated with increased pneumonitis-related mortality, as well as greater loss of both BMD and cortical thickness at both in- and out-of-field vertebrae. Older NHPs exhibited rapid loss of BMD and cortical thickness from in-field vertebrae, but only loss of BMD in out-of-field vertebrae. Bone is sensitive to high-dose radiation, and rapid loss of bone structure and density increases the risk of fractures.