Hotspot Exon 15 Mutations in BRAF Are Uncommon in Feline Tumours.

BRAF is one of multiple RAF proteins responsible for the activation of the MAPK cell signalling cascade involved in cell growth, differentiation, and survival. A hotspot BRAFV600E mutation, in exon 15, was determined to be a driver in 100% hairy cell leukaemias, 50%-60% of human melanomas, 30%-50% of human thyroid carcinomas and 10%-20% of human colorectal carcinomas. The orthologous BRAFV595E mutation was seen in 67% and 80% of canine bladder transitional cell carcinomas and prostatic adenocarcinomas, respectively. Since veterinary and human cancers exploit similar pathways and BRAF is highly conserved across species, BRAF can be expected to be a driver in a feline cancer. Primers were developed to amplify exon 15 of feline BRAF. One hundred ninety-six feline tumours were analysed. Sanger sequencing of the 211 bp PCR amplicon was done. A BRAF mutation was found in one tumour, a cutaneous melanoma. The mutation was a BRAFV597E mutation, orthologous to the canine and human hotspot mutations. A common synonymous variant, BRAFT586T, was seen in 23% (47/196) of tumours. This variant was suspected to be a single nucleotide polymorphism. BRAF was not frequently mutated in common feline tumours or in tumour types that frequently harbour BRAF mutations in human and canine cancers. As is seen in canine cancer genomics, the mutational profile in feline tumours may not parallel the histologic equivalent in human oncology.

Translational History and Hope of Immunotherapy of Canine Tumors.

Companion dogs have served an important role in cancer immunotherapy research. Sharing similar environments and diets with humans, dogs naturally develop many of the same cancers. These shared exposures, coupled with dogs' diverse genetic makeup, make them ideal subjects for studying cancer therapies. Tumors like osteosarcoma, hemangiosarcoma, soft-tissue sarcoma, and non-Hodgkin lymphoma occur with greater frequency than their counterpart disease in humans. Canine brain tumors allow the study of therapy strategies with imaging, surgery, and radiotherapy equipment in veterinary patients with near-human geometry. Nonspecific immunostimulants, autologous and allogeneic vaccines, immune checkpoint inhibitors, and cellular therapies used in treating canine cancers have been tested in veterinary clinical trials. These treatments have not only improved outcomes for dogs but have also provided valuable insights for human cancer treatment. Advancements in radiation technology and the development of tools to characterize canine immune responses have further facilitated the ability to translate veterinary clinical trial results to human applications. Advancements in immunotherapy of canine tumors have directly supported translation to human clinical trials leading to approved therapies for patients with cancer around the world. The study of immunotherapy in dogs has been and will continue to be a promising avenue for advancing human cancer treatment.

Single-cell T-cell receptor repertoire profiling in dogs.

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

Pigs: Large Animal Preclinical Cancer Models.

Pigs are playing an increasingly vital role as translational biomedical models for studying human pathophysiology. The annotation of the pig genome was a huge step forward in translatability of pigs as a biomedical model for various human diseases. Similarities between humans and pigs in terms of anatomy, physiology, genetics, and immunology have allowed pigs to become a comprehensive preclinical model for human diseases. With a diverse range, from craniofacial and ophthalmology to reproduction, wound healing, musculoskeletal, and cancer, pigs have provided a seminal understanding of human pathophysiology. This review focuses on the current research using pigs as preclinical models for cancer research and highlights the strengths and opportunities for studying various human cancers.

The role of companion animal models in radiopharmaceutical development and translation.

Advancements in molecular imaging and drug targeting have created a renaissance in the development of radiopharmaceuticals for therapy and theranostics. While some radiopharmaceuticals, such as Na[131I]I, have been used clinically for decades, new agents are being approved using small-molecules, peptides, and antibodies for targeting. As these agents are being developed, the need to understand dosimetry and biologic effects of the systemically delivered radiotherapy becomes more important, particularly as highly potent radiopharmaceuticals using targeted alpha therapy become clinically utilized. As the processes being targeted become more complex, and the radiobiology of different particulate radiation becomes more diverse, models that better recapitulate human cancer and geometry are necessary. Companion animals develop many of the same types of cancer, carrying many of the same genetic drivers as those seen in people, and the scale and geometry of tumours in dogs more closely mimics those in humans than murine tumour models. Key translational challenges in oncology, such as alterations in tumour microenvironment, hypoxia, heterogeneity, and geometry are addressed by companion animal models. This review paper will provide background on radiopharmaceutical targeting techniques, review the use of radiopharmaceuticals in companion animal oncology, and explore the translational value of treating these patients in terms of dosimetry, treatment outcomes, and normal tissue complication rates.

Differentiation between atypical anorexia nervosa and anorexia nervosa using machine learning.

OBJECTIVE: Body mass index (BMI) is the primary criterion differentiating anorexia nervosa (AN) and atypical anorexia nervosa despite prior literature indicating few differences between disorders. Machine learning (ML) classification provides us an efficient means of accurately distinguishing between two meaningful classes given any number of features. The aim of the present study was to determine if ML algorithms can accurately distinguish AN and atypical AN given an ensemble of features excluding BMI, and if not, if the inclusion of BMI enables ML to accurately classify between the two. METHODS: Using an aggregate sample from seven studies consisting of individuals with AN and atypical AN who completed baseline questionnaires (N = 448), we used logistic regression, decision tree, and random forest ML classification models each trained on two datasets, one containing demographic, eating disorder, and comorbid features without BMI, and one retaining all features and BMI. RESULTS: Model performance for all algorithms trained with BMI as a feature was deemed acceptable (mean accuracy = 74.98%, mean area under the receiving operating characteristics curve [AUC] = 74.75%), whereas model performance diminished without BMI (mean accuracy = 59.37%, mean AUC = 59.98%). DISCUSSION: Model performance was acceptable, but not strong, if BMI was included as a feature; no other features meaningfully improved classification. When BMI was excluded, ML algorithms performed poorly at classifying cases of AN and atypical AN when considering other demographic and clinical characteristics. Results suggest a reconceptualization of atypical AN should be considered. PUBLIC SIGNIFICANCE: There is a growing debate about the differences between anorexia nervosa and atypical anorexia nervosa as their diagnostic differentiation relies on BMI despite being similar otherwise. We aimed to see if machine learning could distinguish between the two disorders and found accurate classification only if BMI was used as a feature. This finding calls into question the need to differentiate between the two disorders.

Effects of neoadjuvant zoledronate and radiation therapy on cell survival, cell cycle distribution, and clinical status in canine osteosarcoma.

Introduction: Zoledronic acid (ZOL) is a third-generation bisphosphonate with a higher affinity for bone resorption areas than earlier bisphosphonates (i.e., pamidronate, PAM). In human medicine, ZOL provides improved bone pain relief and prolonged time to skeletal-related events compared to its older generational counterparts. Preclinical studies have investigated its role as an anti-neoplastic agent, both independently and synergistically, with radiation therapy (RT). ZOL and RT act synergistically in several neoplastic human cell lines: prostate, breast, osteosarcoma, and fibrosarcoma. However, the exact mechanism of ZOL's radiosensitization has not been fully elucidated. Methods: We investigated ZOL's ability to induce apoptosis in canine osteosarcoma cell lines treated with various doses of megavoltage external beam radiotherapy. Second, we evaluated cell cycle arrest in ZOL-treated cells to assess several neo-adjuvant time points. Finally, we treated 20 dogs with naturally occurring appendicular OS with 0.1 mg/kg ZOL IV 24 h before receiving 8 Gy of RT (once weekly fraction x 4 weeks). Results: We found that apoptosis was increased in all ZOL-treated cell lines compared to controls, and the combination of ZOL and RT resulted in dissimilar apoptosis between Abrams and D-17 and HMPOS cell lines. Cell cycle arrest (G2/M phase) was minimal and variable between cell lines but perhaps greatest at 48 h post-ZOL treatment. Only 10% of dogs treated with ZOL and RT developed pathologic fractures, compared to 44% of dogs historically treated with PAM and RT (p = 0.027). Discussion: ZOL and RT appear to be a well-tolerated combination treatment scheme for non-surgical candidates; future studies must elucidate the ideal timing of ZOL.

Benefits and opportunities of the transgenic Oncopig cancer model.

Cancer remains a leading cause of morbidity and mortality, and a paradigm shift is needed to fundamentally revisit drug development efforts. Pigs share close similarities to humans and may serve as an alternative model. Recently, a transgenic 'Oncopig' line has been generated to induce solid tumors with organ specificity, opening the potential of Oncopigs as a platform for developing novel therapeutic regimens.

Updates in Osteosarcoma.

Clinical care of osteosarcoma (OSA) in dogs has seen little change during the past 2 decades, relying on amputation and platinum-based chemotherapy for pain control and survival. Recent advancements offer hope for improved outcomes. Genomic research reveals shared genetic abnormalities between canine and human OSA. Multidimensional imaging provides valuable staging and prognostic information. Limb-sparing approaches including stereotactic body radiation therapy are routine. Ablative therapies such as microwave ablation and histotripsy show promise. Immunotherapy including cell therapy and immune checkpoint inhibition are available. Radiopharmaceuticals are tuned to target OSA cells directly. These innovations may enhance treatment and prognosis for dogs with OSA.

Therapeutic efficacy of an alpha-particle emitter labeled anti-GD2 humanized antibody against osteosarcoma-a proof of concept study.

PURPOSE: Current treatments for osteosarcoma (OS) have a poor prognosis, particularly for patients with metastasis and recurrence, underscoring an urgent need for new targeted therapies to improve survival. Targeted alpha-particle therapy selectively delivers cytotoxic payloads to tumors with radiolabeled molecules that recognize tumor-associated antigens. We have recently demonstrated the potential of an FDA approved, humanized anti-GD2 antibody, hu3F8, as a targeted delivery vector for radiopharmaceutical imaging of OS. The current study aims to advance this system for alpha-particle therapy of OS. METHODS: The hu3F8 antibody was radiolabeled with actinium-225, and the safety and therapeutic efficacy of the [225Ac]Ac-DOTA-hu3F8 were evaluated in both orthotopic murine xenografts of OS and spontaneously occurring OS in canines. RESULTS: Significant antitumor activity was proven in both cases, leading to improved overall survival. In the murine xenograft's case, tumor growth was delayed by 16-18 days compared to the untreated cohort as demonstrated by bioluminescence imaging. The results were further validated with magnetic resonance imaging at 33 days after treatment, and microcomputed tomography and planar microradiography post-mortem. Histological evaluations revealed radiation-induced renal toxicity, manifested as epithelial cell karyomegaly and suggestive polyploidy in the kidneys, suggesting rapid recovery of renal function after radiation damage. Treatment of the two canine patients delayed the progression of metastatic spread, with an overall survival time of 211 and 437 days and survival beyond documented metastasis of 111 and 84 days, respectively. CONCLUSION: This study highlights the potential of hu3F8-based alpha-particle therapy as a promising treatment strategy for OS.

Red cabbage juice-mediated gut microbiota modulation improves intestinal epithelial homeostasis and ameliorates colitis.

Gut microbiota plays a crucial role in inflammatory bowel disease (IBD) and has therapeutic benefits. Thus, targeting the gut microbiota is a promising therapeutic approach for IBD treatment. We recently found that red cabbage juice (RCJ) ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. However, the underlying mechanisms remain unknown. The current study investigated the modulation of gut microbiota in response to treatment with RCJ to ameliorate the DSS colitis. The initial results demonstrated that mice treated with DSS + RCJ showed increased body weight and decreased diarrhea and blood in feces compared to the DSS alone group. RCJ ameliorated colitis by regulating the intestinal barrier function by reducing the number of apoptotic cells, improving colonic protective mucin, and increasing tight junction protein in RCJ + DSS groups compared to the DSS group. Short-gun metagenomic analysis revealed significant enrichment of short-chain fatty acid (SCFAs)-producing bacteria (Butyrivibrio, Ruminococcaceae, Acetatifactor muris, Rosburia Sp. CAG:303 , Dorea Sp. 5-2) increased PPAR-© activation, leading to repression of the nuclear factor κB (NFκB) signaling pathway, thus decreasing the production of crucial inflammatory cytokines and chemokines in the RCJ + DSS groups compared to the DSS group. Pathway abundance analysis showed an increased abundance of the SCFA pathway, reduced histidine degradation ( Bacteroides sartorii, and Bacteroides caecimuris ), and LCFA production in the RCJ+DSS treated group, suggesting the promotion of good colonic health. Furthermore, increased T-reg (FOXP3+) cells in the colon were due to SCFAs produced by the gut microbiota, which was corroborated by an increase in IL-10, a vital anti-inflammatory cytokine. Thus, our study provides the first evidence that RCJ ameliorates colonic inflammation by modulating the gut microbiota.

Phase I evaluation of CycloSam® (Sm-153-DOTMP) bone seeking radiopharmaceutical in dogs with spontaneous appendicular osteosarcoma.

153 Sm-DOTMP (CycloSam® ) is a newly-patented radiopharmaceutical for bone tumor treatment. DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylene-phosphonate) is a macrocyclic chelating agent with superior binding properties to 153 Sm when compared with EDTMP (Quadramet™, used for palliative treatment of bone cancer). CycloSam® was administered at 1 mCi/kg (37 MBq/kg) in a prospective pilot study to seven dogs with bone cancer resulting in no myelosuppression. Then, 13 dogs were enrolled in a prospective clinical trial study using traditional 3+3 dose escalation and starting at 1.5 mCi/kg. Baseline evaluation included hematologic and biochemical testing, diagnosis confirmation, thoracic and limb radiographs, technetium-99 m-HDP bone scintigraphy, and 18 F-FDG PET scan (SUVmax). Toxicity (primary endpoint) was assessed through weekly blood counts and adverse events. Dogs received 1.5 mCi/kg (n = 4), 1.75 mCi/kg (n = 6), and 2 mCi/kg (n = 3) of 153 Sm-DOTMP. Dose-limiting neutropenia and thrombocytopenia were seen at 2 mCi/kg. No dose-limiting nonhematologic toxicities occurred. Efficacy (secondary endpoint) was assessed by objective lameness measurement (body-mounted inertial sensors), owner quality-of-life (QoL) questionnaire, and repeat PET scan. Objective lameness measurement improved in four dogs (53%-60% decrease) was equivocal in three dogs, and worsened in four dogs (66%-115% increase); two dogs were not evaluable. Repeat 18 F-FDG PET scan results varied and change in lameness did not consistently correlate with SUVmax changes. QoL score worsened (n = 5) or was improved/stable (n = 7). Carboplatin chemotherapy (300 mg/m2 IV every 3 weeks ×4) started 4 weeks after 153 Sm-DOTMP injection. No dog died of chemotherapy-related complications. All dogs completed study monitoring. The recommended dose for CycloSam® in dogs is 1.75 mCi/kg, which resulted in some pain control with minimal toxicity and was safely combined with chemotherapy.

Genomic landscape and gene expression profiles of feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a cancer of the squamous cell lining in the oral cavity and represents up to 80% of all oral cancers in cats, with a poor prognosis. We have used whole exome sequencing (WES) and RNA sequencing of the tumor to discover somatic mutations and gene expression changes that may be associated with FOSCC occurrence. FOSCC offers a potential comparative model to study human head and neck squamous cell carcinoma (HNSCC) due to its similar spontaneous formation, and morphological and histological features. In this first study using WES to identify somatic mutations in feline cancer, we have identified tumor-associated gene mutations in six cats with FOSCC and found some overlap with identified recurrently mutated genes observed in HNSCC. Four samples each had mutations in TP53, a common mutation in all cancers, but each was unique. Mutations in other cellular growth control genes were also found such as KAT2B and ARID1A. Enrichment analysis of FOSCC gene expression profiles suggests a molecular similarity to human OSCC as well, including alterations in epithelial to mesenchymal transition and IL6/JAK/STAT pathways. In this preliminary study, we present exome and transcriptome results that further our understanding of FOSCC.

Metagenomic Analysis of DNA Viruses with Targeted Sequence Capture of Canine Lobular Orbital Adenomas and Normal Conjunctiva.

Our study aims are: (1) to evaluate phenotypically normal canine conjunctival and orbital tissue and tissue from canine lobular orbital adenomas (CLOAs) for the presence of viral genomic material and (2) phylogenetically classify detected DNA viruses to determine if a DNA virus is associated with CLOAs. A total of 31 formalin fixed paraffin embedded CLOA tissue samples, 4 papillomas or sarcoid, and 10 fresh clinically normal conjunctival tissues were included in this study. Genomic DNA was isolated from all samples and sequencing libraries were prepared. The libraries were molecularly indexed and pooled and viral DNA was enriched via targeted sequence capture utilizing ViroCap. The libraries were sequenced on the Illumina HiSeq platform and compared to known viral DNA reference genomes to identify viral DNA. Carnivore parvovirus was identified in 6.4% and 20% of CLOA tissue and normal conjunctival samples, respectively. This study showed that conjunctival tissue from healthy dogs and CLOAs uncommonly harbor DNA viruses, and no DNA virus was associated with these tumors. Further studies are needed to evaluate the etiologic cause of CLOAs.

A multi-institutional epidemiologic study evaluating environmental risk factors for feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive cancer in domestic cats that has no effective treatment option when advanced. Preventative or early diagnostic measures are thus crucial. FOSCC is also a model for human head and neck SCC (HNSCC); strong risk factors in HNSCC include exposure to alcohol, tobacco, areca nut, and high-risk human papillomavirus. Previous studies have identified flea collar and tobacco smoke exposure, feeding canned tuna, canned cat food and cat foods with chemical additives, living in a rural environment, and having outdoor access as risk factors for FOSCC but there was no overlap in the risk factors between studies. In our study, risks for FOSCC were evaluated in an online epidemiologic survey study in 67 cats with FOSCC and 129 control cats. Clumping clay cat litter and flea collar use were significant risk factors for FOSCC on multiple logistic regression with odds ratios of 1.66 (95% CI 1.20-2.30) and 4.48 (95% CI 1.46-13.75) respectively. Crystalline silica is a carcinogen that may be present in all clay cat litters and tetrachlorvinphos is a carcinogen that is present in the most commonly used flea collars in our study. We recommend further investigation into the association between FOSCC and clay-based litter and/or flea collars containing tetrachlorvinphos.

Red Cabbage Juice-Mediated Gut Microbiota Modulation Improves Intestinal Epithelial Homeostasis and Ameliorates Colitis.

Gut microbiota plays a crucial role in inflammatory bowel diseases (IBD) and can potentially prevent IBD through microbial-derived metabolites, making it a promising therapeutic avenue. Recent evidence suggests that despite an unclear underlying mechanism, red cabbage juice (RCJ) alleviates Dextran Sodium Sulfate (DSS)-induced colitis in mice. Thus, the study aims to unravel the molecular mechanism by which RCJ modulates the gut microbiota to alleviate DSS-induced colitis in mice. Using C57BL/6J mice, we evaluated RCJ's protective role in DSS-induced colitis through two cycles of 3% DSS. Mice were daily gavaged with PBS or RCJ until the endpoint, and gut microbiota composition was analyzed via shotgun metagenomics. RCJ treatment significantly improved body weight (p ≤ 0.001), survival in mice (p < 0.001) and reduced disease activity index (DAI) scores. Further, RCJ improved colonic barrier integrity by enhancing the expression of protective colonic mucins (p < 0.001) and tight junction proteins (p ≤ 0.01) in RCJ + DSS-treated mice compared to the DSS group. Shotgun metagenomic analysis revealed an enrichment of short-chain fatty acids (SCFAs)-producing bacteria (p < 0.05), leading to increased Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) activation (p ≤ 0.001). This, in turn, resulted in repression of the nuclear factor κB (NFκB) signaling pathway, causing decreased production of inflammatory cytokines and chemokines. Our study demonstrates colitis remission in a DSS-induced mouse model, showcasing RCJ as a potential modulator for gut microbiota and metabolites, with promising implications for IBD prevention and treatment.

Exploring approaches for predictive cancer patient digital twins: Opportunities for collaboration and innovation.

We are rapidly approaching a future in which cancer patient digital twins will reach their potential to predict cancer prevention, diagnosis, and treatment in individual patients. This will be realized based on advances in high performance computing, computational modeling, and an expanding repertoire of observational data across multiple scales and modalities. In 2020, the US National Cancer Institute, and the US Department of Energy, through a trans-disciplinary research community at the intersection of advanced computing and cancer research, initiated team science collaborative projects to explore the development and implementation of predictive Cancer Patient Digital Twins. Several diverse pilot projects were launched to provide key insights into important features of this emerging landscape and to determine the requirements for the development and adoption of cancer patient digital twins. Projects included exploring approaches to using a large cohort of digital twins to perform deep phenotyping and plan treatments at the individual level, prototyping self-learning digital twin platforms, using adaptive digital twin approaches to monitor treatment response and resistance, developing methods to integrate and fuse data and observations across multiple scales, and personalizing treatment based on cancer type. Collectively these efforts have yielded increased insights into the opportunities and challenges facing cancer patient digital twin approaches and helped define a path forward. Given the rapidly growing interest in patient digital twins, this manuscript provides a valuable early progress report of several CPDT pilot projects commenced in common, their overall aims, early progress, lessons learned and future directions that will increasingly involve the broader research community.

Genome wide exploration of the methylome in aggressive B-cell lymphoma in Golden Retrievers reveals a conserved hypermethylome.

Few recurrent DNA mutations are seen in aggressive canine B cell lymphomas (cBCL), suggesting other frequent drivers. The methylated island recovery assay (MIRA-seq) or methylated CpG-binding domain sequencing (MBD-seq) was used to define the genome-wide methylation profiles in aggressive cBCL in Golden Retrievers to determine if cBCL can be better defined by epigenetic changes than by DNA mutations. DNA hypermethylation patterns were relatively homogenous within cBCL samples in Golden Retrievers, in different breeds and in geographical regions. Aberrant hypermethylation is thus suspected to be a central and early event in cBCL lymphomagenesis. Distinct subgroups within cBCL in Golden Retrievers were not identified with DNA methylation profiles. In comparison, the methylome profile of human DLBCL (hDLBCL) is relatively heterogeneous. Only moderate similarity between hDLBCL and cBCL was seen and cBCL likely cannot be accurately classified into the subtypes seen in hDLBCL. Genes with hypermethylated regions in the promoter-TSS-first exon of cBCL compared to normal B cells often also had additional hyper- and hypomethylated regions distributed throughout the gene suggesting non-randomized repeat targeting of key genes by epigenetic mechanisms. The prevalence of hypermethylation in transcription factor families in aggressive cBCL may represent a fundamental step in lymphomagenesis.

Comparative Oncology Assessment of a Novel Inhibitor of Valosin-Containing Protein in Tumor-Bearing Dogs.

Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.