Rare germline variants in childhood cancer patients suspected of genetic predisposition to cancer.

Identification of cancer-predisposing germline variants in childhood cancer patients is important for therapeutic decisions, disease surveillance and risk assessment for patients, and potentially, also for family members. We investigated the spectrum and prevalence of pathogenic germline variants in selected childhood cancer patients with features suggestive of genetic predisposition to cancer. Germline DNA was subjected to exome sequencing to filter variants in 1048 genes of interest including 176 known cancer predisposition genes (CPGs). An enrichment burden analysis compared rare deleterious germline CPG variants in the patient cohort with those in a healthy aged control population. A subset of predicted deleterious variants in novel candidate CPGs was investigated further by examining matched tumor samples, and the functional impact of AXIN1 variants was analyzed in cultured cells. Twenty-two pathogenic/likely pathogenic (P/LP) germline variants detected in 13 CPGs were identified in 19 of 76 patients (25.0%). Unclear association with the diagnosed cancer types was observed in 11 of 19 patients carrying P/LP CPG variants. The burden of rare deleterious germline variants in autosomal dominant CPGs was significantly higher in study patients versus healthy aged controls. A novel AXIN1 frameshift variant (Ser321fs) may impact the regulation of ß-catenin levels. Selection of childhood cancer patients for germline testing based on features suggestive of an underlying genetic predisposition could help to identify carriers of clinically relevant germline CPG variants, and streamline the integration of germline genomic testing in the pediatric oncology clinic.

Dextran-Curcumin Nanosystems Inhibit Cell Growth and Migration Regulating the Epithelial to Mesenchymal Transition in Prostate Cancer Cells.

Functional nanocarriers which are able to simultaneously vectorize drugs to the site of interest and exert their own cytotoxic activity represent a significant breakthrough in the search for effective anticancer strategies with fewer side effects than conventional chemotherapeutics. Here, we propose previously developed, self-assembling dextran-curcumin nanoparticles for the treatment of prostate cancer in combination therapy with Doxorubicin (DOXO). Biological effectiveness was investigated by evaluating the cell viability in either cancer and normal cells, reactive oxygen species (ROS) production, apoptotic effect, interference with the cell cycle, and the ability to inhibit cell migration and reverse the epithelial to mesenchymal transition (EMT). The results proved a significant enhancement of curcumin efficiency upon immobilization in nanoparticles: IC50 reduced by a half, induction of apoptotic effect, and improved ROS production (from 67 to 134%) at low concentrations. Nanoparticles guaranteed a pH-dependent DOXO release, with a more efficient release in acidic environments. Finally, a synergistic effect between nanoparticles and Doxorubicin was demonstrated, with the free curcumin showing additive activity. Although in vivo studies are required to support the findings of this study, these preliminary in vitro data can be considered a proof of principle for the design of an effective therapy for prostate cancer treatment.

Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma.

Driver mutations in the CTNNB1 gene (encoding ß-catenin) are a hallmark of sporadic hepatoblastoma (HBL). Our results show that CTNNB1 circulating tumour DNA (ctDNA) is readily detected in patients diagnosed with localised HBL, with serial sampling along the course of therapy and follow up providing a sensitive mechanism to monitor tumour dynamics and response to treatment. This exciting potential for CTNNB1 ctDNA to serve as a biomarker for treatment response in HBL holds clinical value, and requires assessment in a larger cohort of mixed tumour stages and recurrent disease.

Functionalized Carbon Nanostructures Versus Drug Resistance: Promising Scenarios in Cancer Treatment.

Carbon nanostructures (CN) are emerging valuable materials for the assembly of highly engineered multifunctional nanovehicles for cancer therapy, in particular for counteracting the insurgence of multi-drug resistance (MDR). In this regard, carbon nanotubes (CNT), graphene oxide (GO), and fullerenes (F) have been proposed as promising materials due to their superior physical, chemical, and biological features. The possibility to easily modify their surface, conferring tailored properties, allows different CN derivatives to be synthesized. Although many studies have explored this topic, a comprehensive review evaluating the beneficial use of functionalized CNT vs G or F is still missing. Within this paper, the most relevant examples of CN-based nanosystems proposed for MDR reversal are reviewed, taking into consideration the functionalization routes, as well as the biological mechanisms involved and the possible toxicity concerns. The main aim is to understand which functional CN represents the most promising strategy to be further investigated for overcoming MDR in cancer.

Identification of nonferritin mitochondrial iron deposits in a mouse model of Friedreich ataxia.

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich ataxia (FA). This disease is due to decreased expression of the mitochondrial protein, frataxin, which leads to alterations in mitochondrial iron (Fe) metabolism. The identification of potentially toxic mitochondrial Fe deposits in FA suggests Fe plays a role in its pathogenesis. Studies using the muscle creatine kinase (MCK) conditional frataxin knockout mouse that mirrors the disease have demonstrated frataxin deletion alters cardiac Fe metabolism. Indeed, there are pronounced changes in Fe trafficking away from the cytosol to the mitochondrion, leading to a cytosolic Fe deficiency. Considering Fe deficiency can induce apoptosis and cell death, we examined the effect of dietary Fe supplementation, which led to body Fe loading and limited the cardiac hypertrophy in MCK mutants. Furthermore, this study indicates a unique effect of heart and skeletal muscle-specific frataxin deletion on systemic Fe metabolism. Namely, frataxin deletion induces a signaling mechanism to increase systemic Fe levels and Fe loading in tissues where frataxin expression is intact (i.e., liver, kidney, and spleen). Examining the mutant heart, native size-exclusion chromatography, transmission electron microscopy, Mössbauer spectroscopy, and magnetic susceptibility measurements demonstrated that in the absence of frataxin, mitochondria contained biomineral Fe aggregates, which were distinctly different from isolated mammalian ferritin molecules. These mitochondrial aggregates of Fe, phosphorus, and sulfur, probably contribute to the oxidative stress and pathology observed in the absence of frataxin.

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-ß signaling pathways in cancer.

BACKGROUND: Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-ß levels and EMT signaling. Given that many drugs targeting TGF-ß have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-ß/EMT axis. RESULTS: Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-ß and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-ß levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-ß/SMAD2&3) and non-canonical (TGF-ß/PI3K/AKT, TGF-ß/RAS/RAF/MEK/ERK, and TGF-ß/WNT/ß-catenin) TGF-ß signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, ß-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. CONCLUSIONS: Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-ß and inhibiting EMT in a diverse range of cancers.

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer.

BACKGROUND: Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. METHODS: We combined immunohistochemistry (IHC) with RNA sequencing and whole-genome sequencing across a diverse spectrum of high-risk paediatric cancers to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME. Furthermore, we explored transcriptional features of immune archetypes and T-cell receptor sequencing diversity, assessed the relationship between CD8+ and CD4+ abundance by IHC and deconvolution predictions and assessed the common adult biomarkers such as neoantigen load and TMB. RESULTS: A novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), was identified. Using this signature, we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. In addition, we showed that PD-L1 protein expression is poorly correlated with PD-L1 RNA expression and TMB and neoantigen load are not predictive of T-cell infiltration in paediatrics. Furthermore, deconvolution algorithms are only weakly correlated with IHC measurements of T-cells. CONCLUSIONS: Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

Cellular iron depletion and the mechanisms involved in the iron-dependent regulation of the growth arrest and DNA damage family of genes.

Iron plays a crucial part in proliferation while iron deficiency results in G(1)/S arrest, DNA damage, and apoptosis. However, the precise role of iron in cell cycle control remains unclear. We showed that iron depletion using the iron chelators, desferrioxamine (DFO), or 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311), increased the mRNA levels of the growth arrest and DNA damage 45α gene, GADD45α (Darnell, G. and Richardson, D. R. (1999) Blood 94, 781-792). In this study, we examined the effect of iron depletion on up-regulating GADD family members involved in growth control, including cell cycle arrest, apoptosis, and DNA repair, making them therapeutic targets for tumor suppression. We showed the GADD family members were up-regulated by cellular iron depletion. Further, up-regulation of GADD45α after iron deprivation was independent of hypoxia-inducible factor-1α (HIF-1α), octamer-1 (Oct-1), p53 and early growth response 1 (Egr1). We then analyzed the regulatory elements responsible for iron depletion-mediated regulation of GADD45α and identified the specific transcription factor/s involved. This region was within -117 bp and -81 bp relative to the start codon where the consensus sequences of three transcription factors are located: the CCAAT-binding factor/nuclear factor-Y (NF-Y), the stabilizing molecule v-MYB and the enhancer, CCAAT enhancer-binding protein (CEBPα). Mutation analysis, shRNA studies, Western blotting, and electrophoretic mobility shift assays led to the identification of NF-Y in the transcriptional up-regulation of GADD45α after iron depletion. Furthermore, like GADD45α, NF-YA was up-regulated after iron chelation and down-regulated by iron supplementation. These results are important for understanding the mechanisms of iron depletion-mediated cell cycle arrest, DNA damage repair, and apoptosis.

SLC7A8 coding for LAT2 is associated with early disease progression in osteosarcoma and transports doxorubicin.

Background: Despite (neo) adjuvant chemotherapy with cisplatin, doxorubicin and methotrexate, some patients with primary osteosarcoma progress during first-line systemic treatment and have a poor prognosis. In this study, we investigated whether patients with early disease progression (EDP), are characterized by a distinctive pharmacogenetic profile. Methods and Findings: Germline DNA from 287 Dutch high-grade osteosarcoma patients was genotyped using the DMET Plus array (containing 1,936 genetic markers in 231 drug metabolism and transporter genes). Associations between genetic variants and EDP were assessed using logistic regression models and associated variants (p <0.05) were validated in independent cohorts of 146 (Spain and United Kingdom) and 28 patients (Australia). In the association analyses, EDP was significantly associated with an SLC7A8 locus and was independently validated (meta-analysis validation cohorts: OR 0.19 [0.06-0.55], p = 0.002). The functional relevance of the top hits was explored by immunohistochemistry staining and an in vitro transport models. SLC7A8 encodes for the L-type amino acid transporter 2 (LAT2). Transport assays in HEK293 cells overexpressing LAT2 showed that doxorubicin, but not cisplatin and methotrexate, is a substrate for LAT2 (p < 0.0001). Finally, SLC7A8 mRNA expression analysis and LAT2 immunohistochemistry of osteosarcoma tissue showed that the lack of LAT2 expression is a prognostic factor of poor prognosis and reduced overall survival in patients without metastases (p = 0.0099 and p = 0.14, resp.). Conclusion: This study identified a novel locus in SLC7A8 to be associated with EDP in osteosarcoma. Functional studies indicate LAT2-mediates uptake of doxorubicin, which could give new opportunities to personalize treatment of osteosarcoma patients.

In vitro and in vivo drug screens of tumor cells identify novel therapies for high-risk child cancer.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid high-throughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

The translational regulator eIF3a: the tricky eIF3 subunit!

Regulation of gene expression is a fundamental step in cellular physiology as abnormalities in this process may lead to de-regulated growth and cancer. Translation of mRNA is mainly regulated at the rate-limiting initiation step, where many eukaryotic initiation factors (eIFs) are involved. The largest and most complex initiation factor is eIF3 which plays a role in translational regulation, cell growth and cancer. The largest subunit of eIF3 is eIF3a, although it is not required for the general function of eIF3 in translation initiation. However, eIF3a may play a role as a regulator of a subset of mRNAs and has been demonstrated to regulate the expression of p27(kip1), tyrosinated α-tubulin and ribonucleotide reductase M2 subunit. These molecules have a pivotal role in the regulation of the cell cycle. Moreover, the eIF3a mRNA is ubiquitously expressed in all tissues at different levels and is found elevated in a number of cancer types. eIF3a can modulate the cell cycle and may be a translational regulator for proteins important for entrance into S phase. The expression of eIF3a is decreased in differentiated cells in culture and the suppression of eIF3a expression can reverse the malignant phenotype and change the sensitivity of cells to cell cycle modulators. However, the role of eIF3a in cancer is still unclear. In fact, some studies have identified eIF3a to be involved in cancer development, while other results indicate that it could provide protection against evolution into higher malignancy. Together, these findings highlight the "tricky" and interesting nature of eIF3a.

Chimeric Antigen Receptor-modified T cells targeting EphA2 for the immunotherapy of paediatric bone tumours.

Chimeric Antigen Receptor (CAR) T-cell therapy, as an approved treatment option for patients with B cell malignancies, demonstrates that genetic modification of autologous immune cells is an effective anti-cancer regimen. Erythropoietin-producing Hepatocellular receptor tyrosine kinase class A2 (EphA2) is a tumour associated antigen expressed on a range of sarcomas, including paediatric osteosarcoma (OS) and Ewing sarcoma (ES). We tested human EphA2 directed CAR T cells for their capacity to target and kill human OS and ES tumour cells using in vitro and in vivo assays, demonstrating that EphA2 CAR T cells have potent anti-tumour efficacy in vitro and can eliminate established OS and ES tumours in vivo in a dose and delivery route dependent manner. Next, in an aggressive metastatic OS model we demonstrated that systemically infused EphA2 CAR T cells can traffic to and eradicate tumour deposits in murine livers and lungs. These results support further pre-clinical evaluation of EphA2 CAR T cells to inform the design of early phase clinical trial protocols to test the feasibility and safety of this immune cell therapy in paediatric bone sarcoma patients.

Neuroblastoma Molecular Risk-Stratification of DNA Copy Number and ALK Genotyping via Cell-Free Circulating Tumor DNA Profiling.

BACKGROUND: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation. METHODS: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR. RESULTS: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6-8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment. CONCLUSIONS: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.

Copper: An Intracellular Achilles' Heel Allowing the Targeting of Epigenetics, Kinase Pathways, and Cell Metabolism in Cancer Therapeutics.

Copper is an essential transition metal frequently increased in cancer known to strongly influence essential cellular processes. Targeted therapy protocols utilizing both novel and repurposed drug agents initially demonstrate strong efficacy, before failing in advanced cancers as drug resistance develops and relapse occurs. Overcoming this limitation involves the development of strategies and protocols aimed at a wider targeting of the underlying molecular changes. Receptor Tyrosine Kinase signaling pathways, epigenetic mechanisms and cell metabolism are among the most common therapeutic targets, with molecular investigations increasingly demonstrating the strong influence each mechanism exerts on the others. Interestingly, all these mechanisms can be influenced by intracellular copper. We propose that copper chelating agents, already in clinical trial for multiple cancers, may simultaneously target these mechanisms across a wide variety of cancers, serving as an excellent candidate for targeted combination therapy. This review summarizes the known links between these mechanisms, copper, and copper chelation therapy.

Iron chelator-mediated alterations in gene expression: identification of novel iron-regulated molecules that are molecular targets of hypoxia-inducible factor-1 alpha and p53.

Iron deficiency affects 500 million people, yet the molecular role of iron in gene expression remains poorly characterized. In addition, the alterations in global gene expression after iron chelation remain unclear and are important to assess for understanding the molecular pathology of iron deficiency and the biological effects of chelators. Considering this, we assessed the effect on whole genome gene expression of two iron chelators (desferrioxamine and 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone) that have markedly different permeability properties. Sixteen genes were significantly regulated by both ligands, whereas a further 50 genes were significantly regulated by either compound. Apart from iron-mediated regulation of expression via hypoxia inducible factor-1 alpha, it was noteworthy that the transcription factor p53 was also involved in iron-regulated gene expression. Examining 16 genes regulated by both chelators in normal and neoplastic cells, five genes (APP, GDF15, CITED2, EGR1, and PNRC1) were significantly differentially expressed between the cell types. In view of their functions in tumor suppression, proliferation, and apoptosis, these findings are important for understanding the selective antiproliferative effects of chelators against neoplastic cells. Most of the genes identified have not been described previously to be iron-regulated and are important for understanding the molecular and cellular effects of iron depletion.

Natural Polysaccharide Carriers in Brain Delivery: Challenge and Perspective.

Targeted drug delivery systems represent valuable tools to enhance the accumulation of therapeutics in the brain. Here, the presence of the blood brain barrier strongly hinders the passage of foreign substances, often limiting the effectiveness of pharmacological therapies. Among the plethora of materials used for the development of these systems, natural polysaccharides are attracting growing interest because of their biocompatibility, muco-adhesion, and chemical versatility which allow a wide range of carriers with tailored physico-chemical features to be synthetized. This review describes the state of the art in the field of targeted carriers based on natural polysaccharides over the last five years, focusing on the main targeting strategies, namely passive and active transport, stimuli-responsive materials and the administration route. In addition, in the last section, the efficacy of the reviewed carriers in each specific brain diseases is summarized and commented on in terms of enhancement of either blood brain barrier (BBB) permeation ability or drug bioavailability in the brain.

Intratumoral Copper Modulates PD-L1 Expression and Influences Tumor Immune Evasion.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1-driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. SIGNIFICANCE: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4129/F1.large.jpg.

Exposure to the tobacco smoke constituent 4-aminobiphenyl induces chromosomal instability in human cancer cells.

The relationships between environmental factors and the genetic abnormalities that drive carcinogenesis are supported by experimental and epidemiologic evidence but their molecular basis has not been fully elucidated. At the genomic level, most human cancers display either chromosomal (CIN) or microsatellite (MIN) instability. The molecular mechanisms through which normal cells acquire these forms of instability are largely unknown. The arylamine 4-aminobiphenyl (4-ABP) is a tobacco smoke constituent, an environmental contaminant, and a well-established carcinogen in humans. Among others, bladder, lung, colon, and breast cancers have been associated with 4-ABP. We have investigated the effects of 4-ABP and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on genetically stable colorectal (HCT116) and bladder (RT112) cancer cells. Cells were treated with carcinogens to generate resistant clones that were then subjected to genetic analysis to assess whether they displayed either CIN or MIN. We found that 50% to 60% of cells treated with 4-ABP developed CIN but none developed MIN as confirmed by their ability to gain and lose chromosomes. In contrast, all MNNG-treated clones (12/12) developed MIN but none developed CIN as shown by the microsatellite assay. The mismatch repair protein expression analysis suggests that the acquired mechanism of MIN resistance in the HCT116 MNNG-treated cells is associated with the reduction or the complete loss of MLH1 expression. By providing a mechanistic link between exposure to a tobacco constituent and the development of CIN, our results contribute to a better understanding of the origins of genetic instability, one of the remaining unsolved problems in cancer research.

In vivo [64Cu]CuCl2 PET imaging reveals activity of Dextran-Catechin on tumor copper homeostasis.

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels in vivo is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [64Cu]CuCl2 PET imaging protocol to monitor its effect on copper homeostasis in tumors. Methods: Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin in vivo and its effect on tumor copper homeostasis. PET imaging with [64Cu]CuCl2 was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment. Results: CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. In vivo studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [64Cu]CuCl2 retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. Ex vivo analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity. Conclusion: Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments.

Integration of genomics, high throughput drug screening, and personalized xenograft models as a novel precision medicine paradigm for high risk pediatric cancer.

Pediatric high grade gliomas (HGG) are primary brain malignancies that result in significant morbidity and mortality. One of the challenges in their treatment is inter- and intra-tumoral heterogeneity. Precision medicine approaches have the potential to enhance diagnostic, prognostic and/or therapeutic information. In this case study we describe the molecular characterization of a pediatric HGG and the use of an integrated approach based on genomic, in vitro and in vivo testing to identify actionable targets and treatment options. Molecular analysis based on WGS performed on initial and recurrent tumor biopsies revealed mutations in TP53, TSC1 and CIC genes, focal amplification of MYCN, and copy number gains in SMO and c-MET. Transcriptomic analysis identified increased expression of MYCN, and genes involved in sonic hedgehog signaling proteins (SHH, SMO, GLI1, GLI2) and receptor tyrosine kinase pathways (PLK, AURKA, c-MET). HTS revealed no cytotoxic efficacy of SHH pathway inhibitors while sensitivity was observed to the mTOR inhibitor temsirolimus, the ALK inhibitor ceritinib, and the PLK1 inhibitor BI2536. Based on the integrated approach, temsirolimus, ceritinib, BI2536 and standard therapy temozolomide were selected for further in vivo evaluation. Using the PDX animal model (median survival 28 days) we showed significant in vivo activity for mTOR inhibition by temsirolimus and BI2536 (median survival 109 and 115.5 days respectively) while ceritinib and temozolomide had only a moderate effect (43 and 75.5 days median survival respectively). This case study demonstrates that an integrated approach based on genomic, in vitro and in vivo drug efficacy testing in a PDX model may be useful to guide the management of high risk pediatric brain tumor in a clinically meaningful timeframe.