Investigation of fingolimod-induced lymphocyte sequestration on inflammatory response and neurological damages after cardiac arrest.

BACKGROUND: A sepsis-like syndrome is known to occur after cardiac arrest, leading to cerebral infiltration by white blood cells (WBC). We hypothesized that pharmacological sequestration of WBC, and more specifically lymphocytes within lymphoid tissues, could reduce the cerebral infiltration by these inflammatory cells and subsequent acute brain injury in a porcine model of cardiac arrest. Lymphocyte sequestration was induced by the sphingosine-1 phosphate receptors agonist fingolimod. METHODS: In a first set of experiments, anesthetized pigs underwent a sham instrumentation with no cardiac arrest (n = 4). They received an administration of fingolimod (1 mg/kg, i.v.) in order to confirm its effect on WBC. In a second set of experiments, animals randomly received fingolimod or saline two hours prior to an episode of ventricular fibrillation (14 min) with subsequent resuscitation (n = 6 in each group). Neurological injury was assessed 24 h after resuscitation. RESULTS: In the first set of experiments, WBC and blood lymphocyte counts were significantly reduced by - 61 ± 10% and - 75 ± 6% two hours after fingolimod administration. In the second set of experiments, blood lymphocyte counts, but not WBC, were also significantly reduced after cardiac arrest in Fingolimod vs Control group. However, most cytokine blood levels were not different among groups, including Interleukin (IL)-1ra, IL-8 or IL-18 blood levels. A difference was only observed for IL-6, which decreased in Fingolimod vs Control (e.g., 5.6 ± 4.8 vs 59.4 ± 20.6 pg/ml at 2 h after cardiac arrest, respectively; p = 0.126). Neurofilament light chain (NFL) blood levels were not different among groups (57 ± 25 vs 84 ± 41 pg/ml in Fingolimod vs Control at 6 h after resuscitation, respectively). After awakening, 3 and 2 animals were prematurely euthanized for ethical reasons due to recurrent seizures in Fingolimod and Control groups, respectively. At Day 1, neurological dysfunction score was not different between groups (87 ± 7 vs 87 ± 5% in Fingolimod vs Control, respectively). Conversely, a decrease in the number of CD3 + cells was observed in the brain of surviving animals in Fingolimod vs Control group (3.10 ± 0.50 vs 7.53 ± 0.57 CD3 + cells/field, respectively; p = 0.0286). CONCLUSION: Fingolimod-induced WBC sequestration, and more specifically lymphocytes sequestration, did not improve clinical neurological dysfunction following cardiac arrest although it reduced cerebral infiltration by lymphocytes.

Homologous recombination deficiency derived from whole-genome sequencing predicts platinum response in triple-negative breast cancers.

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

High-Mobility Group Box 1-Signaling Inhibition With Glycyrrhizin Prevents Cerebral T-Cell Infiltration After Cardiac Arrest.

Background High-mobility group box 1 (HMGB1) is a major promotor of ischemic injuries and aseptic inflammatory responses. We tested its inhibition on neurological outcome and systemic immune response after cardiac arrest (CA) in rabbits. Methods and Results After 10 minutes of ventricular fibrillation, rabbits were resuscitated and received saline (control) or the HMGB1 inhibitor glycyrrhizin. A sham group underwent a similar procedure without CA. After resuscitation, glycyrrhizin blunted the successive rises in HMGB1, interleukin-6, and interleukin-10 blood levels as compared with control. Blood counts of the different immune cell populations were not different in glycyrrhizin versus control. After animal awakening, neurological outcome was improved by glycyrrhizin versus control, regarding both clinical recovery and histopathological damages. This was associated with reduced cerebral CD4+ and CD8+ T-cell infiltration beginning 2 hours after CA. Conversely, granulocytes' attraction or loss of microglial cells or cerebral monocytes were not modified by glycyrrhizin after CA. These modifications were not related to the blood-brain barrier preservation with glycyrrhizin versus control. Interestingly, the specific blockade of the HMGB1 receptor for advanced glycation end products by FPS-ZM1 recapitulated the neuroprotective effects of glycyrrhizin. Conclusions Our findings support that the early inhibition of HMGB1-signaling pathway prevents cerebral chemoattraction of T cells and neurological sequelae after CA. Glycyrrhizin could become a clinically relevant therapeutic target in this situation.

HORMAD1 overexpression predicts response to anthracycline-cyclophosphamide and survival in triple-negative breast cancers.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are associated with higher recurrence and distant metastasis rate. Standard of care for early stage TNBC is anthracyclines combined with cyclophosphamide (AC) followed by taxanes, in the neo-adjuvant or adjuvant setting. This work aimed to identify predictive biomarkers of AC response in patient-derived xenograft (PDX) models of TNBC and to validate them in the clinical setting. By gene and protein expression analysis of 39 PDX with different responses to AC, we found that high expression of HORMAD1 was associated with better response to AC. Both gene and protein expression were associated with promoter hypomethylation. In a cohort of 526 breast cancer patients, HORMAD1 was overexpressed in 71% of TNBC. In a second cohort of 186 TNBC patients treated with AC, HORMAD1 expression was associated with longer metastasis-free survival (MFS). In summary, HORMAD1 overexpression was predictive of an improved response to AC in PDX and is an independent prognostic factor in TNBC patients treated with AC.

HRAS is a therapeutic target in malignant chemo-resistant adenomyoepithelioma of the breast.

Malignant adenomyoepithelioma (AME) of the breast is an exceptionally rare form of breast cancer, with a significant metastatic potential. Chemotherapy has been used in the management of advanced AME patients, however the majority of treatments are not effective. Recent studies report recurrent mutations in the HRAS Q61 hotspot in small series of AMEs, but there are no preclinical or clinical data showing H-Ras protein as a potential therapeutic target in malignant AMEs. We performed targeted sequencing of tumours' samples from new series of 13 AMEs, including 9 benign and 4 malignant forms. Samples from the breast tumour and the matched axillary metastasis of one malignant HRAS mutated AME were engrafted and two patient-derived xenografts (PDX) were established that reproduced the typical AME morphology. The metastasis-derived PDX was treated in vivo by different chemotherapies and a combination of MEK and BRAF inhibitors (trametinib and dabrafenib). All malignant AMEs presented a recurrent mutation in the HRAS G13R or G12S hotspot. Mutation of PIK3CA were found in both benign and malignant AMEs, while AKT1 mutations were restricted to benign AMEs. Treatment of the PDX by the MEK inhibitor trametinib, resulted in a marked anti-tumor activity, in contrast to the BRAF inhibitor and the different chemotherapies that were ineffective. Overall, these findings further expand on the genetic features of AMEs and suggest that patients carrying advanced HRAS-mutated AMEs could potentially be treated with MEK inhibitors.

Analysis of genomic and non-genomic signaling of estrogen receptor in PDX models of breast cancer treated with a combination of the PI3K inhibitor alpelisib (BYL719) and fulvestrant.

BACKGROUND: Endocrine therapies targeting estrogen signaling have significantly improved breast cancer (BC) patient survival, although 40% of ERα-positive BCs do not respond to those therapies. Aside from genomic signaling, estrogen triggers non-genomic pathways by forming a complex containing methylERα/Src/PI3K, a hallmark of aggressiveness and resistance to tamoxifen. We aimed to confirm the prognostic value of this complex and investigated whether its targeting could improve tumor response in vivo. METHODS: The interaction of ERα/Src and ERα/PI3K was studied by proximity ligation assay (PLA) in a cohort of 440 BC patients. We then treated patient-derived BC xenografts (PDXs) with fulvestrant or the PI3K inhibitor alpelisib (BYL719) alone or in combination. We analyzed their anti-proliferative effects on 6 ERα+ and 3 ERα- PDX models. Genomic and non-genomic estrogen signaling were assessed by measuring ERα/PI3K interaction by PLA and the expression of estrogen target genes by RT-QPCR, respectively. RESULTS: We confirmed that ERα/Src and ERα/PI3K interactions were associated with a trend to poorer survival, the latter displaying the most significant effects. In ERα+ tumors, the combination of BYL719 and fulvestrant was more effective than fulvestrant alone in 3 models, irrespective of PI3K, PTEN status, or ERα/PI3K targeting. Remarkably, resistance to fulvestrant was associated with non-genomic ERα signaling, since genomic degradation of ERα was unaltered in these tumors, whereas the treatment did not diminish the level of ERα/PI3K interaction. Interestingly, in 2 ERα- models, fulvestrant alone impacted tumor growth, and this was associated with a decrease in ERα/PI3K interaction. CONCLUSIONS: Our results demonstrate that ERα/PI3K may constitute a new prognostic marker, as well as a new target in BC. Indeed, resistance to fulvestrant in ERα+ tumors was associated with a lack of impairment of ERα/PI3K interaction in the cytoplasm. In addition, an efficient targeting of ERα/PI3K in ERα- tumors could constitute a promising therapeutic option.

Spontaneous mouse lymphoma in patient-derived tumor xenografts: The importance of systematic analysis of xenografted human tumor tissues in preclinical efficacy trials.

Patient-derived tumor xenograft (PDX) is now largely recognized as a key preclinical model for cancer research, mimicking patient tumor phenotype and genotype. Immunodeficient mice, well-known to develop spontaneous lymphoma, are required for PDX growth. As for all animal models used for further clinical translation, a robust experimental design is strongly required to lead to conclusive results. Here we briefly report unintentional co-engraftment of mouse lymphoma during expansion of well-established PDXs to illustrate the importance of systematic check of the PDX identity to avoid misinterpretation. Besides, this quality control based on complementary approaches deserves a more detailed description in materials and methods section to ensure experimental validity and reproducibility.

High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient's outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

Health Impact and Cost-Effectiveness of Implementing Gender-Neutral Vaccination With the 9-Valent Human Papillomavirus Vaccine in Belgium.

Background: Routine human papillomavirus (HPV) immunization in Belgium is currently regionally managed, with school-aged girls receiving the 9-valent HPV (9vHPV) vaccine in Flanders and Wallonia-Brussels with a national catch-up program for females only. This study will assess whether expanding these programs to gender-neutral vaccination (GNV) with the 9vHPV vaccine is a cost-effective strategy in Belgium. Methods: A validated HPV-type transmission dynamic model estimated the potential health and economic impact of regional vaccination programs, comparing GNV versus female-only vaccination (FOV) with the 9vHPV vaccine in individuals aged 11-12 years in Flanders, GNV with the 9vHPV vaccine versus FOV with the 2-valent HPV (2vHPV) vaccine in individuals aged 12-13 years in Wallonia-Brussels, and national catch-up GNV versus FOV with the 9vHPV vaccine for those aged 12-18 years. Vaccination coverage rates of 90, 50, and 50% in both males and females were used in the base cases for the three programs, respectively, and sensitivity analyses were conducted. All costs are from the third-party payer perspective, and outcome measures were reported over a 100-year time horizon. Results: GNV with the 9vHPV vaccine was projected to decrease the cumulative incidence of HPV 6/11/16/18/31/33/45/52/58-related diseases relative to FOV in both Flanders and Wallonia-Brussels. Further reductions were also projected for catch-up GNV with the 9vHPV vaccine, including reductions of 6.8% (2,256 cases) for cervical cancer, 7.1% (386 cases) and 18.8% (2,784 cases) for head and neck cancer in females and males, respectively, and 30.3% (82,103 cases) and 44.6% (102,936 cases) for genital warts in females and males, respectively. As a result, a GNV strategy would lead to reductions in HPV-related deaths. Both regional and national catch-up GNV strategies were projected to reduce cumulative HPV-related disease costs and were estimated to be cost-effective compared with FOV with incremental cost-effectiveness ratios of €8,062, €4,179, and €6,127 per quality-adjusted life-years in the three programs, respectively. Sensitivity analyses were consistent with the base cases. Conclusions: A GNV strategy with the 9vHPV vaccine can reduce the burden of HPV-related disease and is cost-effective compared with FOV for both regional vaccination programs and the national catch-up program in Belgium.

YAP and ß-Catenin Cooperate to Drive Oncogenesis in Basal Breast Cancer.

Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in ß-catenin target genes, suggesting that YAP is required for nuclear ß-catenin activity. Mechanistically, nuclear YAP interacted with ß-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, ß-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/ß-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. SIGNIFICANCE: These findings show that YAP cooperates with ß-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2116/F1.large.jpg.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts.

Luminal androgen receptor (LAR) breast cancer accounts for 10% of all triple-negative breast cancers (TNBC). Anti-androgen therapy for this subtype is in development, but yields only partial clinical benefits. In this study, we aimed to characterize the genomic alterations of LAR TNBC, to analyze activation of the PI3K signaling pathway and to compare the response to PI3K pathway inhibitors with that to anti-androgen therapy in patient-derived xenografts (PDX) of LAR TNBC. Methods: Four LAR PDX models were identified, on the basis of their transcriptomic profiles, in a cohort of 57 PDX models of TNBC. The expression of AR-related genes, basal and luminal cytokeratins and EMT genes was analyzed by RT-PCR and IHC. AKT1 and PIK3CA mutations were identified by targeted NGS, and activation of the PI3K pathway was analyzed with a reverse-phase protein array. Three LAR PDXs with a PIK3CA or AKT1 mutation were treated with the AR inhibitor enzalutamide, a PI3K inhibitor, a dual PI3K-mTOR inhibitor and a mTORC1-mTORC2 inhibitor. Finally, we screened a clinical cohort of 329 TNBC for PIK3CA and AKT1 hotspot mutations. Results: LAR TNBC PDXs were significantly enriched in PIK3CA and AKT1 mutations, and had higher levels of luminal-androgen-like gene expression and a higher PI3K pathway protein activation score than other TNBC subtypes. Immunohistochemistry analysis revealed strong expression of the luminal cytokeratin CK18 and AR in three LAR PDX models. We found that mTOR and PI3K inhibitors had marked antitumor activity in vivo in PDX harboring genomic alterations of PIK3CA and AKT1 genes that did not respond to the AR antagonist enzalutamide. PIK3CA mutations were detected in more than one third of AR+ TNBC from patients (38%), and only 10% of AR-negative TNBC. Conclusion: Our results for PDX models of LAR TNBC resistant to enzalutamide indicate that PIK3CA and AKT1 are potential therapeutic targets.

Correction to: Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer.

After publication of the original article [1], we were notified that an author's surname has been erroneously spelled. Elisabetta Maragoni's family name should be replaced with Marangoni.

Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer.

BACKGROUND: Endocrine therapies are still the main strategy for the treatment of oestrogen receptor-positive (ER+) breast cancers (BC), but resistance remains problematic. Cross-talk between ER and PI3K/AKT/mTORC has been associated with ligand-independent transcription of ER. We have previously reported the anti-proliferative effects of the combination of everolimus (an mTORC1 inhibitor) with endocrine therapy in resistance models, but potential routes of escape via AKT signalling can lead to resistance; therefore, the use of dual mTORC1/2 inhibitors has met with significant interest. METHODS: To address this, we tested the effect of vistusertib, a dual mTORC1 and mTORC2 inhibitor, in a panel of endocrine-resistant and endocrine-sensitive ER+ BC cell lines, with varying PTEN, PIK3CA and ESR1 mutation status. End-points included proliferation, cell signalling, cell cycle and effect on ER-mediated transcription. Two patient-derived xenografts (PDX) modelling endocrine resistance were used to assess the efficacy of vistusertib, fulvestrant or the combination on tumour progression, and biomarker studies were conducted using immunohistochemistry and RNA-seq technologies. RESULTS: Vistusertib caused a dose-dependent decrease in proliferation of all the cell lines tested and reduced abundance of mTORC1, mTORC2 and cell cycle markers, but caused an increase in abundance of EGFR, IGF1R and ERBB3 in a context-dependent manner. ER-mediated transcription showed minimal effect of vistusertib. Combined therapy of vistusertib with fulvestrant showed synergy in two ER+ PDX models of resistance to endocrine therapy and delayed tumour progression after cessation of therapy. CONCLUSIONS: These data support the notion that models of acquired endocrine resistance may have a different sensitivity to mTOR inhibitor/endocrine therapy combinations.

Improving breast cancer sensitivity to paclitaxel by increasing aneuploidy.

Predictive biomarkers for tumor response to neoadjuvant chemotherapy are needed in breast cancer. This study investigates the predictive value of 280 genes encoding proteins that regulate microtubule assembly and function. By analyzing 3 independent multicenter randomized cohorts of breast cancer patients, we identified 17 genes that are differentially regulated in tumors achieving pathological complete response (pCR) to neoadjuvant chemotherapy. We focused on the MTUS1 gene, whose major product, ATIP3, is a microtubule-associated protein down-regulated in aggressive breast tumors. We show here that low levels of ATIP3 are associated with an increased pCR rate, pointing to ATIP3 as a predictive biomarker of breast tumor chemosensitivity. Using preclinical models of patient-derived xenografts and 3-dimensional models of breast cancer cell lines, we show that low ATIP3 levels sensitize tumors to the effects of taxanes but not DNA-damaging agents. ATIP3 silencing improves the proapoptotic effects of paclitaxel and induces mitotic abnormalities, including centrosome amplification and multipolar spindle formation, which results in chromosome missegregation leading to aneuploidy. As shown by time-lapse video microscopy, ATIP3 depletion exacerbates cytokinesis failure and mitotic death induced by low doses of paclitaxel. Our results favor a mechanism by which the combination of ATIP3 deficiency and paclitaxel treatment induces excessive aneuploidy, which in turn results in elevated cell death. Together, these studies highlight ATIP3 as an important regulator of mitotic integrity and a useful predictive biomarker for a population of chemoresistant breast cancer patients.

Profiling 58 compounds including cosmetic-relevant chemicals using ToxRefDB and ToxCast.

Safety assessment for cosmetic-relevant chemicals (CRCs) in the European Union has been reshaped by restrictions on animal testing, and new approach methodologies (NAMs) for predicting toxicity are critical to ensure new cosmetic product safety. To demonstrate NAMs for safety assessment, we surveyed in vitro bioactivity and in vivo systemic toxicity data in the US Environmental Protection Agency's (EPA's) Toxicity Forecaster (ToxCast) and Toxicity Reference databases (ToxRefDB), respectively, for 58 chemicals identified as CRCs, including cosmetic ingredients as well as trace contaminants. CRCs were diverse in use types as suggested by broad chemical use categories. In terms of both target organ effects and study type, the median of the lowest effect level (LEL) doses in ToxRefDB for CRCs tended to be slightly higher than the median for the remaining 928 chemicals with study data in ToxRefDB, though the ranges of LELs were similar. For 17 of the 58 CRCs, high-throughput toxicokinetic data were used to calculate administered equivalent doses (AEDs) in mg/kg/day units for the in vitro bioactivity observed, and these AEDs served as conservative estimators of the systemic LELs observed in vivo. This work suggests that NAMs for bioactivity may inform a conservative point-of-departure estimate for diverse CRCs.

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

Expression of MT4-MMP, EGFR, and RB in Triple-Negative Breast Cancer Strongly Sensitizes Tumors to Erlotinib and Palbociclib Combination Therapy.

PURPOSE: Here, we investigated the clinical relevance of an unprecedented combination of three biomarkers in triple-negative breast cancer (TNBC), both in human samples and in patient-derived xenografts of TNBC (PDX-TNBC): EGFR, its recently identified partner (MT4-MMP), and retinoblastoma protein (RB).Experimental Design: IHC analyses were conducted on human and PDX-TNBC samples to evaluate the production of the three biomarkers. The sensitivity of cancer cells expressing or not MT4-MMP to anti-EGFR (erlotinib) or anti-CDK4/6 inhibitor (palbociclib) was evaluated in vitro in 2D and 3D proliferation assays and in vivo using xenografts and PDX-TNBC displaying different RB, MT4-MMP, and EGFR status after single (erlotinib or palbociclib) or combined (erlotinib + palbociclib) treatments. RESULTS: EGFR and MT4-MMP were coexpressed in >70% of TNBC samples and PDX-TNBC, among which approximately 60% maintained RB expression. Notably, approximately 50% of all TNBC and PDX-TNBC expressed the three biomarkers. Single erlotinib and palbociclib treatments drastically reduced the in vitro proliferation of cells expressing EGFR and MT4-MMP when compared with control cells. Both TNBC xenografts and PDX expressing MT4-MMP, EGFR, and RB, but not PDX-TNBC with RB loss, were sensitive to erlotinib and palbociclib with an additive effect of combination therapy. Moreover, this combination was efficient in another PDX-TNBC expressing the three biomarkers and resistant to erlotinib alone. CONCLUSIONS: We defined a new association of three biomarkers (MT4-MMP/EGFR/RB) expressed together in 50% of TNBC and demonstrated its usefulness to predict the TNBC response to anti-EGFR and anti-CDK4/6 drugs used in single or combined therapy.

Inhibition of mTOR downregulates expression of DNA repair proteins and is highly efficient against BRCA2-mutated breast cancer in combination to PARP inhibition.

Breast cancer is a complex disease in which each patient could present several genetic alterations that are therapeutically relevant in cancers. Here we explored the therapeutic benefit of combining PARP and mTOR inhibitors in a context of DNA repair deficiency and PI3K pathway activation. The combination of everolimus and olaparib was tested in BRCA2-mutated patient-derived xenografts (PDX) carrying alterations in the PI3K/AKT/mTOR pathway. An RPPA analysis of different signalling pathways was performed in untreated and treated xenografts. Everolimus and olaparib showed marked anti-tumor activities in the monotherapy setting and high efficacy when given in combination with 100% of mice showing tumor regressions. The fraction of P-H2AX positive cells was increased in both monotherapy arms and strongly increased in the combination setting. Everolimus given as monotherapy resulted in downregulation of different proteins involved in DNA damage repair, including FANCD2, RAD50 and SUV39H1. In the combination setting, expression of these proteins was almost completely abolished, suggesting convergence of PARP and mTOR in downregulation of DNA damage repair components. In conclusion, our results suggest that combining mTOR and DNA repair inhibition could be a successful strategy to treat a subset of breast cancer with BRCA2 mutation and alterations in the PI3K/AKT/mTOR pathway.