Olivier Deas

NOTCH1 Nuclear Interactome Reveals Key Regulators of Its Transcriptional Activity and Oncogenic Function

Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). Despite important advances in our understanding of Notch signal transduction, the regulation of Notch functions in the nucleus remains unclear. Using immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL cells and showed that, beyond the well-characterized core activation complex (ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and the histone demethylases LSD1 and PHF8 acting through their demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation. Our work provides new insights into the molecular mechanisms that govern Notch transcriptional activity and represents glimpse into NOTCH1 interaction landscape, which will help in deciphering mechanisms of NOTCH1 functions and regulation.

The MET inhibitor AZD6094 (Savolitinib, HMPL-504) induces regression in papillary renal cell carcinoma patient derived xenograft models

Purpose: Papillary renal cell carcinoma (PRCC) is the second most common cancer of the kidney and carries a poor prognosis for patients with nonlocalized disease. The HGF receptor MET plays a central role in PRCC and aberrations, either through mutation, copy number gain, or trisomy of chromosome 7 occurring in the majority of cases. The development of effective therapies in PRCC has been hampered in part by a lack of available preclinical models. We determined the pharmacodynamic and antitumor response of the selective MET inhibitor AZD6094 in two PRCC patient-derived xenograft (PDX) models. Experimental Design: Two PRCC PDX models were identified and MET mutation status and copy number determined. Pharmacodynamic and antitumor activity of AZD6094 was tested using a dose response up to 25 mg/kg daily, representing clinically achievable exposures, and compared with the activity of the RCC standard-of-care sunitinib (in RCC43b) or the multikinase inhibitor crizotinib (in RCC47). Results: AZD6094 treatment resulted in tumor regressions, whereas sunitinib or crizotinib resulted in unsustained growth inhibition. Pharmacodynamic analysis of tumors revealed that AZD6094 could robustly suppress pMET and the duration of target inhibition was dose related. AZD6094 inhibited multiple signaling nodes, including MAPK, PI3K, and EGFR. Finally, at doses that induced tumor regression, AZD6094 resulted in a dose- and time-dependent induction of cleaved PARP, a marker of cell death. Conclusions: Data presented provide the first report testing therapeutics in preclinical in vivo models of PRCC and support the clinical development of AZD6094 in this indication. Clin Cancer Res; 21(12); 2811–9. ©2015 AACR.

Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

Background:Oestrogen receptor-negative (ER−) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer.Methods:Gene and protein expression profiles were analysed in a panel of ER− breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively.Results:The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells.Conclusions:Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER− breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

Intracellular STING inactivation sensitizes breast cancer cells to genotoxic agents

Activation of the IFN/STAT1 pathway is closely associated with drug response and recurrence of breast cancer treated by chemotherapy. The aim of the current study was to elucidate the molecular mechanisms involved upstream and downstream of this pathway in order to identify distinct entities that might be manipulated to improve treatment efficacy. Four breast cancer cell lines (T-47D, MCF7, MDA-MB-231 and HBCx-19 established from the eponymous PDX) were treated in vitro with mafosfamide, a DNA damage inducer. In two of these cell lines (MCF7 and HBCx-19), genotoxic treatment upregulated type I IFN expression leading to paracrine activation of IFN/STAT1 signaling pathway after 6–8 days. We show that STING, a well-characterized inducer of IFN in immune cells, is rapidly triggered in MCF7 cells under genotoxic stress and forms nuclear foci that co-localize with phosphorylated IRF-3 and γH2AX. STING silencing abrogated chemotherapy-induced type I IFN production and signaling and potentiated genotoxic treatment efficacy as it promoted cell death extent and delayed cell colony regrowth. Similar results were obtained after silencing PARP12, one selected gene of the IFN/STAT1 pathway fingerprint. In summary, this study provides the first demonstration of STING activation in breast cancer cells. Our data suggest that genotoxic-induced, STING-mediated type I IFN signaling is a cell-intrinsic mechanism of breast cancer cell survival and regrowth.

Patient‐derived mouse xenografts from pediatric liver cancer predict tumor recurrence and advise clinical management

Identification of new treatments for relapsing pediatric cancer is an unmet clinical need and a societal challenge. Liver cancer occurrence in infancy, 1.5 for million children per year, falls far below the threshold of interest for dedicated drug development programs, and this disease is so rare that it is very difficult to gather enough children into a phase II clinical trial. Here, we present the establishment of an unprecedented preclinical platform of 24 pediatric liver cancer patient‐derived xenografts (PLC‐PDXs) from 20 hepatoblastomas (HBs), 1 transitional liver cell tumor (TCLT), 1 hepatocellular carcinoma, and 2 malignant rhabdoid tumors. Cytogenetic array and mutational analysis of the parental tumors and the corresponding PLC‐PDXs show high conservation of the molecular features of the parental tumors. The histology of PLC‐PDXs is strikingly similar to that observed in primary tumors and recapitulates the heterogeneity of recurrent disease observed in the clinic. Tumor growth in the mouse is strongly associated with elevated circulating alpha‐fetoprotein (AFP), low rate of necrosis/fibrosis after treatment, and gain of chromosome 20, all indicators of resistance to chemotherapy and poor outcome. Accordingly, the ability of a tumor to generate PLC‐PDX is predictive of poor prognosis. Exposure of PLC‐PDXs to standards of care or therapeutic options already in use for other pediatric malignancies revealed unique response profiles in these models. Among these, the irinotecan/temozolomide combination induced strong tumor regression in the TCLT and in a model derived from an AFP‐negative relapsing HB. Conclusion: These results provide evidence that PLC‐PDX preclinical platform can strongly contribute to accelerate the identification and diversification of anticancer treatment for aggressive subtypes of pediatric liver cancer. (Hepatology 2016;64:1121‐1135)

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

IntroductionChemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells.MethodsWe performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations.ResultsHigh expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial–mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy.ConclusionsIn this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Abstract P5-06-04: The PARP inhibitor niraparib demonstrated activity in patient-derived triple-negative breast cancer xenograft models with high homologous recombination deficiency (HRD) score

Triple negative breast cancer (TNBC), which comprises 15% of all breast cancers, has a poor prognosis and currently lacks effective treatment. TNBCs are highly proliferative, genomically unstable and share molecular characteristics with that of BRCA1/2 mutation driven breast cancer. Poly(ADP-ribose) polymerase-1 (PARP) is a key DNA repair enzyme that mediates single strand break (SSB) repair through the base excision repair (BER) pathway. PARP inhibitors have been demonstrated to selectively kill tumor cells that harbor BRCA1 and BRCA2 mutations. In addition, pre-clinical and preliminary clinical data suggest that PARP inhibitors are selectively cytotoxic for tumors with homologous recombination repair deficiency caused by dysfunction of genes other than BRCA1 or BRCA2. Niraparib is a potent, orally active PARP inhibitor that is being evaluated in Phase 3 clinical studies for ovarian cancer and BRCA related breast cancer. Previously, we demonstrated that a subset of basal breast cancer (BBC) patient-derived xenograft (PDX) models responded robustly to single agent niraparib treatment. To understand the selectivity observed, the samples from a collection of 37 BBC PDX models have been subjected to homologous recombination deficiency (HRD) analysis. HRD analysis is a DNA-based assay that is capable of detecting homologous recombination deficiency independent of its etiology. Genome-wide SNP data was generated from a custom Agilent SureSelect XT capture followed by sequencing on an Illumina HiSeq2500. SNP data was analyzed using three algorithms (LOH, TAI and LST scores), and the final HRD score is the sum of the LOH+TAI+LAST scores. Niraparib’s antitumor activity was investigated in patient derived BBC models with various HRD scores. The correlation between niraparib efficacy, HRD score and BRCA deficiency will be discussed. Citation Format: Yan Wang, Stefano Cairo, Olivier Deas, Anne-Renee Hartman, Joshua Jones, Alexander Gutin, Jerry Lanchbury, Zaina Sangale, Cara Solimeno, Jean-Gabriel Judde, Kirsten Timms, Keith Wilcoxen. The PARP inhibitor niraparib demonstrated activity in patient-derived triple-negative breast cancer xenograft models with high homologous recombination deficiency (HRD) score [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-04.

An NF90/NF110-mediated feedback amplification loop regulates dicer expression and controls ovarian carcinoma progression

Reduced expression of DICER, a key enzyme in the miRNA pathway, is frequently associated with aggressive, invasive disease, and poor survival in various malignancies. Regulation of DICER expression is, however, poorly understood. Here, we show that NF90/NF110 facilitates DICER expression by controlling the processing of a miRNA, miR-3173, which is embedded in DICER pre-mRNA. As miR-3173 in turn targets NF90, a feedback amplification loop controlling DICER expression is established. In a nude mouse model, NF90 overexpression reduced proliferation of ovarian cancer cells and significantly reduced tumor size and metastasis, whereas overexpression of miR-3173 dramatically increased metastasis in an NF90- and DICER-dependent manner. Clinically, low NF90 expression and high miR-3173-3p expression were found to be independent prognostic markers of poor survival in a cohort of ovarian carcinoma patients. These findings suggest that, by facilitating DICER expression, NF90 can act as a suppressor of ovarian carcinoma.

Abstract 5273: XenTech patient-derived xenograft (PDX) panels: a clinically relevant platform for drug efficacy, target validation and predictive biomarker discovery studies

Despite considerable efforts in understanding the biology and genetics of cancer, most currently available treatments fail to achieve tumor eradication in the majority of patients. Key to more effective therapies is adequate disease classification and subsequent patient stratification. In addition, it is important to understand the mechanisms of drug-response or resistance and identify novel targets amenable to therapeutic intervention. It is increasingly recognized that at the preclinical stage, testing therapeutic strategies and validating target relevance in more predictive models closely mimicking clinical disease such as patient derived xenografts (PDXs), may translate into improved clinical efficacy and lower rate of drug attrition. XenTech collection of over 120 runing PDX models is one of the largest in the world. PDX models were established by grafting post-surgery human tumor fragments in the interscapular region of immunodeficient mice. These deeply characterized PDX models can be used for in vivo preclinical assays. Such preclinical platform is a reliable surrogate of patient cohorts and can address several aims: 1. Evaluate tumor response to treatment. PDXs can be subjected to parallel evaluation of tumor response to various treatment protocols. Drug-response profile is linked to tumor histotype and molecular features in order to identify predictive markers of drug response to assist treatment choice. 2. Assess treatment-driven tumor eradication. The ability of a treatment to induce complete tumor response is assessed by monitoring tumor regression over a long period. Most tumors, despite complete macroscopic regression, are still present as latent microscopic nodular islands that may give rise to tumor recurrence. Molecular characterization of tumor foci responsible for tumor relapse may be performed to identify genes/pathways involved in residual tumor cell survival, which may provide new diagnostic and/or therapeutic targets for designing novel adjuvant treatment strategies. 3. Development of bioluminescent metastatic models to study the mechanisms of tumor invasion and to test anti-metastatic therapy. 4. Non-invasive molecular imaging technology to monitor tumor metabolism, vascularization and apoptosis. 5. Constitution of preclinical panels of rare malignancies to obtain phase II-like tumor cohorts. Development of new therapies for rare tumors is rendered difficult by the unavailability of patient cohorts wide enough to set up robust clinical trials. To assist the clinical need, these panels would allow the evaluation of new and more efficient therapies. We describe here in detail our PDX collection and illustrate how it represents a powerful tool to identify preferential therapeutic options for patients by exploring and improving anti-cancer therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5273. doi:1538-7445.AM2012-5273

MiR-205 as predictive biomarker and adjuvant therapeutic tool in combination with trastuzumab

Trastuzumab is the standard treatment for HER2+ breast cancer (BC) patients, and even though it significantly improved their clinical outcome, 50% of them do not benefit from this drug and disease recurs, underlining the need of reliable predictive biomarkers and new therapeutic strategies. Strikingly, despite all the molecular analyses performed to identify the escape mechanisms behind this resistance, it still represents a question point. MiRNAs have been correlated with occurrence and progression of human cancer, and their potential as clinical tools has emerged in the last years. We previously reported that oncosuppressive miR-205 targets HER3, thus increasing the responsiveness to TKIs lapatinib and gefitinib in preclinical models. Here we demonstrate that HER3 inhibition by miR-205 ectopic expression or siRNA-mediated silencing improves the responsiveness to Trastuzumab in vitro in HER2+ BC cell lines, and that this effect is exerted through impairment of AKT-mediated pathway. Moreover, evaluating a series of 52 HER2+ BC patients treated with adjuvant Trastuzumab, we observed that higher miR-205 expression is significantly associated with better outcome (disease-free survival). In summary, our data indicate that miR-205 could predict Trastuzumab efficacy and that its modulation might be useful as adjuvant treatment to improve the response to the drug.