Paulien Cornelissen-Steijger

Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma

Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas.

Bmi1 Regulates Stem Cells and Proliferation and Differentiation of Committed Cells in Mammary Epithelium

PolycombGroup (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer [1]. Mice without the PcG gene Bmi1 are runted and suffer from progressive loss of hematopoietic and neural stem cells [2-4]. Here, we assess the effects of Bmi1 on stem cells and differentiation of an epithelial tissue in vivo. We chose the mammary gland because it allows limiting dilution transplantations [5, 6] and because Bmi1 is overexpressed in breast cancer [7, 8]. Our analyses show that Bmi1 is expressed in all cells of the mouse mammary gland and is especially high in luminal cells. Loss of Bmi1 results in a severe mammary-epithelium growth defect, which can be rescued by codeletion of the Ink4a/Arf locus or pregnancy. Even though mammary stem cells are present in the absence of Bmi1, their activity is reduced, and this is only partially due to Ink4a/Arf expression. Interestingly, loss of Bmi1 causes premature lobuloalveolar differentiation, whereas overexpression of Bmi1 inhibits lobuloalveolar differentiation induced by pregnancy hormones. Because Bmi1 affects not only mammary stem cells but also more committed cells, our data warrant a more detailed analysis of the different roles of Bmi1 in breast-cancer etiology.

BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A

IntroductionTreatment of breast cancer is becoming more individualized with the recognition of tumor subgroups that respond differently to available therapies. Breast cancer 1 gene (BRCA1)-deficient tumors are usually of the basal subtype and associated with poor survival rates, highlighting the need for more effective therapy.MethodsWe investigated a mouse model that closely mimics breast cancer arising in BRCA1-mutation carriers to better understand the molecular mechanism of tumor progression and tested whether targeting of the Polycomb-group protein EZH2 would be a putative therapy for BRCA1-deficient tumors.ResultsGene expression analysis demonstrated that EZH2 is overexpressed in BRCA1-deficient mouse mammary tumors. By immunohistochemistry we show that an increase in EZH2 protein levels is also evident in tumors from BRCA1-mutation carriers. EZH2 is responsible for repression of genes driving differentiation and could thus be involved in the undifferentiated phenotype of these tumors. Importantly, we show that BRCA1-deficient cancer cells are selectively dependent on their elevated EZH2 levels. In addition, a chemical inhibitor of EZH2, 3-deazaneplanocin A (DZNep), is about 20-fold more effective in killing BRCA1-deficient cells compared to BRCA1-proficient mammary tumor cells.ConclusionsWe demonstrate by specific knock-down experiments that EZH2 overexpression is functionally relevant in BRCA1-deficient breast cancer cells. The effectiveness of a small molecule inhibitor indicates that EZH2 is a druggable target. The overexpression of EZH2 in all basal-like breast cancers warrants further investigation of the potential for targeting the genetic make-up of this particular breast cancer type.

Akt-mediated phosphorylation of Bmi1 modulates its oncogenic potential, E3 ligase activity, and DNA damage repair activity in mouse prostate cancer.

Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.

Intra‐ and inter‐tumor heterogeneity in a vemurafenib‐resistant melanoma patient and derived xenografts

The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAFV600E metastatic melanoma. However, resistance to such compounds represents a formidable problem. Using whole‐exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug‐resistant metastases. Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway. In addition to three independent amplifications and an aberrant form of BRAFV600E, we identified a new activating insertion in MEK1. This MEK1T55delinsRT mutation could be traced back to a fraction of the pre‐treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas. Analysis of patient‐derived xenografts (PDX) from therapy‐refractory metastases revealed that multiple resistance mechanisms were present within one metastasis. This heterogeneity, both inter‐ and intra‐tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient. In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting. Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patients melanoma.

Polycomb group gene Ezh2 regulates mammary gland morphogenesis and maintains the luminal progenitor pool

Specification of the cellular hierarchy in the mammary gland involves complex signaling that remains poorly defined. Polycomb group proteins are known to contribute to the maintenance of stem cell identity through epigenetic modifications, leading to stable alterations in gene expression. The polycomb protein family member EZH2 is known to be important for stem cell maintenance in multiple tissues, but its role in mammary gland development and differentiation remains unknown. Our analyses show that EZH2 is predominantly expressed in luminal cells of the mouse mammary epithelium. As mammary gland development occurs mostly after birth, the analysis of EZH2 gene function in postnatal development is precluded by embryonic lethality of conventional EZH2 knockout mice. To investigate the role of EZH2 in normal mammary gland epithelium, we have generated novel transgenic mice that express doxycycline‐regulatable short hairpin (sh) RNAs directed against Ezh2. Knockdown of EZH2 results in delayed outgrowth of the mammary epithelium during puberty, due to impaired terminal end bud formation and ductal elongation. Furthermore, our results demonstrate that EZH2 is required to maintain the luminal cell pool and may limit differentiation of luminal progenitors into CD61+ differentiated luminal cells, suggesting a role for EZH2 in mammary luminal cell fate determination. Consistent with this, EZH2 knockdown reduced lobuloalveolar expansion during pregnancy, suggesting EZH2 is required for the differentiation of luminal progenitors to alveolar cells.Stem Cells 2013;31:1910‐1920

BRAF(V600E) Kinase Domain Duplication Identified in Therapy-Refractory Melanoma Patient-Derived Xenografts.

Summary The therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established a large collection of patient-derived xenografts (PDXs), derived from BRAFV600E, NRASQ61, or BRAFWT/NRASWT melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. Taking advantage of PDXs as a limitless source, we screened tumor lysates for resistance mechanisms. We identified a BRAFV600E protein harboring a kinase domain duplication (BRAFV600E/DK) in ∼10% of the cases, both in PDXs and in an independent patient cohort. While BRAFV600E/DK depletion restored sensitivity to BRAF inhibition, a pan-RAF dimerization inhibitor effectively eliminated BRAFV600E/DK-expressing cells. These results illustrate the utility of this PDX platform and warrant clinical validation of BRAF dimerization inhibitors for this group of melanoma patients.

Deletion of Polycomb Repressive Complex 2 From Mouse Intestine Causes Loss of Stem Cells

BACKGROUND & AIMS The polycomb repressive complex 2 (PRC2) regulates differentiation by contributing to repression of gene expression and thereby stabilizing the fate of stem cells and their progeny. PRC2 helps to maintain adult stem cell populations, but little is known about its functions in intestinal stem cells. We studied phenotypes of mice with intestine-specific deletion of the PRC2 proteins embryonic ectoderm development (EED) (a subunit required for PRC2 function) and enhancer of zeste homolog 2 (EZH2) (a histone methyltransferase). METHODS We performed studies of AhCre;EedLoxP/LoxP (EED knockout) mice and AhCre;Ezh2LoxP/LoxP (EZH2 knockout) mice, which have intestine-specific disruption in EED and EZH2, respectively. Small intestinal crypts were isolated and subsequently cultured to grow organoids. Intestines and organoids were analyzed by immunohistochemical, in situ hybridization, RNA sequence, and chromatin immunoprecipitation methods. RESULTS Intestines of EED knockout mice had massive crypt degeneration and lower numbers of proliferating cells compared with wild-type control mice. Cdkn2a became derepressed and we detected increased levels of P21. We did not observe any differences between EZH2 knockout and control mice. Intestinal crypts from EED knockout mice had signs of aberrant differentiation of uncommitted crypt cells-these differentiated toward the secretory cell lineage. Furthermore, crypts from EED-knockout mice had impaired Wnt signaling and concomitant loss of intestinal stem cells, this phenotype was not reversed upon ectopic stimulation of Wnt and Notch signaling in organoids. Analysis of gene expression patterns from intestinal tissues of EED knockout mice showed dysregulation of several genes involved in Wnt signaling. Wnt signaling was regulated directly by PRC2. CONCLUSIONS In intestinal tissues of mice, PRC2 maintains small intestinal stem cells by promoting proliferation and preventing differentiation in the intestinal stem cell compartment. PRC2 controls gene expression in multiple signaling pathways that regulate intestinal homeostasis. Sequencing data are available in the genomics data repository GEO under reference series GSE81578; RNA sequencing data are available under subseries GSE81576; and ChIP sequencing data are available under subseries GSE81577.

GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors.

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16INK4A/Rb rather than on regulation of p19ARF/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.

Large variety in a panel of human colon cancer organoids in response to EZH2 inhibition

EZH2 inhibitors have gained great interest for their use as anti-cancer therapeutics. However, most research has focused on EZH2 mutant cancers and recently adverse effects of EZH2 inactivation have come to light. To determine whether colorectal cancer cells respond to EZH2 inhibition and to explore which factors influence the degree of response, we treated a panel of 20 organoid lines derived from human colon tumors with different concentrations of the EZH2 inhibitor GSK126. The resulting responses were associated with mutation status, gene expression and responses to other drugs. We found that the response to GSK126 treatment greatly varied between organoid lines. Response associated with the mutation status of ATRX and PAX2, and correlated with BIK expression. It also correlated well with response to Nutlin-3a which inhibits MDM2-p53 interaction thereby activating p53 signaling. Sensitivity to EZH2 ablation depended on the presence of wild type p53, as tumor organoids became resistant when p53 was mutated or knocked down. Our exploratory study provides insight into which genetic factors predict sensitivity to EZH2 inhibition. In addition, we show that the response to EZH2 inhibition requires wild type p53. We conclude that a subset of colorectal cancer patients may benefit from EZH2-targeting therapies.