Paul Savage

YAP-induced resistance of cancer cells to antitubulin drugs is modulated by a Hippo-independent pathway

Although antitubulin drugs are used widely to treat human cancer, many patients display intrinsic or acquired drug resistance that imposes major obstacles to successful therapy. Mounting evidence argues that cancer cell apoptosis triggered by antitubulin drugs relies upon activation of the cell-cycle kinase Cdk1; however, mechanistic connections of this event to apoptosis remain obscure. In this study, we identified the antiapoptotic protein YAP, a core component of the Hippo signaling pathway implicated in tumorigenesis, as a critical linker coupling Cdk1 activation to apoptosis in the antitubulin drug response. Antitubulin drugs activated Cdk1, which directly phosphorylated YAP on five sites independent of the Hippo pathway. Mutations in these phosphorylation sites on YAP relieved its ability to block antitubulin drug-induced apoptosis, further suggesting that YAP was inactivated by Cdk1 phosphorylation. Notably, we found that YAP was not phosphorylated and inactivated after antitubulin drug treatment in taxol-resistant cancer cells. Our findings suggest YAP and its phosphorylation status as candidate prognostic markers in predicting antitubulin drug response in patients.

Sumoylation of Krüppel-like Factor 4 Inhibits Pluripotency Induction but Promotes Adipocyte Differentiation

Background: It remains largely unexplored how post-translational modifications regulate reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Results: Substitution of the sole sumoylation site of the well known reprogramming factor KLF4 promotes iPS cell formation. Conclusion: KLF4 sumoylation inhibits iPS cell induction but stimulates adipocyte differentiation. Significance: The study highlights the importance of KLF4 sumoylation in regulating pluripotency and cell fate determination. Ectopic expression of transcription factors has been shown to reprogram somatic cells into induced pluripotent stem (iPS) cells. It remains largely unexplored how this process is regulated by post-translational modifications. Several reprogramming factors possess conserved sumoylation sites, so we investigated whether and how this modification regulates reprogramming of fibroblasts into iPS cells. Substitution of the sole sumoylation site of the Krüppel-like factor (KLF4), a well known reprogramming factor, promoted iPS cell formation. In comparison, much smaller effects on reprogramming were observed for sumoylation-deficient mutants of SOX2 and OCT4, two other classical reprogramming factors. We also analyzed KLF2, a KLF4 homolog and a member of the KLF family of transcription factors with a known role in reprogramming. KLF2 was sumoylated at two conserved neighboring motifs, but substitution of the key lysine residues only stimulated reprogramming slightly. KLF5 is another KLF member with an established link to embryonic stem cell pluripotency. Interestingly, although it was much more efficiently sumoylated than either KLF2 or KLF4, KLF5 was inactive in reprogramming, and its sumoylation was not responsible for this deficiency. Furthermore, sumoylation of KLF4 but not KLF2 or KLF5 stimulated adipocyte differentiation. These results thus demonstrate the importance KLF4 sumoylation in regulating pluripotency and adipocyte differentiation.

The SUMO Conjugating Enzyme Ubc9 Is Required for Inducing and Maintaining Stem Cell Pluripotency

Sumoylation adds a small ubiquitin‐like modifier (SUMO) polypeptide to the ε‐amino group of a lysine residue. Reminiscent of ubiquitination, sumoylation is catalyzed by an enzymatic cascade composed of E1, E2, and E3. For sumoylation, this cascade uses Ubc9 (ubiquitin conjugating enzyme 9, now officially named ubiquitin conjugating enzyme E2I [UBE2I]) as the sole E2 enzyme. Here, we report that expression of endogenous Ubc9 increases during reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. In addition, this E2 enzyme is required for reprogramming as its suppression dramatically inhibits iPS cell induction. While Ubc9 knockdown does not affect survival of MEFs and immortalized fibroblasts, Ubc9 is essential for embryonic stem cell (ESC) survival. In addition, we have found that Ubc9 knockdown stimulates apoptosis in ESCs but not in MEFs. Furthermore, the knockdown decreases the expression of the well‐known pluripotency marker Nanog and the classical reprogramming factors Klf4, Oct4, and Sox2 in ESCs. Together, these observations indicate that while dispensable for fibroblast survival, the sole SUMO E2 enzyme Ubc9 plays a critical role in reprogramming fibroblasts to iPS cells and maintaining ESC pluripotency. Stem Cells 2014;32:1012–1020

ERRα mediates metabolic adaptations driving lapatinib resistance in breast cancer

Despite the initial benefits of treating HER2-amplified breast cancer patients with the tyrosine kinase inhibitor lapatinib, resistance inevitably develops. Here we report that lapatinib induces the degradation of the nuclear receptor ERRα, a master regulator of cellular metabolism, and that the expression of ERRα is restored in lapatinib-resistant breast cancer cells through reactivation of mTOR signalling. Re-expression of ERRα in resistant cells triggers metabolic adaptations favouring mitochondrial energy metabolism through increased glutamine metabolism, as well as ROS detoxification required for cell survival under therapeutic stress conditions. An ERRα inverse agonist counteracts these metabolic adaptations and overcomes lapatinib resistance in a HER2-induced mammary tumour mouse model. This work reveals a molecular mechanism by which ERRα-induced metabolic reprogramming promotes survival of lapatinib-resistant cancer cells and demonstrates the potential of ERRα inhibition as an effective adjuvant therapy in poor outcome HER2-positive breast cancer.

A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer

Therapies targeting epidermal growth factor receptor (EGFR) have variable and unpredictable responses in breast cancer. Screening triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), we identify a subset responsive to EGFR inhibition by gefitinib, which displays heterogeneous expression of wild-type EGFR. Deep single-cell RNA sequencing of 3,500 cells from an exceptional responder identified subpopulations displaying distinct biological features, where elevated EGFR expression was significantly enriched in a mesenchymal/stem-like cellular cluster. Sorted EGFRhi subpopulations exhibited enhanced stem-like features, including ALDH activity, sphere-forming efficiency, and tumorigenic and metastatic potential. EGFRhi cells gave rise to EGFRhi and EGFRlo cells in primary and metastatic tumors, demonstrating an EGFR-dependent expansion and hierarchical state transition. Similar tumorigenic EGFRhi subpopulations were identified in independent PDXs, where heterogeneous EGFR expression correlated with gefitinib sensitivity. This provides new understanding for an EGFR-dependent hierarchy in TNBC and for patient stratification for therapeutic intervention.

The Receptor Tyrosine Kinase AXL is Required at Multiple Steps of the Metastatic Cascade during HER2-positive Breast Cancer Progression

AXL is activated by its ligand GAS6 and is expressed in triple-negative breast cancer cells. In the current study, we report AXL expression in HER2-positive (HER2+) breast cancers where it correlates with poor patient survival. Using murine models of HER2+ breast cancer, Axl, but not its ligand Gas6, was found to be essential for metastasis. We determined that AXL is required for intravasation, extravasation, and growth at the metastatic site. We found that AXL is expressed in HER2+ cancers displaying epithelial-to-mesenchymal transition (EMT) signatures where it contributes to sustain EMT. Interfering with AXL in a patient-derived xenograft (PDX) impaired transforming growth factor β (TGF-β)-induced cell invasion. Last, pharmacological inhibition of AXL specifically decreased the metastatic burden of mice developing HER2+ breast cancer. Our data identify AXL as a potential anti-metastatic co-therapeutic target for the treatment of HER2+ breast cancers.

Targeting EZH2 reactivates a breast cancer subtype-specific anti-metastatic transcriptional program

Emerging evidence has illustrated the importance of epigenomic reprogramming in cancer, with altered post-translational modifications of histones contributing to pathogenesis. However, the contributions of histone modifiers to breast cancer progression are unclear, and how these processes vary between molecular subtypes has yet to be adequately addressed. Here we report that genetic or pharmacological targeting of the epigenetic modifier Ezh2 dramatically hinders metastatic behaviour in both a mouse model of breast cancer and patient-derived xenografts reflective of the Luminal B subtype. We further define a subtype-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the FOXC1 gene, thereby inactivating a FOXC1-driven, anti-invasive transcriptional program. We demonstrate that higher FOXC1 is predictive of favourable outcome specifically in Luminal B breast cancer patients and establish the use of EZH2 methyltransferase inhibitors as a viable strategy to block metastasis in Luminal B breast cancer, where options for targeted therapy are limited.Histone modifications in cancer can contribute to pathogenesis. Here, the authors demonstrate that targeting epigenetic modifier Ezh2 hinders metastatic behaviour in Luminal B breast cancer models, and highlight a mechanism where Ezh2 contributes to metastatic behaviour by repression of FOXC1.

Dual MAPK Inhibition Is an Effective Therapeutic Strategy for a Subset of Class II BRAF Mutant Melanomas

Purpose: Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi. Experimental Design: Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi. Results: BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi. Conclusions: Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma. See related commentary by Johnson and Dahlman, p. 6107.

Abstract 2177: Sensitive single cell copy number profiling using a novel microfluidic droplet based platform

Intratumoral genetic heterogeneity may relate to aggressiveness and treatment-resistance. However, the genomic abnormalities underlie this heterogeneity remain poorly characterized. To better describe the picture of tumour heterogeneity, single cell approaches to accommodate larger number of cells are highly in demand. Here, we present results of single cell DNA sequencing by using a novel microfluidic droplet system. We applied this approach to discover copy number profiles in two common and therapeutically challenging cancers, isocitrate dehydrogenase wild-type glioblastoma (GBM) and breast cancer. The GBM cells were extracted from patients operated on site and cultured for 2 months in restricted media to isolate glioma stem cells. The breast cancer cells were extracted from patient-derived xenografts (PDXs). A novel microfluidic droplet based platform (10x GenomicsTM) was used to perform sensitive copy number profiling of thousands of single cells simultaneously. Briefly, the platform integrates single cell encapsulation, cell lysis, genomic amplification, and barcoding with oligonucleotides. The barcoded genomic DNA fragments from individual cells were pooled and converted into libraries that are compatible with Illumina sequencers. Thus far, we sequenced one glioma stem cell sample and two breast cancer PDXs. Uniquely mapped reads were counted in variable-bins with GC bias corrected by lowess fit. The Circular Binary Segmentation was performed and the integer copy number was estimated by least square fitting. The single cell copy number profiles were clustered by hierarchical clustering. We achieved approximately 2% and 6% coverage of the genome per cell for the glioblastoma and the breast tumors, respectively. The uniformity of coverage was comparable to other whole genome amplification and single cell multiplex approaches, while requiring 700k-2.1mi reads/single cell with the median absolute deviation of pair-wise differences in read counts between neighboring bins between 0.17-0.21 vs 0.16)1. We identified two major clonal subpopulations in the glioblastoma sample. One subclone had chr1q amplification and the other subclone had a 9.8Mb deletion on chr4. The clonal CNVs observed were validated using single-cell RNA-seq-based CNV analysis, and the frequent CNVs using fluorescent in situ hybridization. We also identified the HER2 copy number profile within a basal like triple-negative breast cancer as 40% of 2.5N and 25% of 3N. This was previously validated by fluorescence in situ hybridization2. By applying this new microfluidic system, our work will provide more insights into genomic heterogeneity for malignant properties of cancers. 1. Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90-94 (2011). 2. Savage P. et al. A Targetable EGFR-Dependent Tumor-Initiating Program in Breast Cancer. Cell Rep. 21, 1140-1149 (2017). Citation Format: Rui Li, Charles Couturier, Paul Savage, Jean Monlong, Guillaume Bourque, Kevin Petrecca, Morag Park, Jiannis Ragoussis. Sensitive single cell copy number profiling using a novel microfluidic droplet based platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2177.

Abstract 2386: Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition

Human breast tumors have been shown to exhibit extensive inter- and intra-tumor heterogeneity. While recent advances in genomic technologies have allowed us to deconvolute this heterogeneity, few studies have addressed the functional consequences of diversity within tumor populations. Here, we identified an index case for which we have derived a patient-derived xenograft (PDX) as a renewable tissue source to identify subpopulations and perform functional assays. On pathology, the tumor was an invasive ductal carcinoma which was hormone receptor-negative, HER2-positive (IHC 2+, FISH average HER2/CEP17 2.4), though the FISH signal was noted to be heterogeneous. On gene expression profiling of bulk samples, the primary tumor and PDX were classified as basal-like. We performed single cell RNA and exome sequencing of the PDX to identify population structure. Using a single sample predictor of breast cancer subtype, we have identified single basal-like, HER2-enriched and normal-like cells co-existing within the PDX tumor, a finding replicated in several independent experiments. Genes differentially expressed between these subpopulations are involved in proliferation and differentiation. One population was characterized by high MYC and the other high EGFR/KRT14. These findings were validated using immunohistochemistry on PDX and primary tumor material. Further functional experiments showed that that EGFR subpopulation has stem cell character istics and forms metastasis in the animal model. Microfluidic whole genome amplification followed by whole exome capture of 81 single cells, along with exome sequencing of the germline, primary and post treatment tumor and whole PDX showed that BRCA1 and TP53 are mutated in all single cells, as well as a number of sub-clonal mutations that are being investigated further. Loss of heterozygocity was observed in 16 TCGA cancer driver genes and novel mutations in 7 known cancer driver genes. Careful comparison of the exome sequencing data allowed the association of driver gene mutation prevalence with tumor progression. These findings are important in our understanding the functional consequences of intra-tumor heterogeneity with respect to clinically important phenotypes such as invasion, metastasis and drug-resistance. Citation Format: Ioannis Ragoussis, Paul Savage, Yu-Chang Wang, Timothee Revil, Dunarel Badescu, Sadiq Saleh, Ernesto Iacucci, Nicolas Bertos, Anie Monast, Attila Omeroglou, Dongmei Zuo, Morag Park. Microfluidic single cell exome-seq and RNA-seq analysis of tumor composition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2386.