High throughput spheroid screen results (z scores) in the MCF10 progression series. Gene rank for the validation screen (average z scores across cell lines).

Enrichment analysis from TCGA RNA-seq and RPPA data comparing CREBBPaltered versus wild-type patients.

Supplementary Materials and Methods

Supplementary Figures and legends

Gene list for the spheroid screen

Phosphorylated, acetylated peptides and total proteins identified in HAP1 CREBBP WT and mutant spheroids.

Validation screen of the top 50 ranked genes in 2D and 3D.

Abstract Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo–like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. Significance: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Solid tumors display significant histological, genetic and micro-environmental intra-tumor heterogeneity that can change substantially over the course of their evolutionary trajectory. In particular, changes in the micro-environmental complexity within breast cancer such as hypoxic and nutrient deplete environments are associated with aggressive disease and a poor patient outcome. We sought to identify novel driver alterations in aggressive disease by employing a functional genomics screen in a 3-dimensional model of breast cancer progression that more accurately recapitulates in vivo micro-environmental heterogeneity. Screening of the top 200 recurrently mutated genes in breast cancer in cancer cell line spheroids identified several genes whose silencing impacted growth. A second targeted validation screen in a larger panel of triple negative cell line models showed that silencing of the histone acetyltransferase CREBBP, promoted growth in 3D but had limited effect under traditional 2D culture conditions. Investigation of TCGA and METABRIC datasets showed that CREBBP was more frequently mutated in triple negative breast cancers (TNBCs) and at least a third of TNBCs also displayed gene haploinsufficiency or complete loss of CREBBP. Interrogation of expression and proteomic datasets showed that loss of CREBBP resulted in the upregulation of the pro-proliferative transcription factor FOXM1. Significantly, this conserved FOXM1-driven transcriptional programme was also seen in multiple solid tumors with CREBBP alterations including lung, oesophageal, bladder and endometrial cancers. This was recapitulated in several CREBBP deficient cells where we identified that FOXM1 is driving altered metabolism, allowing cancer cells to grow under nutrient stress conditions. In summary, CREBBP is a bona fide tumor suppressor in up to a third of TNBCs, as well as a wide range of other solid tumors. CREBBP-altered tumors display up-regulation of FOXM1, which alters cancer cell metabolism under nutrient stress conditions. Moreover, CREBBP-altered tumors are selectively sensitive to small molecule inhibitors that target FOXM1 activity, suggesting that this maybe a viable targeted therapeutic approach for CREBBP altered cancers. Citation Format: Barrie Peck, Philip J. Bland, Patty T. Wai, Hannah Cottom, Sarah L. Maguire, Eamonn Morrison, Holly E. Barker, Divya Kriplani, Rebecca Marlow, Kalnisha Naidoo, Gareth Muirhead, Syed Haider, Frances Daley, Frederik Wallberg, Andrew N. Tutt, Rachael C. Natrajan. Modeling tumor microenvironmental heterogeneity identifies CREBBP as a novel tumor suppressor in breast cancer [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 788.

Recent next generation sequencing studies have comprehensively mapped the genetic landscape of breast cancer and revealed that only a small number of genes are recurrently mutated in more than 10% of unselected tumors (i.e. TP53, PIK3CA and GATA3), and that the vast majority of recurrent mutations occur at low frequencies. Although some have been shown to be drivers (i.e. confer a selective advantage), such as oncogenic ERBB2 mutations, there is a myriad of significantly altered lower frequency mutations whose functional impact is unknown. We utilized a functional genomics approach silencing the 200 most frequently mutated genes in breast cancer in 3D spheroid cultures that more accurately recapitulate in vivo like conditions, using the MCF10A progression series cell line panel to identify novel loss of function mutations that affect breast cancer progression from non-malignant to highly invasive disease. Genes whose silencing significantly altered spheroid growth were integrated with comprehensive copy number and mutation data in order to analyze the impact of these genes in concert with additional driver alterations in genes such as TP53 and PIK3CA mutations. We identified 11 genes whose silencing with siRNA had a significant effect on growth in two or more cell lines in 3D, including FMN2, FOXA1, NIPBL and CREBBP. Silencing of FMN2 increased spheroid growth in the invasive cell lines only, suggesting loss of function mutations are a later event in breast cancer progression. A second targeted validation screen showed that silencing of a cohort of these genes had limited effect under traditional 2D culture conditions, for example, silencing of maltase-glucoamylase (MGAM) resulted in increased growth in AT1 and DCIS.com cells in 3D while having no effect in 2D; an effect that was recapitulated by treating cells with an established MGAM inhibitor. Furthermore, loss of NIPBL significantly increased spheroid growth in cells harboring TP53 nuclear accumulation, and was significantly co-mutated in TP53 mutant primary tumors, suggestive of epistasis. Integrating genes that when silenced decreased spheroid growth with mutation status in the cell lines identified 3D specific oncogenic dependencies with PIK3CA and novel SZT2 mutations. Using a functional genomic approach in 3D models we have identified recurrently mutated genes whose loss or gain of function contribute to breast cancer progression and furthermore may be epistatic or cooperate with established driver mutations in breast cancer. Citation Format: Barrie Peck, Sarah Maguire, Eamonn Morrison, Patty Wai, Rachael Natrajan. Modeling the tumor microenvironment to identify novel loss of function mutations in breast cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR06.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials. Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.