Larissa S. Carnevalli

AstraZeneca (United Kingdom)

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Simon T. Barry

AstraZeneca (United Kingdom)

Pablo Morentin Gutierrez

AstraZeneca (United Kingdom)

Teresa Klinowska

AstraZeneca (United Kingdom)

Mandy Lawson

AstraZeneca (United Kingdom)

Adina Hughes

AstraZeneca (United Kingdom)

Susan E. Critchlow

AstraZeneca (United Kingdom)

Oona Delpuech

AstraZeneca (United Kingdom)

Neha Wali

Wellcome Sanger Institute

Azadeh Cheraghchi-Bashi

AstraZeneca (United Kingdom)

Daniel Sutton

AstraZeneca (United Kingdom)

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AstraZeneca (United Kingdom)

182

University of Cambridge

108

AstraZeneca (United States)

Cancer Research UK

Cancer Research UK Cambridge Center

AstraZeneca (Brazil)

Engineering Arts (United States)

Howard Hughes Medical Institute

Addenbrooke's Hospital

Cambridge University Hospitals NHS Foundation Trust

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Supplementary Materials and Methods and Supplementary Tables 1-2 from Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition

Larissa S. Carnevalli Molly A. Taylor Matthew King Anna Coenen-Stass Adina Hughes

Supplementary Materials and Methods. Suplementary Table 1. Tumor Immuno-phenotyping. Suplementary Table 2. Flow cytometry panels.

Table (database)

10.1158/1535-7163.22522330.v1

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Dissecting the early steps of MLL induced leukaemogenic transformation using a mouse model of AML

Nature Communications (2020)

Silvia Basilico Xiaonan Wang Alison Kennedy Konstantinos Tzelepis George Giotopoulos

Abstract Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 ( Kmt2a ) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.

Malignant Transformation

10.1038/s41467-020-15220-0

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Citations (54)

PO-491 Single-cell phenotypic profiling of breast cancerpatient-derived tumour xenografts using mass cytometry

ESMO Open (2018)

Dimitra Georgopoulou Maurizio Callari Alistair Martin Oscar M. Rueda Wendy Greenwood

Introduction Despite the advances in patient stratification and the understanding of basic molecular mechanisms underlining breast cancer (BC), 30% of patients with early stages of the disease later relapse. To offer more effective treatments to these patients, we need to adapt pre-clinical drug development to account for the main feature of human cancer: heterogeneity. Our group has established a biobank of 160 BC patient-derived tumour xenografts (PDTXs) and has robustly shown that PDTXs and their matching patient-derived tumour cells (PDTCs) preserve most of the originating sample’s molecular features, including intra-tumour clonal diversity (Bruna et al. Cell 2016). This integrated platform permits high-throughput (HT) assessment of drug responses ex vivo in PDTCs and their validation in vivo in PDTXs. Thus, this PDTX/PDTC biobank represents a powerful resource for pre-clinical drug development in BC. Material and methods Here, we aim to add a crucial layer of molecular characterisation at single-cell resolution by studying the phenotypic tumour heterogeneity in BC PDTXs and its role in drug response mechanisms. To achieve this goal, we employed the state-of-the-art HT single-cell method called mass cytometry (CYTOF), which combines the principles of flow cytometry and mass spectrometry allowing the simultaneous analysis of about 40 molecular events using metal-conjugated antibodies. Results and discussions We designed and validated a BC-specific panel of 35 antibodies to investigate: a)the epithelial tumour compartment; b)the stroma tumour compartment; c)oncogenic signalling; d)cell cycle and apoptosis. We used this panel in CyTOF experiments to perform an in-depth phenotypic characterisation of a representative collection of BC cell lines and molecularly distinct PDTXs. Using data-driven unsupervised methods to analyse the CyTOF data focusing on the epithelial compartment, we show that we capture different degrees of both inter- and intra-tumour functional heterogeneity. Importantly, we show that CyTOF can be used to characterise the heterogeneity in signalling dynamics within the different tumour compartments under the perturbation of therapy. Conclusion CyTOF is a novel approach to characterise heterogeneity and drug responses in BC PDTXs. The integration of high-level features, such as single-cell phenotypic profiles, and functional features, such as drug response data, obtained with CyTOF will facilitate the pre-clinical development of novel therapeutic strategies and will help to uncover mechanisms of drug resistance.

Mass cytometry

Profiling (computer programming)

Cytometry

10.1136/esmoopen-2018-eacr25.508

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Figure 4 from The Next-Generation Oral Selective Estrogen Receptor Degrader Camizestrant (AZD9833) Suppresses ER+ Breast Cancer Growth and Overcomes Endocrine and CDK4/6 Inhibitor Resistance

Mandy Lawson Natalie Cureton Susana R�os Azadeh Cheraghchi-Bashi Jelena Urosevic

Camizestrant has superior in vivo activity to fulvestrant in ESR1wt and ESR1m PDX models (<a href="#bib2" target="_blank">2</a>). A, Characteristics of ER+ breast cancer models used. B, Change in ER pathway gene activation after treatment, expressed as change in ER pathway gene score and in cell-cycle G1–S checkpoint genes. See Supplementary Methods for details. Statistical analysis comparing fulvestrant and camizestrant was done using one-way analysis of covariance (n ≥ 4 animals per group). Models shown in bold (x-axis) are fulvestrant sensitive; those in regular type are fulvestrant resistant. ***, P < 0.001; ****, P < 0.0001. Amp, amplification; CCND1, cyclin D1; Del, deletion; MET, metastasis; Mut, mutation; PIK3CA, phosphatidylinositol 3-kinase subunit α; PR, progesterone receptor; PRIM, primary; RESIST, resistant; RB1, retinoblastoma gene; SENS, sensitive. Patient treatment reported: Be, bevacizumab; Ch, chemotherapy; E, exemestane; Ev, everolimus; F, fulvestrant; I, investigational; L, letrozole, T, tamoxifen; X, radiotherapy.

Fulvestrant

Palbociclib

10.1158/0008-5472.24710686.v1

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Clonal somatic copy number altered driver events inform drug sensitivity in high-grade serous ovarian cancer

Zenodo (CERN European Organization for Nuclear Research) (2022)

Filipe Correia Martins Dominique‐Laurent Couturier Ines de Santiago Carolin M. Sauer Maria Vias

Serous ovarian cancer

10.5281/zenodo.7094277

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Functional significance of co-occurring mutations in PIK3CA and MAP3K1 in breast cancer

Oncotarget (2018)

Alvaro Avivar‐Valderas Robert McEwen Amir Taheri‐Ghahfarokhi Larissa S. Carnevalli Elizabeth Hardaker

The PI3Kα signaling pathway is frequently hyper-activated in breast cancer (BrCa), as a result of mutations/amplifications in oncogenes (e.g. HER2), decreased function in tumor suppressors (e.g. PTEN) or activating mutations in key components of the pathway. In particular, activating mutations of PIK3CA (~45%) are frequently found in luminal A BrCa samples. Genomic studies have uncovered inactivating mutations in MAP3K1 (13-20%) and MAP2K4 (~8%), two upstream kinases of the JNK apoptotic pathway in luminal A BrCa samples. Further, simultaneous mutation of PIK3CA and MAP3K1 are found in ~11% of mutant PIK3CA tumors. How these two alterations may cooperate to elicit tumorigenesis and impact the sensitivity to PI3K and AKT inhibitors is currently unknown. Using CRISPR gene editing we have genetically disrupted MAP3K1 expression in mutant PIK3CA cell lines to specifically create in vitro models reflecting the mutational status of PIK3CA and MAP3K1 in BrCa patients. MAP3K1 deficient cell lines exhibited ~2.4-fold increased proliferation rate and decreased sensitivity to PI3Kα/δ(AZD8835) and AKT (AZD5363) inhibitors (~2.61 and ~5.23-fold IC50 increases, respectively) compared with parental control cell lines. In addition, mechanistic analysis revealed that MAP3K1 disruption enhances AKT phosphorylation and downstream signaling and reduces sensitivity to AZD5363-mediated pathway inhibition. This appears to be a consequence of deficient MAP3K1-JNK signaling increasing IRS1 stability and therefore promoting IRS1 binding to p85, resulting in enhanced PI3Kα activity. Using 3D-MCF10A-PI3KαH1047R models, we found that MAP3K1 depletion increased overall acinar volume and counteracted AZD5363-mediated reduction of acinar growth due to enhanced proliferation and reduced apoptosis. Furthermore, in vivo efficacy studies revealed that MAP3K1-deficient MCF7 tumors were less sensitive to AKT inhibitor treatment, compared with parental MCF7 tumors. Our study provides mechanistic and in vivo evidence indicating a role for MAP3K1 as a tumor suppressor gene at least in the context of PIK3CA-mutant backgrounds. Further, our work predicts that MAP3K1 mutational status may be considered as a predictive biomarker for efficacy in PI3K pathway inhibitor trials.

10.18632/oncotarget.25118

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Combination of dual mTORC1/2 inhibition and immune-checkpoint blockade potentiates anti-tumour immunity

OncoImmunology (2018)

Sophie Langdon Adina Hughes Molly A. Taylor Elizabeth A. Kuczynski Deanna A. Mele

mTOR inhibition can promote or inhibit immune responses in a context dependent manner, but whether this will represent a net benefit or be contraindicated in the context of immunooncology therapies is less understood. Here, we report that the mTORC1/2 dual kinase inhibitor vistusertib (AZD2014) potentiates anti-tumour immunity in combination with anti-CTLA-4 (αCTLA-4), αPD-1 or αPD-L1 immune checkpoint blockade. Combination of vistusertib and immune checkpoint blocking antibodies led to tumour growth inhibition and improved survival of MC-38 or CT-26 pre-clinical syngeneic tumour models, whereas monotherapies were less effective. Underlying these combinatorial effects, vistusertib/immune checkpoint combinations reduced the occurrence of exhausted phenotype tumour infiltrating lymphocytes (TILs), whilst increasing frequencies of activated Th1 polarized T-cells in tumours. Vistusertib alone was shown to promote a Th1 polarizing proinflammatory cytokine profile by innate primary immune cells. Moreover, vistusertib directly enhanced activation of effector T-cell and survival, an effect that was critically dependent on inhibitor dose. Therefore, these data highlight direct, tumour-relevant immune potentiating benefits of mTOR inhibition that complement immune checkpoint blockade. Together, these data provide a clear rationale to investigate such combinations in the clinic.

Immune checkpoint

Proinflammatory cytokine

10.1080/2162402x.2018.1458810

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Citations (45)

S6K1 Plays a Critical Role in Early Adipocyte Differentiation

Developmental Cell (2010)

Larissa S. Carnevalli Kouhei Masuda Francesca Frigerio Olivier Le Bacquer Sung Hee Um

Lipid droplet

Progenitor

Perilipin

10.1016/j.devcel.2010.02.018

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Citations (186)

Genome engineering for estrogen receptor mutations reveals differential responses to anti-estrogens and new prognostic gene signatures for breast cancer

Oncogene (2022)

Alison Harrod Chun‐Fui Lai Isabella Goldsbrough Georgia M. Simmons Natasha Oppermans

Abstract Mutations in the estrogen receptor (ESR1) gene are common in ER-positive breast cancer patients who progress on endocrine therapies. Most mutations localise to just three residues at, or near, the C-terminal helix 12 of the hormone binding domain, at leucine-536, tyrosine-537 and aspartate-538. To investigate these mutations, we have used CRISPR-Cas9 mediated genome engineering to generate a comprehensive set of isogenic mutant breast cancer cell lines. Our results confirm that L536R, Y537C, Y537N, Y537S and D538G mutations confer estrogen-independent growth in breast cancer cells. Growth assays show mutation-specific reductions in sensitivities to drugs representing three classes of clinical anti-estrogens. These differential mutation- and drug-selectivity profiles have implications for treatment choices following clinical emergence of ER mutations. Our results further suggest that mutant expression levels may be determinants of the degree of resistance to some anti-estrogens. Differential gene expression analysis demonstrates up-regulation of estrogen-responsive genes, as expected, but also reveals that enrichment for interferon-regulated gene expression is a common feature of all mutations. Finally, a new gene signature developed from the gene expression profiles in ER mutant cells predicts clinical response in breast cancer patients with ER mutations.

Estrogen receptor alpha

10.1038/s41388-022-02483-8

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STAT3Antisense Oligonucleotide Remodels the Suppressive Tumor Microenvironment to Enhance Immune Activation in Combination with Anti–PD-L1

Clinical Cancer Research (2020)

Theresa A. Proia Maneesh Singh Richard Woessner Larissa S. Carnevalli Gayathri Bommakanti

Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action.Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen.Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti-PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti-PD-L1.STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Proinflammatory cytokine

10.1158/1078-0432.ccr-20-1066

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Citations (41)