Cancer Biology Dynamical Cell Systems

The Institute of Cancer Research PHD STUDENTSHIP PROJECT PROPOSAL PROJECT DETAILS Project Title: SUPERVISORY TEAM Primary Supervisor: The forces behind pancreatic cancer; and changing them as a therapeutic approach. Chris Bakal Other supervisory team members: Fernando Calvo, Vicky Bousgouni, Caroline Springer Lead contact person for the project: DIVISIONAL AFFILIATION Primary Division: Primary Team: PROJECT PROPOSAL Chris Bakal Cancer Biology Dynamical Cell Systems BACKGROUND TO THE PROJECT (up to 300 words) My lab is studying how signalling networks regulate the fate decisions made by cancer cells. We are particularly interested in understanding how tumour microenvironments drive tumorigenesis, and metastasis, by activating the nuclear factor kappab/rela transcription factor. Although NF-kappaB is clearly a driver of oncogenesis [1], attempts to target this pathway directly have to date proven largely unsuccessful [2]. Using quantitative single-cell imaging approaches we recently discovered that NF-kappaB activity is regulated by mechanical cues such as adhesion, contractility, actin polymerization, as well as by cell shape [3]. These mechanical and geometrical thus regulate processes controlled by NF-kappaB, such survival, proliferation, stemness, and the Epithelial-to-Mesenchymal Transition (EMT). Because remodelling of the extracellular matrix (ECM), disruption of tissue architecture, alteration of mechanical forces, and changes in cell shape are characteristic of almost all solid tumours [4], we have proposed that mechanical activation of NF-kappaB may represent an important axis underpinning disease progression. Supporting this idea, through a series of bioinformatic studies, we have shown that NF-kappaB activation by mechanotransduction correlates with increased tumour aggressiveness, and poor prognosis in patients (Sailem et al. Genome Research, accepted). In this PhD project we aim to describe the role of mechanical forces and cell shape in regulating NF-kappaB activity in Pancreatic Ductal Adenocarcinoma (PDAC). NF-kappaB has a dual role in the progression to PDAC. Early in the development of disease, NF-kappaB acts as a tumour suppressor by promoting oncogene induced senescence (OIS), but then in full-blown PDAC NF-kappaB is an oncogene [5]. Because of the highly fibrotic nature of PDAC, and that mechanical forces clearly have a role in regulating PDAC progression [6], studying the role of NF-kappaB as an effector of mechanotransduction is warranted, and will open up therapeutic avenues. PROJECT AIMS Page 1 of 5

Aim 1a: Quantify the dynamics of NF-kappaB (p65rela) nuclear translocation, and thus activity, in single living pancreatic tumour cells in mouse models of PDAC by intravital imaging. Aim 1b: Validate models of NF-kappaB activation by mechanical forces in genetically engineered mouse models (GEMMs) of PDAC. Aim 2: Determine if the role of NF-kappaB in promoting senescence, and/or tumour proliferation, is regulated by mechanical forces. Aim 3: Identify clinically relevant small-molecules targeting mechanical signals that affect NF-kappaB dynamics in tumour cells in vivo. Aim 4: Determine whether manipulation of mechanical forces that promote NF-kappaB activation during PDAC can enhance immunotherapy. RESEARCH PROPOSAL We have the following questions regarding NF-kappaB activation in pancreatic cancer: How is the activation of NF-kappaB in both pancreatic cancer cells in vivo, affected by mechanical cues in tumour microenvironments such as; i) the stiffness of the extracellular matrix (ECM); ii) cell-cell adhesion; iii) cell shape and cytoskeletal organization? Do mechanical signals differentially affect the threshold and/or dynamics of NF-kappaB activity to promote senescence versus proliferation? Does therapeutic manipulation of mechanical forces alter NF-kappaB activity to inhibit tumour cell survival and/or promote senescence? Can alteration of the mechanical forces that promote NF-kappaB activity render PDAC tumour more sensitive to immune checkpoint inhibitors? To answer these questions this project has 4 aims: Aim 1a: Quantify the dynamics of NF-kappaB (p65rela) nuclear translocation, and thus activity, in single living pancreatic tumour cells in mouse models of PDAC by intravital imaging. In this aim, the student will generate tumours of pancreatic cancer cells that express a fluorescently tagged version of NF-kappaB/p65RelA, and image NF-kappa dynamics in single, living tumour cells in vivo by intravital microscopy. Using CRISPR/Cas9-based methods we have already generated reporter cell lines where the endogenous NF-kappaB (p65rela) protein has been tagged with fluorescent proteins. Page 2 of 5

To complete this aim, the student will also develop new automated image analysis methods to quantify single cell phenotypes in 2D and 3D cultures to quantify NF-kappaB nuclear translocation in single cells, the shape of these cells, and microenvironmental factors (such as number of neighbours, amount of free space). By understanding how NF-kappaB activity correlates with cell shape, cell-cell/cell-ecm adhesions, and cell position in the tumour (i.e. whether the tumour cell is in the cell body, near the invasive front, in contact with stromal cells, or attached to ECM such as collagen), we can gain insight into how mechanical signals regulate NF-kappaB in vivo. These experiments in this aim will be done in orthotopic models of PDAC because this will facilitate the imaging of different reporter lines which can be created rapidly. In contrast, to perform live cell imaging experiments in genetically engineered mouse models (GEMMs) of PDAC would first require derivation of a mouse where all cells express a tagged version of NF-kappaB, which would then be crossed with PDAC models. All mouse imaging experiments will be performed in collaboration with Caroline Springer and Fernando Calvo. We will collaborate with the Springer laboratory to both generate and treat (Aim 3) PDAC tumours, and we will collaborate with Calvo laboratory to perform intravital imaging. Aim 1b: Validate models of NF-kappaB activation by mechanical forces in genetically engineered mouse models (GEMMs) of PDAC. Although performing live tumour cell imaging of NF-kappaB dynamics in PDAC GEMMs is currently unfeasible, we will perform quantitative imaging of fixed tumours generating in GEMMs to validate findings in Aim 1a. Specifically, the student will quantify the relationship between NF-kappaB nuclear translocation, and cell-cell/cell- ECM adhesion, cell shape, and position by imaging fixed tumours generated in LSL-Kras G12D Ptf1a-Cre ex1 mice Outcome: The completion of this aim will result in understanding of how NF-kappaB dynamics are regulated by mechanical and geometrical cues to either suppress, or enhance, pancreatic tumorigenesis. Aim 2: Determine if the role of NF-kappaB in promoting senescence, and/or tumour proliferation, is regulated by mechanical forces. We hypothesize that NF-kappaB may respond differentially to changes in mechanical forces to promote senescence in one context (low fibrosis, and thus low tension/stiffness), but proliferation in others (high fibrosis, high tension/stiffness). The student will make use of NF-kappaB reporter lines generated in Aim 1, and monitor the expression of markers of senescence (i.e. senescence-associated β-galactosidase (SA-β-Gal), p16, p18, p53), or proliferation (Cyclin D/E), following the manipulation of mechanical forces in 2D and 3D settings. Many of these experiments will be similar to those we have previously performed on breast cancer cells [7]. Briefly the student will: manipulate ECM geometry, stiffness, and type (collagen vs. fibronectin) force changes in cell shape by micropatterns alter cytoskeletal tension and adhesion by well-characterized small-molecules inhibitors (i.e. ROCK, FAK, Src, inhibitors) or by genetic depletion of mechanotransducers Page 3 of 5

To facilitate completion of this aim, we have excellent relationships with experts in both senescence (Jesus Gil, MRC Hammersmith) and biological engineering (Molly Stevens, Imperial College) who can provide reagents, assistance and expertise. The outcome of these experiments will be a mechanistic understanding of how mechanical forces regulate NFkappaB dynamics to determine cell fates. Aim 3: Identify clinically relevant small-molecules targeting mechanical signals that affect NF-kappaB dynamics in tumour cells in vivo. We will test if the treatment of NF-kappaB reporter line tumours with small-molecules which affect different mechanical forces affect NF-kappaB dynamics in vivo in a similar matter as predicted by our cell culture studies (Aim 2). In particular, the student will also determine whether treatments that inhibit the highly fibrotic nature of PDAC tumours, and thus are expected to decrease ECM stiffness and tumour cell spreading, inhibit NF-kappaB as predicted by our cell line studies. These experiments will initially be performed in GEMMs of PDAC, and expanded to orthotopic models to perform live cell imaging of NF-kappaB dynamics in response to small molecule inhibitors (as in Aim 1a). Aim 4: Determine whether manipulation of mechanical forces that promote NF-kappaB activation during PDAC can enhance immunotherapy. Single agent immunotherapy has largely been ineffective in treating PDAC [8], which is likely due in part to the highly fibrotic nature of the disease that both limits immune infiltration but also promotes tumour cell survival in inflammatory environments via activation of pro-survival pathways such as STAT3 [6] and NF-kappaB. However, there is already evidence that inhibition of mechanotransduction signalling can markedly improve the efficacy of checkpoint immunotherapy [9]. In this aim the student will test if inhibition of signalling pathways that promote pro-survival signalling mediated by NF-kappaB enhances the efficacy of checkpoint immunotherapy. To perform these experiments, PDAC-bearing mice will treated checkpoint immunotherapy, and with inhibitors of NF-kappaB activation downstream of mechanical signals that we have identified in previous studies [3], and/or characterized in Aim 3. Outcomes: By completing Aim 3 and 4 we will gain insight into whether inhibiting the mechanical signals that promote NF-kappaB may be clinically useful, and/or whether this can enhance the efficacy of checkpoint immunotherapy. LITERATURE REFERENCES (Please use the Harvard system of referencing and provide up to 10 key references) 1 Staudt LM. 2010. Oncogenic activation of NF-kappaB. Cold Spring Harbor perspectives in biology 2: a000109. 2 DiDonato JA, Mercurio F, Karin M. 2012. NF-kappaB and the link between inflammation and cancer. Immunological reviews 246: 379-400. 3 Sero JE, Sailem HZ, Ardy RC, Almuttaqi H, et al. 2015. Cell shape and the microenvironment regulate nuclear translocation of NF-kappaB in breast epithelial and tumor cells. Molecular systems biology 11: 790. 4 Butcher DT, Alliston T, Weaver VM. 2009. A tense situation: forcing tumour progression. Nature reviews Cancer Page 4 of 5

9: 108-22. 5 Lesina M, Wormann SM, Morton J, Diakopoulos KN, et al. 2016. RelA regulates CXCL1/CXCR2-dependent oncogene-induced senescence in murine Kras-driven pancreatic carcinogenesis. The Journal of clinical investigation 126: 2919-32. 6 Laklai H, Miroshnikova YA, Pickup MW, Collisson EA, et al. 2016. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression. Nature medicine 22: 497-505. 7 Sailem HZ, Sero JE, Bakal C. 2015. Visualizing cellular imaging data using PhenoPlot. Nature communications 6: 5825. 8 Royal RE, Levy C, Turner K, Mathur A, et al. 2010. Phase 2 trial of single agent Ipilimumab (anti-ctla-4) for locally advanced or metastatic pancreatic adenocarcinoma. Journal of immunotherapy 33: 828-33. 9 Jiang H, Hegde S, Knolhoff BL, Zhu Y, et al. 2016. Targeting focal adhesion kinase renders pancreatic cancers responsive to checkpoint immunotherapy. Nature medicine 22: 851-60. Page 5 of 5