URPP Fellow: Lorenza Feretti

Genome instability is a cancer hallmark that leads to a wide spectrum of genetic changes, including mutations in epigenetic modifiers. Disruption of the “epigenome” as a result of alterations in epigenetic regulators such as the ‘writers’, ‘readers’, or ‘editors’ of DNA methylation and/or chromatin states, is a fundamental mechanism in oncogenesis. In fact, changes in the epigenome can profoundly influence many hallmarks of cancer as well as clinical responses to anticancer therapies. Moreover, epigenetic mechanisms modulate a variety of transcriptional pathways resulting in a dynamic heterogeneous tumor cell population and alerted tumor microenvironments. Despite a more sophisticated understanding of the relationship between the epigenome and cancer, many epigenetic drugs in use have not been linked to specific cancer biomarkers that could guide therapies to maximize patient benefit. The observation that epigenetic inhibitors lead to dramatic effects in malignant cells, although their normal counterparts remain largely unaltered, underlines their potential as anti-cancer therapeutics. Elucidating the networks of epigenetic regulators in different cancer types provides further insights into the mode of action and thus the interplay between genetic and epigenetic alterations allowing the development of novel epigenome-targeted therapeutic strategies.
During phase II of this URPP funding, we carried out an epigenetic drug screen in 3D melanoma cultures to identify a possible epigenetic mechanism of MAPK inhibitor (MAPKi), melanoma’s first-line treatments, resistance. We demonstrated that in 3D patient-derived cultures a PARP inhibitor (PARPi) restore MAPKi sensitivity in BRAF and NRAS mutated melanomas. It is not surprising that the synergistic activity of PARPi and MAPKi was not observed in monolayer (2D) cultures since 3D culturing better represent in vivo tumors by preserving chemical gradients and cell-cell interactions. Currently, we are uncovering the molecular mechanism for this synergistic combination by RNA-sequencing, MS-based proteomic studies and functional assays and are testing it in both mouse models and other RAF or RAS mutated cancer cultures types. In addition, we are investigating by cell surface markers screening of 3D cancer cultures whether epigenetic alterations induce tumor antigens that can serve as targets for immunotherapies or even are responsible for targeted therapy resistance.
In the last decade, immunotherapies, such as checkpoint blockade, have become a powerful weapon for a number of cancers. However, a large proportion of patients fail to respond to these therapies mostly because of the absence of tumor-infiltrating lymphocytes (TILs) within the tumor microenvironments. Indeed, cancers with few or no immune cells within the tumor are classified as ‘cold’. Moreover, tumor-infiltrating T cells are often exhausted and thus unable to maintain an inflamed tumor state. Epigenetic modulators are responsible for altering diverse immunogenic proprieties in both malignant cells and in the tumor microenvironments, such as immune and fibroblast cells. For instance, DNA methylation drives T cell exhaustion and thus multiple clinical trials are currently testing the combination of DNA methylation inhibitors with various immune checkpoint blockade compounds. Nevertheless, the regulation of T cell infiltration, as well as tumor immune landscapes, are poorly understood, at least in part, due to the lack of in vitro systems that faithfully recapitulate the heterogeneity and the 3D structure of the tumor and its microenvironment.
To understand the molecular basis of ‘cold’ tumors and to better characterize how epigenetic drugs can affect cancer survival and progression in this regard, we plan to screen for signalling factors released by un-inflamed tumors that block T cell infiltration and sensitivity to immunotherapy as well as to understand whether such chemokine(s) could be epigenetically repressed. To better preserve diversity and cell heterogeneity of the tumor, we will use, when possible, patient-derived 3D culture cancer cells and/or co-culture with its patient-matched fibroblast. The obtained secretion profiles will be linked to detailed epigenetic characterization to describe the molecular mechanisms of ‘cold’ tumor as well as to design epigenetic treatments to convert ‘cold’ tumors into ‘hot’.
As in Phase II, we will initially focus on melanoma, for which many characterized cell lines and linked clinical and molecular data are already available in the URPP biobank. Later, we will include other cancer types, which are currently being collected and characterized as part of the URPP tumor cell biobank platform-developing project of this URPP funding.

Aims

1. 1. Identifying the secretome of ‘hot’ and ‘cold’ live-cell cancer co-cultures of patient-derived tumor samples provided by the URPP biobank.
   a)    Live-cell cultures of different cancer types will be categorized accordingly to their immune status and thus as ‘hot’ or ‘cold’ by:
   •    Immunohistochemistry for activated T-cells markers, such as CD8+/CD4+, granzyme B and TIA-1, of the corresponding formalin-fixed biopsy section
   •    IFN-γ–related gene expression signatures of the isolated live-cells
   b)    In order to identify signalling molecules responsible for tumor immune escape the secretomes of those cancer cultures will be elucidated by beads-based immunoassay, which reliably measure the amount of secreted proteins. Indeed, it has been demonstrated that the correlation between mRNA concentrations and actual secreted protein levels is inaccurate and variable mostly due because post-transcriptional and post-translation regulations are not taken into account.
   c)    Live-cell cultures for which patient-matched fibroblast are present/extractable will be culture as mono- (either only cancer or fibroblast) as well as co-culture to assess whether fibroblast can secrete chemo- and cytokines per se or can modulate their production by cancer cells. When possible those mono- and co-culture will be grown in 3D and compared to 2D.
2. Defining whether clinically evaluated epigenetic compounds can convert ‘cold’ into ‘hot’ tumors. The impact of epigenetic regulation on the expression of the identified immune suppressing or promoting molecule (from aim 1) will be investigated by analysing the secretome of selected ‘cold’ cancer mono- or co-cultures (depending on aim 1) treated with several epigenetic inhibitors alone or in the presence of immune checkpoints blockade. Afterward and depending on the epigenetic regulator the mode of action affecting immune factor secretion will be defined. If there will be no epigenetic mechanisms involved, we will explore whether its modulation could be post-translationally regulated (e.g. phosphorylations, proteasome mediated degradation etc.) or linked to clinical available data (e.g. cancer stage, aggressiveness, response to approved targeted or immune therapies, etc.).
3. Functional ex vivo and in vivo validation. The identified secreted immune suppressing/promoting factor(s) will be first validated in a functional assay. We set out to establish whether autologous T cell can be co-cultured with cancer/fibroblast cells as recently established from and Dijkstra and colleagues. To address this, we will first need to obtain tumor-reactive T cells from peripheral blood samples available in the URPP biobank. Thus, selected cell lines from aim 1 and 2 for which patient-matched blood samples are available, will be tested for T cell extraction, 3D co-culture and extent of T cell infiltration. The secretomes of those tumor organoid co-cultures will be analysed before and after epigenetic treatments alone or synergized with immune checkpoints blockades. If this method will fail, we would perform such validation in an easier functional assay, for instance co-culture with engineered T cell or other immune cell types. Finally, the above-analyzed epigenetic inhibitor will be tested in patient-derived xenografts of melanoma using nude mice and early passage melanoma cultures from the biobank.

Although growing and screening primary cells as 3D co-cultures is challenging, we provided very strong evidence during phase II that this approach is key to increase the quality of preclinical cancer research (manuscript in preparation). Indeed, besides strengthening the overall therapeutic potential of epigenetic drugs and the understanding of the cancer epigenome, our innovative strategy involving 3D patient-derived cell cultures will strengthen the translation to the clinic and the success of epigenetic drugs. Thus, we foresee that these projects (URPP phase II and phase III) will contribute to better patient stratification and lead to the development of more personalized and thus even more effective anticancer therapies.