URPP Fellow: Lorenza Feretti
Epigenetics refers to a number of modifications of chromatin, either to DNA directly or to its associated histone complexes that affect DNA-based processes, such as transcription, DNA repair, and replication without altering the primary sequence of DNA (Jones and Baylin, 2007; Robertson and Wolffe, 2000). The biochemical modification underlying this process involves DNA methylation or histone posttranslational modifications, including acetylation, phosphorylation, methylation, ADP-ribosylation, sumoylation and ubiquitination of distinct amino acids.
It is becoming increasingly clear that disruption of the “epigenome” as a result of alterations in epigenetic regulators, that have roles as ‘writers’, ‘readers’ or ‘editors’ of DNA methylation and/or chromatin states, is a fundamental mechanism in cancer. The principal tenet in oncology, that cancer is a disease initiated and driven by genetic anomalies, remains uncontested, but epigenetic pathways also play a significant role in oncogenesis. In fact, it is now irrefutable that many of the hallmarks of cancer, such as malignant self-renewal, differentiation blockade, evasion of cell death, and tissue invasiveness are profoundly influenced by changes in the epigenome.
High-resolution genome-sequencing efforts have discovered a wealth of mutations in genes encoding epigenetic regulators (Dawson and Kouzarides, 2012). Moreover, a less studied but recently emerged concept is that information about a cell’s metabolic state is also integrated into the regulation of epigenetics. The activities of many chromatin-modifying enzymes are regulated in part by the concentrations of their required metabolic substrates or cofactors (Lu and Thompson, 2012). Such substrates or cofactors are capable of diffusing through nuclear pores, therefore providing a way for the cell to deliver metabolic information to nuclear transcription. It is now appreciated that cells constantly adjust their metabolic state in response to extracellular signaling and/or nutrient availability (Vander Heiden et al., 2009). As a classical example, while quiescent cells fully oxidize glucose to carbon dioxide in the mitochondrial electron transport chain; proliferative cells and tumor cells consume much larger quantities of glucose, secreting excess carbon as lactate even when oxygen is abundant, a process termed ‘‘aerobic glycolysis.’’
Elucidating the networks of epigenetic regulators will provide further mechanistic understanding of the interplay between genetic and epigenetic alterations, and will allow developing novel epigenome-targeted therapeutic strategies. Although epigenetic inhibitors are thought to have global effects and thus to act in an unselective manner, recent studies with BET protein inhibitors revealed that this inhibitors alter only a few hundred genes depending on cell type (Dawson et al., 2011; Nicodeme et al., 2010). Thus, these drugs can disrupt a selective set of genes. The observation that epigenetic inhibitors lead to dramatic effects in malignant cells, although their normal counterparts remain largely unaltered suggests that, during normal homeostasis, epigenetic regulators function in a semi-redundant manner, but in cancer, they may be required to maintain the expression of a few key target genes, whereas normal cells have alternative compensating pathways to rely on (Weinstein, 2002).
In fact, recent work from our consortium showed an essential role of the epigenetic modifier Ezh2 in melanoma progression. Specifically, conditional Ezh2 ablation or treatment with the EZH2-inhibitor GSK503 inhibits tumor progression as well as metastasis formation in an autochthonous mouse melanoma model; those results were validated using primary human melanoma cells in vitro (Zingg et al., 2015). Furthermore, we found that methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognosticator in advanced melanoma (Cheng et al., 2015).
The U.S.FDA has already approved epigenetic inhibitors as anticancer drugs (Mummaneni and Shord, 2014). It is likely that many of these new epigenetic drugs offer synergistic benefits, and these new therapies may also synergize with conventional chemotherapies. This strategy of combination therapy may not only increase therapeutic efficacy but also reduce the likelihood of drug resistance. Another exciting possibility is the use of epigenetic modifiers to induce tumor antigens that can then be used as targets for cancer immunotherapy.
Aim 1: Defining the sensitivities of spheroid cancer cultures to clinically validated epigenetic inhibitors.
a) Establishment of spheroid cancer cultures (compared to 2D cultures) with different media (e.g. cytokine cocktail) under normoxic and hypoxic conditions.
Different tumor types with stratified aggressiveness will be included such as prostate cancer, melanoma, acute myeloid leukemia, and 10 other cancer types represented in the current URPP tumor cell biobank collection of the URPP.
b) Defining the sensitivities of these spheroid cancer cultures to different clinically validated epigenetic inhibitors against important regulators (including DNA methyltransferase inhibitors (e.g. azacitidine or decitabine), histone deacetylase inhibitors (e.g. vorinostat or romidepsin), histone demethylase inhibitors (Tranylcypromine or IBET762), histone methyltransferase inhibitors (e.g. GSK126) or ADP-ribosyltransferase inhibitors (e.g. Olaparib or ABT-888). These inhibitors will be assessed alone or in combination with other drugs. In particular, the most relevant first-line therapies in melanoma include BRAF and MEK inhibitors, so screening will be done in combination with these.
Tumor sensitivity towards these drugs will be assessed by a self-renewal assay, cell viability/death readout, metabolic activity analysis and the investigation of the cellular redox potential.
Aim 2: Establishing the epigenetic profile of the most sensitive spheroid cancer cultures (from aim 1).
Dependent on the sensitivities to a certain epigenetic inhibitors, the epigenome profile of the most sensitive cancer cells will be established. If the number of cancer cells should exceed our capacity, additional clinical selection criteria (e.g. aggressiveness, response to approved inhibitors, or the availability of supportive material such as histology samples) will be taken into account to reduce the number of analyzed tumor cells to a feasible number.
Following epigenetic parameters will be investigated, dependent on the epigenetic inhibitor, i.e. epigenetic regulator):
a) DNA methylation profiling (Bisulfite-sequencing),
b) RNA deep sequencing (including coding/non-coding),
c) Immunohistochemical staining of spheroids using antibodies against different histone modifications,
d) Chromatin immunoprecipitation (ChIP)/Chromatin affinity precipitation (ChAP) analysis for defined histone modifications.
Aim 3: Elucidating the molecular mechanism of the observed spheroid cancer culture sensitivities and confirming these findings in mouse models as a proof of concept.
Dependent on the involved epigenetic regulator, loss and gain of function experiments for a selected regulator will be performed (e.g. siRNA or genetic complementation by retroviral transduction of tumor cells). The cellular readouts will be the same as described in Aim 1 and the epigenetic consequences will be investigated with technologies described in Aim 2 (although not anymore genome-wide but rather focusing at defined genomic regions).
Finally, the above-analyzed epigenetic inhibitor will be tested in different mouse models for cancer including autochthonous mouse models for melanoma.