Investigation of the functional interplay between signalling pathways and epigenetic factors in acute leukaemia

T-ALL frequently harbours genetic alterations in cell signalling pathway components and epigenetic regulators that drive disease progression and resistance to treatment. For example, common loss of function alterations of PRC2 factors are associated with chemoresistance and poor outcomes in T-ALL. These epigenetic alterations often co-occur with activating mutations of cell signalling components such as the IL7R-JAK-STAT cascade, but the functional interactions between epigenetic and signalling pathways in T-ALL are poorly characterised. We used a combination of computational and wet laboratory approaches to tackle this problem. We first used a novel statistical method (DISCOVER) to test genetic interactions (i.e., co-occurrence and mutual exclusivity of mutations and deletions) in a large clinical T-ALL dataset. This approach allowed us to profile the patterns of co-occurrence and mutual exclusivity between epigenetic and signalling alterations in paediatric T-ALL, and identified several novel interactions between genes coding for PRC2 components and WNT pathway molecules (e.g., NLK). To understand the molecular and phenotypic impact of these events, we created a leukaemia cell line model of PRC2 depletion by CRISPR-Cas9 gene editing of PRC2 core component EZH2 in the T-ALL Jurkat cell line. We then used a multi-omic approach with RNA-sequencing and mass spectrometry-based analysis to investigate the molecular effects of epigenetic deregulation on cell signalling. We used the PROGENy algorithm to infer cell signalling activity from transcriptional readouts and found links between EZH2 loss and deregulated activity of several signalling pathways, including MAPK and WNT. These changes were validated at the protein level by both immunoblotting and proteomic analysis. Further, phospho-proteomic profiling and KSEA confirmed significant activity differences in multiple cascades between WT and EZH2-deficient cells, with MAPK and WNT signalling again found to be deregulated. To provide a holistic picture of the molecular effects of PRC2 depletion in this cell line model, all high-throughput data were integrated using the CausalPath algorithm. This allowed us to identify the key nodes responsible for cell signalling differences in EZH2-depleted cells and confirmed a central role for WNT signalling in the PRC2-deficient cellular molecular network. To link these results to the clinic, we compared the efficacy of standard leukaemia therapies in the EZH2-WT and EZH2-KO Jurkat lines. These studies revealed significant differences in asparaginase sensitivity, with increased asparaginase resistance in PRC2-depleted cells, and with pharmaceutical manipulation of PRC2 activity by either enzymatic EZH2 inhibition or PROTAC-based PRC2 degradation in multiple T-ALL cell lines. We further found asparaginase resistance to be correlated with total levels of protein ubiquitination, linking our findings to recent reports of asparaginase sensitivity being predicted by activity of the WNT/STOP pathway. Taken together, our results suggest that PRC2 depletion causes decreased activity of the WNT/STOP pathway, creating a reservoir of ubiquitinated proteins that allows EZH2-deficienct cells to escape asparaginase toxicity. Importantly, analysis of gene expression data from a T-ALL patient cohort revealed similar differences in WNT signalling transcriptional readouts in cases that harboured mutation or deletion of PRC2 core components. Overall, this work provides a comprehensive analysis of signalling pathway changes in epigenetically altered T-ALL, identifies a novel potential mechanism explaining resistance to a key leukaemia treatment, and further suggests potential avenues to overcome this clinical problem. Further work is needed to decipher the epigenetic mechanisms that underpin these observations, and whether pharmaceutical modulation of either WNT signalling, or proteasome activity, might be effective in reversing asparaginase resistance.