Understanding how initiating genetic mutations permit the emergence of full-blown cancer using IPSC models of childhood leukaemia

Primary supervisor: Tariq Enver, UCL

Secondary supervisor: Charles Swanton, The Francis Crick Institute/ UCL

Project

Childhood lymphoblastic leukaemia remains the most common cause of cancer related deaths in children. 1% of children are born with a B-lymphoid clone containing an ETV6-Runx1 (first hit) translocation. Of these, only 1% subsequently develop leukaemia highlighting the importance of additional co-operating mutations; loss of the remaining ETV6 allele being the most common. Evidence, both epidemiolocal and experimental has lent support for a role for infection/inflammation in the aetiology of childhood leukaemia. The goal of this project is to understand how the different genetic and epigenetic events associated with childhood leukaemia contribute individually and collectively to leukemogenesis.

We have developed a model using human induced pluripotent cells which when differentiated along the B-cell lineage authentically recapitulate features of human B-lymphopoiesis in the developing embryo where prior studies have shown childhood leukaemia initiates. Using this system, we have generated cell lines that contain the first (ETV6/Runx1) and second hit (ETV6 deletion) either individually or in combination. Preliminary molecular analysis of these preleukaemic lines shows involvement of pathways that regulate differentiation, cell cycle as well as inflammatory signalling and upregulation of RAG mediated recombination which is thought to underpin the generation of additional co-operating mutations. We now propose to combine analysis of gene expression by RNAseq with that of chromatin accessibility by ATAQseq on single cells across the trajectory of their differentiation from IPS cells into the B-cell lineage to assemble gene regulatory networks (GRN). These will guide functional experiments to test cell intrinsic or microenvironment-derived candidate regulators involved in leukemic progression.

Functional studies will take the form of assays of B-cell expansion, survival, and differentiation using defined cellular compartments in bespoke in vitro culture, colony formation and flow cytometry following: i) targeted genetic expression, or disruption, of cell intrinsic regulators, ii) the engineering a fluorescent reporter of RAG-mediated rearrangement, iii) direct administration of recombinant signalling molecules (e.g. TGFb, TNFa, IFN, IL3, IL7) or small molecule inhibitors (e.g. Ruxolitinib), and iv) simulation of more complex changes in the micro-environment through direct manipulation of the co-generated stroma or individual components thereof. There are many ways to induce inflammatory signalling (e.g. polyDIDC, polyIC, viral infection such as Sendai, LPS) with one tractable model being the exposure of macrophages, readily produced from IPSCs in vitro, to PM2.5 particles. This approach has been used in lung cancer to mimic inflammation linked to air pollution which like electromagnetic fields or radon gas, has been tabled as a tumour promoter in childhood leukaemia.

To further characterise the mechanisms underlying altered environmental responses we will employ a new method developed in the Cancer Institute – Signal-seq – to measure the global transcriptome and intracellular protein post-translational modifications (PTMs) in single cells, enabling simultaneous quantification of changes in signalling activity, cell cycle and apoptosis at the protein-level, and associated changes to the transcriptome. This will allow us to relate altered functional responses to changes to the GRN during leukaemogenesis.

These experiments will reveal how genetic and epigenetic cues combine to transform a normal progenitor and highlight strategies to target that process.

References

1. Wray, J. P., Deltcheva, E. M., Boiers, C., Richardson, S. E., Chhetri, J. B., Brown, J., Gagrica, S., Guo, Y., Illendula, A., Martens, J. H. A., Stunnenberg, H. G., Bushweller, J. H., Nimmo, R., & Enver, T. (2022). Regulome analysis in B-acute lymphoblastic leukemia exposes Core Binding Factor addiction as
   a therapeutic vulnerability. Nature Communications, 13(1), 7124.
2. Turati, V. A., Guerra-Assunção, J. A., Potter, N. E., Gupta, R., Ecker, S., Daneviciute, A., Tarabichi, M., Webster, A. P., Ding, C., May, G., James, C., Brown, J., Conde, L., Russell, L. J., Ancliff, P., Inglott, S., Cazzaniga, G., Biondi, A., Hall, G. W., Enver, T. (2021). Chemotherapy induces canalization of cell state in childhood B-cell precursor acute lymphoblastic leukemia. Nature Cancer, 2(8), 835-852.
3. Böiers C, Richardson SE, Laycock E, Zriwil A, Turati VA, Brown J, Wray JP, Wang D, James C, Herrero J, Sitnicka E, Karlsson S, Smith AJH, Jacobsen SEW, Enver T. A Human IPS Model Implicates Embryonic B-Myeloid Fate Restriction as Developmental Susceptibility to B Acute Lymphoblastic Leukemia-Associated ETV6-RUNX1. Dev Cell. 2018 Feb 5;44(3):362-377.e7.
4. Hong, D., Gupta, R., Ancliff, P., Atzberger, A., Brown, J., Soneji, S., Green, J., Colman, S., Piacibello, W., Buckle, V., Tsuzuki, S., Greaves, M., & Enver, T. (2008). Initiating and cancer-propagating cells in TEL-AML1-associated childhood leukemia. Science, 319(5861):336-9.
5. Greaves, M. A causal mechanism for childhood acute lymphoblastic leukaemia (2018) Nature Reviews Cancer, 18: 471-484