Modelling Down Syndrome Acute Lymphoblastic Leukaemia in Induced Pluripotent Stem Cells
Primary supervisor: Tariq Enver, UCL
Secondary supervisor: Victor Tybulewicz, The Francis Crick Institute
Children with Down Syndrome (DS) have ~150 and ~30 times greater risk of developing acute myeloid (AML) or acute lymphoid leukaemia (ALL), respectively. Trisomy 21 (T21) can therefore be considered a “first-hit” event in leukaemogenesis. The most frequent second-hit mutations in DS-ALL, a disease characterised by an accumulation of immature B-cells, converge on activation of JAK/STAT signalling, including up-regulation of the cytokine receptor, CRLF2, constitutive activation of the IL7-Receptor (IL7R) and activating mutations in JAK2 . Although T21 has previously been shown to alter haematopoiesis, it is not fully understood how it collaborates with second hit events during leukaemogenesis [2,3].
We propose to model the development of DS-ALL using induced pluripotent stem cells (IPSCs). IPSCs can be induced to differentiate in vitro, recapitulating foetal definitive haematopoiesis. Since DS-ALL is thought to have a foetal origin, this provides a unique system in which to study the stages of oncogenic transformation. Isogenic IPSC lines which are di- or tri-somic for chromosome 21  will form the basis of the project and will be further engineered to carry common second-hit events associated with DS-ALL including chromosomal rearrangements that induce expression of CRLF2 and activating mutations of IL7R and JAK2. CRISPR/Cas9 based strategies will be employed to modify the endogenous loci and precisely mimic the mutations that occur in DS-ALL, establishing a panel of isogenic IPSC lines representing stages of oncogenic transformation.
Initially, established protocols for directed B-cell differentiation  will be coupled to detailed immuno-phenotyping by flow-cytometry, and to mass-cytometry facilitating simultaneous interrogation of immuno-phenotype, cell cycle phase, apoptosis, and JAK/STAT signalling. We will complement this with transcriptomic and epigenomic analyses of sorted cell fractions and, using single cell multi-omic approaches, characterise cell states with high resolution. Together these techniques will paint a detailed picture of the impact of T21 and second hit mutations on the differentiation trajectories as cells mature from progenitors to B-cells and associated changes to the epigenome.
Non-DS childhood B-ALL also has foetal origins with the initiating events occurring in utero. The most common of these is the t(12;21) translocation which gives rise to the ETV6-RUNX1 fusion protein. We have previously modelled this translocation and the associated pre-leukaemic phenotype in IPSCs . Interestingly, both DS and ETV6-RUNX1 reduce B-cell output, suggesting that their role in leukaemogenesis is to predispose cells of the B-lineage to additional transforming events. We will compare the data generated during this project to our existing flow-cytometry, mass-cytometry and RNA-seq data for the ETV6-RUNX1 system to explore possible commonalities in these alternative paths to transformation.
We anticipate that this project will reveal the nature of the pre-leukaemic state established by T21 and allow us to infer the likely cell of first impact for subsequent transformation by second hits. Novel immuno-phenotypes associated with DS-ALL transformation facilitating prospective identification may be uncovered facilitating further study and with potential prognostic value. Pathways disrupted by T21, alone or in collaboration with secondary events, will be identified and may suggest therapeutic vulnerabilities. Moreover, by comparison to non-DS-ALL insights with implications across childhood B-ALL will be gained.
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