Identifying novel therapeutic susceptibilities in leukaemia by master regulator ablation

Background:
Around a third of children diagnosed with acute myeloid leukaemia (AML) cannot be cured by current therapy. These children are in urgent need of new treatments. A novel approach is to target master regulators. We recently discovered that a protein called RUVBL2 functions as a master regulator in leukaemia1,2. The guiding hypothesis is that such master regulators represent the ‘Achilles' heel’ of AML and disrupting their action offers an exciting opportunity for novel, targeted anti-AML therapies. The current project will develop a tool to ablate this master regulator, providing vital insights into how master regulator inhibition can be harnessed for new anti-leukaemia therapies. This project offers the opportunity to learn state-of-the-art molecular techniques, bioinformatics analysis, next generation sequencing and sophisticated leukaemia modelling.

Aims/Objectives:
1)  Use CRISPR/Cas9 and dTAG technology to develop a model for inducible RUVBL2 protein depletion;
2) Examine the consequences of acute RUVBL2 ablation on oncogenic programs in AML;
3) Identify clinically approved drugs that target master regulator activity of RUVBL2 in AML.

Methods:
Objective 1 (12-16 months): An inducible RUVBL2 protein depletion model will be generated in AML cells by CRISPR/Cas9-mediated3 inactivation of endogenous RUVBL2 alleles and simultaneous expression of RUVBL2/FKBP12F36V transgene, in which the RUVBL2 cDNA is fused in frame to the single-point (F36V) mutated FKBP12. The FKBP12F36V moiety will render the fusion protein sensitive to degradation by the cereblon (CRBN) E3 ubiquitin ligase complex upon addition of the heterobifunctional degrader dTAG-134. We have all the necessary targeting and expression constructs for this approach in our laboratory.
Objective 2 (10-24 months): The consequences of acute RUVBL2 protein ablation on AML cell growth and survival will be examined using specialized haematopoietic assays. Perturbation of essential leukaemia transcriptional networks will be determined by quantitative RT-PCR and chromatin immunoprecipitation (ChIP) analyses. Immunoprecipitation and western blot experiments will be performed to examine the effect of RUVBL2 loss on critical activating and repressive transcriptional complexes. These experiments will be used to define an optimal time-point for global gene expression analysis by next generation RNA sequencing, to generate a list of significant gene expression changes resulting from acute RUVBL2 loss.
Objective 3 (18-30 months): The resulting gene list will then be used to interrogate the connectivity map (Cmap) database in order to identify and validate the anti-AML activity of clinically approved drugs that induce similar gene expression changes to those observed following RUVBL2 depletion. We have used this approach previously to identify drugs for anti-AML repurposing5.

References:
1. Oasaki et al (2013) Leukemia 27:1461-8.
2. Armenteros-Monterroso et al (2019) Leukemia 33:2817-29.
3. Luo (2016) Trends Cancer 2:313-24.
4. Nabet et al (2018) Nature Chemical Biology 14:431–41.
5. Walf-Vorderwülbecke et al (2018) Leukemia 32:882-9.