Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

Acute lymphoblastic leukaemia (ALL) is the most common malignancy in children and adolescents where treatment is associated with significant morbidity, hence novel therapeutic approaches are required. We are currently performing a genome wide RNAi screen using the Decode™ pGIPZ negative selection library consisting of 7 pools of 10,000 shRNA constructs to identify candidate genes involved in the clonal propagation of ALL.

We have established a serial re-plating protocol where we co-culture the pre-B childhood ALL t(4;11)-positive SEM cell line with feeder cells at low serum concentration to provide a surrogate niche environment.

Results obtained so far have been extremely encouraging with shRNA targeting genes associated with lymphoid differentiation such as AHR (B-cell maturation), IKZF3 (B-cell progenitor differentiation) or CD20 (marker of B-cell differentiation) being enriched and amongst the depleted genes we identified BCL2 (survival), BRD4 (epigenetic reader, therapeutic target in leukaemia), MAX (partner of MYC), BMI1 (polycomb complex 1 member, required for self-renewal) or POT1 (telomere integrity).

These results demonstrate the feasibility of our approach, however further analyses are warranted to functionally characterise key mechanisms by which leukaemic blasts maintain their “stemness” in ALL.