Finding and probing cancer drug targets using advanced RNAi technologies
An optimized microRNA backbone for effective single-copy RNAi
Due to our incomplete understanding of miRNA biogenesis factors, available shRNAmir reagents remain far from perfect and often fail to trigger potent target knockdown. Following recent advances in the design of the synthetic stem, we have over the past year established an optimised miRNA backbone, termed miR-E (Figure 1), which greatly improves knockdown potency by enhancing pri-miRNA processing (~10-fold).
When combined with up-to-date design algorithms and improved expression vectors, miR-E predominantly yields single-copy effective shRNAs required for multiplexed screening, and thereby overcomes a key limitation of previous reagents. To implement these improvements in new shRNA libraries, we have established a robotics/deep-sequencing-supported cloning pipeline which permits rapid construction of custom-optimized shRNA libraries.
Exploring new therapeutic targets in high-risk AML
Despite our advanced genetic understanding, acute myeloid leukaemia (AML) remains incurable in more than 70% of patients. While many targeted therapies (mainly inhibitors of pro-proliferative ‘type-I’ mutations) have failed in clinical studies, all-trans-retinoic acid (ATRA) targeting PML/RARA (a self-renewal promoting ‘type-II’ mutation) has turned a deadly AML subtype into a curable disease. To systematically search entry points for similarly effective targeted therapies, we are pursuing two approaches: a) using a series of AML mouse models harbouring regulatable ‘type-II’ mutations and focused RNAi studies, we seek to gain insight into common factors and pathways involved in maintaining aberrant self-renewal; and b) using an established model of high-risk MLL-AF9;Nras-driven AML, we have performed a multiplexed RNAi screen targeting ~1000 druggable and MLL-regulated candidate genes, which has revealed druggable leukaemia-specific dependencies including several metabolic regulators, which we focus on in mechanistic follow-up studies.
A systematic survey of chromatin-associated cancer dependencies
Altered chromatin landscapes in cancer are believed to open major therapeutic opportunities because epigenetic aberrations are, in principle, reversible and controlled by a machinery that is amenable to drug modulation. To systematically explore this promising target space, we have constructed a comprehensive shRNA library (>5000 miR-E shRNAs) targeting 650 chromatin-associated genes, which we are screening comparatively in several leukaemia models (Figure 2). Using this approach, we intend to establish functional-genetic dependency profiles that, similar to expression profiles developed a decade ago, will provide a new layer of cancer classification with direct translational implications.
Mechanisms of sensitivity and resistance to BRD4 inhibition
In a pioneering screen using an incomplete chromatin shRNA library (242 genes, 1072 shRNAs), we previously identified the epigenetic reader BRD4 as a promising therapeutic target in AML. While several BET bromodomain inhibitors have now entered clinical phase-I trials, the mechanistic basis underlying sensitivity and resistance to BRD4 suppression remains unclear.
To explore genetic and epigenetic determinants of BRD4 dependency, we profiled sensitive and resistant cancers of different tissue origins in great detail and performed multiplexed RNAi screens to systematically probe chromatin regulators for a potential role in modulating the response to BRD4 suppression. From a translational perspective, we hope these studies will help to identify reliable biomarkers which are urgently needed to further develop BRD4 inhibitors in the clinic.
- Rathert, P., Roth, M., Neumann, T., Muerdter, F., Roe, JS., Muhar, M., Deswal, S., Cerny-Reiterer, S., Peter, B., Jude, J., Hoffmann, T., Boryń, ŁM., Axelsson, E., Schweifer, N., Tontsch-Grunt, U., Dow, LE., Gianni, D., Pearson, M., Valent, P., Stark, A., Kraut, N., Vakoc, CR., Zuber, J. (2015). Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature. 525(7570):543-7
- Fellmann, C., Hoffmann, T., Sridhar, V., Hopfgartner, B., Muhar, M., Roth, M., Lai, DY., Barbosa, IA., Kwon, JS., Guan, Y., Sinha, N., Zuber, J. (2013). An optimized microRNA backbone for effective single-copy RNAi. Cell Rep. 5(6):1704-13
- Zuber, J., McJunkin, K., Fellmann, C., Dow, LE., Taylor, MJ., Hannon, GJ., Lowe, SW. (2011). Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol. 29(1):79-83
- Zuber, J., Rappaport, AR., Luo, W., Wang, E., Chen, C., Vaseva, AV., Shi, J., Weissmueller, S., Fellmann, C., Fellman, C., Taylor, MJ., Weissenboeck, M., Graeber, TG., Kogan, SC., Vakoc, CR., Lowe, SW. (2011). An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance. Genes Dev. 25(15):1628-40
- Zuber, J., Shi, J., Wang, E., Rappaport, AR., Herrmann, H., Sison, EA., Magoon, D., Qi, J., Blatt, K., Wunderlich, M., Taylor, MJ., Johns, C., Chicas, A., Mulloy, JC., Kogan, SC., Brown, P., Valent, P., Bradner, JE., Lowe, SW., Vakoc, CR. (2011). RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature. 478(7370):524-8