Johannes Zuber

RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

An oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer

Cancers are highly heterogeneous and contain many passenger and driver mutations. To functionally identify tumor suppressor genes relevant to human cancer, we compiled pools of short hairpin RNAs (shRNAs) targeting the mouse orthologs of genes recurrently deleted in a series of human hepatocellular carcinomas and tested their ability to promote tumorigenesis in a mosaic mouse model. In contrast to randomly selected shRNA pools, many deletion-specific pools accelerated hepatocarcinogenesis in mice. Through further analysis, we identified and validated 13 tumor suppressor genes, 12 of which had not been linked to cancer before. One gene, XPO4, encodes a nuclear export protein whose substrate, EIF5A2, is amplified in human tumors, is required for proliferation of XPO4-deficient tumor cells, and promotes hepatocellular carcinoma in mice. Our results establish the feasibility of in vivo RNAi screens and illustrate how combining cancer genomics, RNA interference, and mosaic mouse models can facilitate the functional annotation of the cancer genome.

Cancer stem cell definitions and terminology: the devil is in the details

The cancer stem cell (CSC) concept has important therapeutic implications, but its investigation has been hampered both by a lack of consistency in the terms used for these cells and by how they are defined. Evidence of their heterogeneous origins, frequencies and their genomic, as well as their phenotypic and functional, properties has added to the confusion and has fuelled new ideas and controversies. Participants in The Year 2011 Working Conference on CSCs met to review these issues and to propose a conceptual and practical framework for CSC terminology. More precise reporting of the parameters that are used to identify CSCs and to attribute responses to them is also recommended as key to accelerating an understanding of their biology and developing more effective methods for their eradication in patients.

A genome-wide survey of RAS transformation targets.

An important aspect of multi-step tumorigenesis is the mutational activation of genes of the RAS family, particularly in sporadic cancers of the pancreas, colon, lung and myeloid system. RAS genes encode small GTP-binding proteins that affect gene expression in a global way by acting as major switches in signal transduction processes, coupling extracellular signals with transcription factors. Oncogenic forms of RAS are locked in their active state and transduce signals essential for transformation, angiogenesis, invasion and metastasis via downstream pathways involving the RAF/MEK/ERK cascade of cytoplasmic kinases, the small GTP-binding proteins RAC and RHO, phosphatidylinositol 3-kinase and others. We have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, to contrast differential gene expression profiles in immortalized, non-tumorigenic rat embryo fibroblasts and in HRAS- transformed cells. Sequence and expression analysis of more than 1,200 subtracted cDNA fragments revealed transcriptional stimulation or repression of 104 ESTs, 45 novel sequences and 244 known genes in HRAS- transformed cells compared with normal cells. Furthermore, we identified common and distinct targets in cells transformed by mutant HRAS, KRAS and NRAS, as well as 61 putative target genes controlled by the RAF/MEK/ERK pathway in reverted cells treated with the MEK-specific inhibitor PD 98059.

Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17–92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17–92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17–92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

Toward understanding and exploiting tumor heterogeneity

The extent of tumor heterogeneity is an emerging theme that researchers are only beginning to understand. How genetic and epigenetic heterogeneity affects tumor evolution and clinical progression is unknown. The precise nature of the environmental factors that influence this heterogeneity is also yet to be characterized. Nature Medicine, Nature Biotechnology and the Volkswagen Foundation organized a meeting focused on identifying the obstacles that need to be overcome to advance translational research in and tumor heterogeneity. Once these key questions were established, the attendees devised potential solutions. Their ideas are presented here.

Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.

TAp63 induces senescence and suppresses tumorigenesis in vivo

p63 is distinct from its homologue p53 in that its role as a tumour suppressor is controversial, an issue complicated by the existence of two classes of p63 isoforms. Here we show that TAp63 isoforms are robust mediators of senescence that inhibit tumorigenesis in vivo. Whereas gain of TAp63 induces senescence, loss of p63 enhances sarcoma development in mice lacking p53. Using a new TAp63-specific conditional mouse model, we demonstrate that TAp63 isoforms are essential for Ras-induced senescence, and that TAp63 deficiency increases proliferation and enhances Ras-mediated oncogenesis in the context of p53 deficiency in vivo. TAp63 induces senescence independently of p53, p19Arf and p16Ink4a, but requires p21Waf/Cip1 and Rb. TAp63-mediated senescence overrides Ras-driven transformation of p53-deficient cells, preventing tumour initiation, and doxycycline-regulated expression of TAp63 activates p21Waf/Cip1, induces senescence and inhibits progression of established tumours in vivo. Our findings demonstrate that TAp63 isoforms function as tumour suppressors by regulating senescence through p53-independent pathways. The ability of TAp63 to trigger senescence and halt tumorigenesis irrespective of p53 status identifies TAp63 as a potential target of anti-cancer therapy for human malignancies with compromised p53.

Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation

Cancer cells frequently depend on chromatin regulatory activities to maintain a malignant phenotype. Here, we show that leukemia cells require the mammalian SWI/SNF chromatin remodeling complex for their survival and aberrant self-renewal potential. While Brg1, an ATPase subunit of SWI/SNF, is known to suppress tumor formation in several cell types, we found that leukemia cells instead rely on Brg1 to support their oncogenic transcriptional program, which includes Myc as one of its key targets. To account for this context-specific function, we identify a cluster of lineage-specific enhancers located 1.7 Mb downstream from Myc that are occupied by SWI/SNF as well as the BET protein Brd4. Brg1 is required at these distal elements to maintain transcription factor occupancy and for long-range chromatin looping interactions with the Myc promoter. Notably, these distal Myc enhancers coincide with a region that is focally amplified in ∼3% of acute myeloid leukemias. Together, these findings define a leukemia maintenance function for SWI/SNF that is linked to enhancer-mediated gene regulation, providing general insights into how cancer cells exploit transcriptional coactivators to maintain oncogenic gene expression programs.

Mouse models of human AML accurately predict chemotherapy response

The genetic heterogeneity of cancer influences the trajectory of tumor progression and may underlie clinical variation in therapy response. To model such heterogeneity, we produced genetically and pathologically accurate mouse models of common forms of human acute myeloid leukemia (AML) and developed methods to mimic standard induction chemotherapy and efficiently monitor therapy response. We see that murine AMLs harboring two common human AML genotypes show remarkably diverse responses to conventional therapy that mirror clinical experience. Specifically, murine leukemias expressing the AML1/ETO fusion oncoprotein, associated with a favorable prognosis in patients, show a dramatic response to induction chemotherapy owing to robust activation of the p53 tumor suppressor network. Conversely, murine leukemias expressing MLL fusion proteins, associated with a dismal prognosis in patients, are drug-resistant due to an attenuated p53 response. Our studies highlight the importance of genetic information in guiding the treatment of human AML, functionally establish the p53 network as a central determinant of chemotherapy response in AML, and demonstrate that genetically engineered mouse models of human cancer can accurately predict therapy response in patients.