Functional exploration of colorectal cancer genomes using Drosophila

The multigenic nature of human tumours presents a fundamental challenge for cancer drug discovery. Here we use Drosophila to generate 32 multigenic models of colon cancer using patient data from The Cancer Genome Atlas. These models recapitulate key features of human cancer, often as emergent properties of multigenic combinations. Multigenic models such as ras p53 pten apc exhibit emergent resistance to a panel of cancer-relevant drugs. Exploring one drug in detail, we identify a mechanism of resistance for the PI3K pathway inhibitor BEZ235. We use this data to identify a combinatorial therapy that circumvents this resistance through a two-step process of emergent pathway dependence and sensitivity we term ‘induced dependence’. This approach is effective in cultured human tumour cells, xenografts and mouse models of colorectal cancer. These data demonstrate how multigenic animal models that reference cancer genomes can provide an effective approach for developing novel targeted therapies

A personalized platform identifies trametinib plus zoledronate for a patient with KRAS-mutant metastatic colorectal cancer

Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor's genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease's genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer

A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila.

Experimental models that capture the genetic complexity of human disease and allow mechanistic explorations of the underlying cell, tissue, and organ interactions are crucial to furthering our understanding of disease biology. Such models require combinatorial manipulations of multiple genes, often in more than one tissue at once. The ability to perform complex genetic manipulations in vivo is a key strength of Drosophila, where many tools for sophisticated and orthogonal genetic perturbations exist. However, combining the large number of transgenes required to establish more representative disease models and conducting mechanistic studies in these already complex genetic backgrounds is challenging. Here we present a design that pushes the limits of Drosophila genetics by allowing targeted combinatorial ectopic expression and knockdown of multiple genes from a single inducible transgene. The polycistronic transcript encoded by this transgene includes a synthetic short hairpin cluster cloned within an intron placed at the 5' end of the transcript, followed by two protein-coding sequences separated by the T2A sequence that mediates ribosome skipping. This technology is particularly useful for modeling genetically complex diseases like cancer, which typically involve concurrent activation of multiple oncogenes and loss of multiple tumor suppressors. Furthermore, consolidating multiple genetic perturbations into a single transgene further streamlines the ability to perform combinatorial genetic manipulations and makes it readily adaptable to a broad palette of transgenic systems. This flexible design for combinatorial genetic perturbations will also be a valuable tool for functionally exploring multigenic gene signatures identified from omics studies of human disease and creating humanized Drosophila models to characterize disease-associated variants in human genes. It can also be adapted for studying biological processes underlying normal tissue homeostasis and development that require simultaneous manipulation of many genes

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-β signaling

In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila

The recent establishment of high-throughput methods for culturing Drosophila provided a unique ability to screen compound libraries against complex disease phenotypes in the context of whole animals. However, as compound studies in Drosophila have been limited so far, the degree of conservation of compound activity between Drosophila and vertebrates or the effectiveness of feeding as a compound delivery system is not well known. Our comprehensive in vivo analysis of 27 small molecules targeting seven signaling pathways in Drosophila revealed a high degree of conservation of compound activity between Drosophila and vertebrates. We also investigated the mechanism of action of AY9944, one of the Hh pathway antagonists that we identified in our compound feeding experiments. Our epistasis analysis of AY9944 provided novel insights into AY9944’s mechanism of action and revealed a novel role for cholesterol transport in Hh signal transduction