B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7 , a transcription factor, and Phip , a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.

Many cases of AML are associated with mutational activation of receptor tyrosine kinases (RTKs) such as FLT3. However, RTK inhibitors have limited clinical efficacy as single agents, indicating that AML is driven by concomitant activation of different signaling molecules. We used a functional genomic approach to identify RET, encoding an RTK, as an essential gene in multiple subtypes of AML, and observed that AML cells show activation of RET signaling via ARTN/GFRA3 and NRTN/GFRA2 ligand/co-receptor complexes. Interrogation of downstream pathways identified mTORC1-mediated suppression of autophagy and subsequent stabilization of leukemogenic drivers such as mutant FLT3 as important RET effectors. Accordingly, genetic or pharmacologic RET inhibition impaired the growth of FLT3-dependent AML cell lines and was accompanied by upregulation of autophagy and FLT3 depletion. RET dependence was also evident in mouse models of AML and primary AML patient samples, and transcriptome and immunohistochemistry analyses identified elevated RET mRNA levels and co-expression of RET and FLT3 proteins in a substantial proportion of AML patients. Our results indicate that RET-mTORC1 signaling promotes AML through autophagy suppression, suggesting that targeting RET or, more broadly, depletion of leukemogenic drivers via autophagy induction provides a therapeutic opportunity in a relevant subset of AML patients.

Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction-digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.

Acute myeloid leukemia (AML) is a heterogeneous disease with diverse leukemogenic driver lesions. The genetic understanding of AML has resulted in major improvements in diagnosis, classification, prognostication, and outcome prediction. However, these insights have not yet translated into molecular mechanism-based therapies in the majority of cases, because AML cells rapidly escape attempts at therapeutic targeting such as small-molecule inhibition of FLT3 internal tandem duplication (FLT3-ITD) mutants, which occur in up to 30% of AML cases and confer a poor prognosis. To identify potential new targets for combinatorial treatment approaches, we performed a series of large-scale short hairpin RNA (shRNA) screens and observed that cell lines representing various AML subtypes were dependent on expression of the RET receptor tyrosine kinase (RTK), which has not previously been implicated in AML pathogenesis. Validation experiments demonstrated that depletion of RET by shRNA knockdown or CRISPR/Cas9-mediated knockout led to cell cycle arrest in the G0/G1 phase, increased apoptosis, and reduced clonogenic activity. RTK profiling using ELISA-based antibody arrays demonstrated that RET is highly phosphorylated in RET-dependent AML cell lines. Analysis of known RET ligand/co-receptor pairs (GDNF/GFRA1, NRTN/GFRA2, ARTN/GFRA3, PSPN/GFRA4) by quantitative real-time PCR and shRNA knockdown indicated that RET signaling is facilitated mainly through NTRN/GFRA2 or ARTN/GFRA3. Interrogation of various signaling pathways known to promote myeloid leukemogenesis showed that RET knockdown resulted in decreased phosphorylation of 4E-BP1 (T37/46), p70S6K (T389), S6RP (S240/244), and ULK1 (S758), pointing to mTORC1-mediated protein synthesis and/or suppression of autophagy as important effectors of RET signaling in AML cells. Based on recent data showing that FLT3-ITD mutants can be degraded by autophagy (Larrue et al. Blood 2016), we reasoned that the RET-mTORC1 signaling axis promotes AML through protection of FLT3-ITD mutants from autophagic degradation. Consistent with this hypothesis, genetic or pharmacologic (vandetanib, danusertib) inhibition of RET predominantly affected FLT3-dependent AML cell lines and were accompanied by upregulation of autophagy and destabilization of FLT3, as evidenced by p62 degradation, LC3B turnover, increased numbers of autophagic vacuoles, and decreased FLT3 protein levels. Furthermore, we observed accumulation of STAT5, a key FLT3-ITD downstream effector, upon pharmacologic autophagy inhibition in low RET-expressing AML cells, underlining the importance of RET-mediated suppression of autophagy for leukemogenic FLT3-ITD signaling. In line with the observations in AML cell lines, preliminary data from a murine bone marrow transplantation model show that Ret is required for AML development and propagation in vivo as we observed a significant survival advantage for mice transplanted with Ret knockdown cells compared with mice transplanted with control cells. Finally, genome-wide transcriptome analysis identified elevated RET mRNA levels in 35 of 260 (13.5%) primary human AML samples. Since there are no known RET copy number alterations or mutations of the RET coding region in AML patients and cell lines, we are currently investigating whether aberrant RET expression in AML can be attributed to perturbed epigenetic regulation. To this end, we are applying chromosome conformation capture combined with high-throughput sequencing (4C-seq) technology to systematically analyze interactions of the RET promoter region with enhancer sequences in high and low RET-expressing AML cell lines. Combined, our results indicate that in a proportion of AML, RET-mTORC1 signaling promotes cell viability and proliferation through suppression of autophagy, suggesting that targeting RET or, more broadly, depletion of critical leukemogenic drivers via induction of autophagy may provide a therapeutic opportunity in this subset of patients. Disclosures No relevant conflicts of interest to declare.

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Transposon screens are powerful in vivo assays used to identify loci driving carcinogenesis. These loci are identified as Common Insertion Sites (CISs), i.e. regions with more transposon insertions than expected by chance. However, the identification of CISs is affected by biases in the insertion behaviour of transposon systems. Here, we introduce Transmicron, a novel method that differs from previous methods by (i) modelling neutral insertion rates based on chromatin accessibility, transcriptional activity and sequence context and (ii) estimating oncogenic selection for each genomic region using Poisson regression to model insertion counts while controlling for neutral insertion rates. To assess the benefits of our approach, we generated a dataset applying two different transposon systems under comparable conditions. Benchmarking for enrichment of known cancer genes showed improved performance of Transmicron against state-of-the-art methods. Modelling neutral insertion rates allowed for better control of false positives and stronger agreement of the results between transposon systems. Moreover, using Poisson regression to consider intra-sample and inter-sample information proved beneficial in small and moderately-sized datasets. Transmicron is open-source and freely available. Overall, this study contributes to the understanding of transposon biology and introduces a novel approach to use this knowledge for discovering cancer driver genes.

Disease-causing mutations in genes encoding transcription factors (TFs) are a recurrent finding in hematopoietic malignancies and might involve key regulators of lineage adherence and cellular differentiation. Such mutations can affect TF-interactions with their cognate DNA-binding motifs. Whether and how TF-mutations impact upon the nature of binding to TF composite elements (CE) and influence their interaction with other TFs is unclear. Here, we report a new mechanism of TF alteration in human lymphomas with perturbed B cell identity. It is caused by a recurrent somatic missense mutation c.295T>C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cell-differentiation and -activation. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF composite elements (CEs). Furthermore, IRF4-C99R thoroughly modifies IRF4 function, by blocking IRF4-dependent plasma cell induction, and up-regulating disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single arginine mutation creates a complex switch of TF specificity and gene regulation. These data open the possibility of designing specific inhibitors to block the neomorphic, disease-causing DNA-binding activities of a mutant transcription factor.

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