EVI1 drives leukemogenesis through aberrant ERG activation.

Chromosomal rearrangements involving the MDS1 and EVI1 complex locus (MECOM) on chromosome 3q26 define an aggressive subtype of acute myeloid leukemia (AML) that is associated with chemotherapy resistance and dismal prognosis. Established treatment regimens commonly fail in these patients, therefore, there is an urgent need for new therapeutic concepts that will require a better understanding of the molecular and cellular functions of the ecotropic viral integration site 1 (EVI1) oncogene. To characterize gene regulatory functions of EVI1 and associated dependencies in AML, we developed experimentally tractable human and murine disease models, investigated the transcriptional consequences of EVI1 withdrawal in vitro and in vivo, and performed the first genome-wide CRISPR screens in EVI1-dependent AML. By integrating conserved transcriptional targets with genetic dependency data, we identified and characterized the ETS transcription factor ERG as a direct transcriptional target of EVI1 that is aberrantly expressed and selectively required in both human and murine EVI1-driven AML. EVI1 controls the expression of ERG and occupies a conserved intragenic enhancer region in AML cell lines and samples from patients with primary AML. Suppression of ERG induces terminal differentiation of EVI1-driven AML cells, whereas ectopic expression of ERG abrogates their dependence on EVI1, indicating that the major oncogenic functions of EVI1 are mediated through aberrant transcriptional activation of ERG. Interfering with this regulatory axis may provide entry points for the development of rational targeted therapies.

Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell-like Program Conserved in HPV-Positive Cancers.

The skin is exposed to viral pathogens, but whether they contribute to the oncogenesis of skin cancers has not been systematically explored. Here we investigated 19 skin tumor types by analyzing off-target reads from commonly available next-generation sequencing data for viral pathogens. We identified human papillomavirus 42 (HPV42) in 96% (n = 45/47) of digital papillary adenocarcinoma (DPA), an aggressive cancer occurring on the fingers and toes. We show that HPV42, so far considered a nononcogenic, "low-risk" HPV, recapitulates the molecular hallmarks of oncogenic, "high-risk" HPVs. Using machine learning, we find that HPV-driven transformation elicits a germ cell-like transcriptional program conserved throughout all HPV-driven cancers (DPA, cervical carcinoma, and head and neck cancer). We further show that this germ cell-like transcriptional program, even when reduced to the top two genes (CDKN2A and SYCP2), serves as a fingerprint of oncogenic HPVs with implications for early detection, diagnosis, and therapy of all HPV-driven cancers. SIGNIFICANCE: We identify HPV42 as a uniform driver of DPA and add a new member to the short list of tumorigenic viruses in humans. We discover that all oncogenic HPVs evoke a germ cell-like transcriptional program with important implications for detecting, diagnosing, and treating all HPV-driven cancers. See related commentary by Starrett et al., p. 17. This article is highlighted in the In This Issue feature, p. 1.

DNA replication timing directly regulates the frequency of oncogenic chromosomal translocations.

Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic Myc translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at Myc causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.

AKIRIN2 controls the nuclear import of proteasomes in vertebrates.

Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.

Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumor microenvironment and cross-resistance to immunotherapy in melanoma.

How targeted therapies and immunotherapies shape tumors, and thereby influence subsequent therapeutic responses, is poorly understood. In the present study, we show, in melanoma patients and mouse models, that when tumors relapse after targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies. We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumor microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response. Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumors to immunotherapy. Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which not only is reactivated, but also exhibits an increased transcriptional output that drives immune evasion. Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.

Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters.

We developed a functional lineage tracing tool termed CaTCH (CRISPRa tracing of clones in heterogeneous cell populations). CaTCH combines precise clonal tracing of millions of cells with the ability to retrospectively isolate founding clones alive before and during selection, allowing functional experiments. Using CaTCH, we captured rare clones representing as little as 0.001% of a population and investigated the emergence of resistance to targeted melanoma therapy in vivo.

LSD1 inhibition induces differentiation and cell death in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly aggressive, neuroendocrine skin cancer that lacks actionable mutations, which could be utilized for targeted therapies. Epigenetic regulators governing cell identity may represent unexplored therapeutic entry points. Here, we targeted epigenetic regulators in a pharmacological screen and discovered that the lysine-specific histone demethylase 1A (LSD1/KDM1A) is required for MCC growth in vitro and in vivo. We show that LSD1 inhibition in MCC disrupts the LSD1-CoREST complex leading to displacement and degradation of HMG20B (BRAF35), a poorly characterized complex member that is essential for MCC proliferation. Inhibition of LSD1 causes derepression of transcriptional master regulators of the neuronal lineage, activates a gene expression signature resembling normal Merkel cells, and induces cell cycle arrest and cell death. Our study unveils the importance of LSD1 for maintaining cellular plasticity and proliferation in MCC. There is also growing evidence that cancer cells exploit cellular plasticity and dedifferentiation programs to evade destruction by the immune system. The combination of LSD1 inhibitors with checkpoint inhibitors may thus represent a promising treatment strategy for MCC patients.

Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.

Canonical NF-κB signaling in myeloid cells promotes lung metastasis in a mouse breast cancer model.

An inflammatory tumor microenvironment is a common characteristic of solid tumors. It is the result of a complex interplay between tumor cells, tumor infiltrating immune cells and other stromal cells. Myeloid cells in the tumor microenvironment are considered major drivers of tumor progression and metastasis and increased numbers of these cells are associated with poor prognosis in various cancer patients. The transcription factor NF-κB is considered the master regulator of inflammatory gene expression and immune cell function. Its activation in various cells of the tumor microenvironment contributes essentially to tumorigenesis. In the present study, the role of canonical NF-κB signaling in myeloid cells in metastatic breast cancer was addressed by myeloid-specific deletion of Ikkß in the MMTV polyoma middle T (PyMT) mouse model. Ikkß deletion in myeloid cells did not affect primary mammary tumor growth but significantly reduced lung metastasis. While dissemination from the primary tumor was unaltered, myeloid-specific Ikkß loss resulted in a strong up-regulation of pro-inflammatory genes and changes in immune cell populations in the lung, creating a tumor-suppressive microenvironment at the distant site. Thus, canonical NF-κB signaling in myeloid cells creates a permissive lung microenvironment that supports breast to lung metastasis.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

Myeloid Cell-Derived Reactive Oxygen Species Induce Epithelial Mutagenesis.

Increased oxidative stress has been suggested to initiate and promote tumorigenesis by inducing DNA damage and to suppress tumor development by triggering apoptosis and senescence. The contribution of individual cell types in the tumor microenvironment to these contrasting effects remains poorly understood. We provide evidence that during intestinal tumorigenesis, myeloid cell-derived H2O2 triggers genome-wide DNA mutations in intestinal epithelial cells to stimulate invasive growth. Moreover, increased reactive oxygen species (ROS) production in myeloid cells initiates tumor growth in various organs also in the absence of a carcinogen challenge in a paracrine manner. Our data identify an intricate crosstalk between myeloid cell-derived ROS molecules, oxidative DNA damage, and tumor necrosis factor α-mediated signaling to orchestrate a tumor-promoting microenvironment causing invasive cancer.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

First clinical evaluation of the C-MAC D-Blade videolaryngoscope during routine and difficult intubation.

In the present preliminary study we evaluated the C-MAC® D-Blade (Karl Storz, Tuttlingen, Germany), a new videolaryngoscopic C-MAC blade for difficult intubation, during both routine and difficult intubations. First, both the conventional direct laryngoscopy and the D-Blade were used in 15 consecutive patients with normal airways during routine induction of anesthesia. Second, the D-Blade was used as a rescue device in 20 of 300 (6.7%) consecutive patients, when conventional direct laryngoscopy failed. In the 15 patients during routine induction of anesthesia, with direct laryngoscopy, a Cormack-Lehane (C/L) grade 1 and grade 2a view was seen in 7 and 8 patients, respectively. It was possible to insert the D-Blade and to get a video view of the glottis on the first attempt in all patients; with the D-Blade, all 15 patients had a C/L 1 view. The time to successful intubation with the D-Blade was 15 (8-26) seconds (median (range)). In the 20 patients, in whom unexpected difficulty with direct laryngoscopy was observed, C/L grades 3 and 4 were present in 15 and 5 patients, respectively. With the use of the D-Blade, indirect C/L video view improved to C/L class 1 in 15 patients, and to 2a in 5 patients, respectively. The time from touching the laryngoscope to optimal laryngoscopic view was 11 (5-45) seconds and for successful intubation 17 (3-80) seconds. In all 35 patients, with the D-Blade no direct view of the glottis was possible and subsequently a semiflexible tube guide was required.