Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties.

Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma.

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS.

Transcriptional Silencing of Confers a Dependency on Fanconi Anemia Proteins in Acute Myeloid Leukemia.

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitination machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T and FANCL as unique dependencies in AML.

Salt-inducible kinase inhibition suppresses acute myeloid leukemia progression in vivo.

Lineage-defining transcription factors (TFs) are compelling targets for leukemia therapy, yet they are among the most challenging proteins to modulate directly with small molecules. We previously used CRISPR screening to identify a salt-inducible kinase 3 (SIK3) requirement for the growth of acute myeloid leukemia (AML) cell lines that overexpress the lineage TF myocyte enhancer factor (MEF2C). In this context, SIK3 maintains MEF2C function by directly phosphorylating histone deacetylase 4 (HDAC4), a repressive cofactor of MEF2C.

A Transcription Factor Addiction in Leukemia Imposed by the MLL Promoter Sequence.

The Mixed Lineage Leukemia gene (MLL) is altered in leukemia by chromosomal translocations to produce oncoproteins composed of the MLL N-terminus fused to the C-terminus of a partner protein. Here, we used domain-focused CRISPR screening to identify ZFP64 as an essential transcription factor in MLL-rearranged leukemia. We show that the critical function of ZFP64 in leukemia is to maintain MLL expression via binding to the MLL promoter, which is the most enriched location of ZFP64 occupancy in the human genome.

TP63-Mediated Enhancer Reprogramming Drives the Squamous Subtype of Pancreatic Ductal Adenocarcinoma.

The aberrant expression of squamous lineage markers in pancreatic ductal adenocarcinoma (PDA) has been correlated with poor clinical outcomes. However, the functional role of this putative transdifferentiation event in PDA pathogenesis remains unclear. Here, we show that expression of the transcription factor TP63 (ΔNp63) is sufficient to install and sustain the enhancer landscape and transcriptional signature of the squamous lineage in human PDA cells.

POU2F3 is a master regulator of a tuft cell-like variant of small cell lung cancer.

Small cell lung cancer (SCLC) is widely considered to be a tumor of pulmonary neuroendocrine cells; however, a variant form of this disease has been described that lacks neuroendocrine features. Here, we applied domain-focused CRISPR screening to human cancer cell lines to identify the transcription factor (TF) POU2F3 (POU class 2 homeobox 3; also known as SKN-1a/OCT-11) as a powerful dependency in a subset of SCLC lines. An analysis of human SCLC specimens revealed that POU2F3 is expressed exclusively in variant SCLC tumors that lack expression of neuroendocrine markers and instead express markers of a chemosensory lineage known as tuft cells.

LKB1, Salt-Inducible Kinases, and MEF2C Are Linked Dependencies in Acute Myeloid Leukemia.

The lineage-specific transcription factor (TF) MEF2C is often deregulated in leukemia. However, strategies to target this TF have yet to be identified. Here, we used a domain-focused CRISPR screen to reveal an essential role for LKB1 and its Salt-Inducible Kinase effectors (SIK3, in a partially redundant manner with SIK2) to maintain MEF2C function in acute myeloid leukemia (AML).

A TFIID-SAGA Perturbation that Targets MYB and Suppresses Acute Myeloid Leukemia.

Targeting of general coactivators is an emerging strategy to interfere with oncogenic transcription factors (TFs). However, coactivator perturbations often lead to pleiotropic effects by influencing numerous TFs. Here we identify TAF12, a subunit of TFIID and SAGA coactivator complexes, as a selective requirement for acute myeloid leukemia (AML) progression.

Enhancer Reprogramming Promotes Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancies, owing in part to its propensity for metastasis. Here, we used an organoid culture system to investigate how transcription and the enhancer landscape become altered during discrete stages of disease progression in a PDA mouse model. This approach revealed that the metastatic transition is accompanied by massive and recurrent alterations in enhancer activity.

NSD3-Short Is an Adaptor Protein that Couples BRD4 to the CHD8 Chromatin Remodeler.

The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain.

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains.

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.

V-fusion: a convenient, nontoxic method for cell fusion.

Cell-to-cell fusion (cell fusion) is a fundamental biological process that also has been used as a versatile experimental tool to dissect a variety of cellular mechanisms, including the consequences of cell fusion itself, and to produce cells with desired properties, such as hybridomas and reprogrammed progenitors. However, current methods of cell fusion are not satisfactory because of their toxicity, inefficiency, or lack of flexibility. We describe a simple, versatile, scalable, and nontoxic approach that we call V-fusion, as it is based on the ability of the vesicular stomatitis virus G protein (VSV-G), a viral fusogen of broad tropism, to become rapidly and reversibly activated.