In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, which recruit much of the cell’s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits it’s H3K27 acetylase binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, suggesting a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy1-3. BET bromodomain inhibitors, which have shown efficacy in several models of cancer4-6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyllysine recognition modules, leading to inhibition of oncogenic transcriptional programs7-9. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

SUMMARY Cyclin D-dependent kinases (CDK4 and CDK6) are positive regulators of cell cycle entry, and they are overactive in the majority of human cancers. However, it is currently not completely understood by which cellular mechanisms CDK4/6 promote tumorigenesis, largely due to the limited number of identified substrates. Here we performed a systematic screen for substrates of cyclin D1-CDK4 and cyclin D3-CDK6. We identified the Forkhead Box M1 (FOXM1) transcription factor as a common critical phosphorylation target. CDK4/6 stabilize and activate FOXM1, thereby maintain expression of G1/S phase genes, suppress the levels of reactive oxygen species (ROS), and protect cancer cells from senescence. Melanoma cells, unlike melanocytes, are highly reliant on CDK4/6-mediated senescence suppression, which makes them particularly susceptible to CDK4/6 inhibition.

Summary Neuroblastoma is a pediatric malignancy that typically arises in early childhood and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumors with excellent outcomes to widely metastatic disease where long-term survival is approximately 40% despite intensive therapy1. A previous genome-wide association study (GWAS) identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumor cells2. Here we sought to discover the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding sites. SNP rs2168101 G>T was the most highly associated variant (combined P=7.47×10-29, Odds Ratio 0.65, 95% CI: 0.60-0.70) and resided in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G-allele that is associated with tumor formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P=0.028) in neuroblastoma primary tumors and ablates GATA3 binding (P<0.0001). We demonstrate allelic imbalance favoring the G-containing strand in tumors heterozygous for this SNP as demonstrated both by RNA sequencing (P<0.0001) and reporter assays (P=0.002). These findings show that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumor cells.

Research over the past quarter century has identified cyclin D-dependent kinases, CDK4 and CDK6, as the major oncogenic drivers among members of the CDK superfamily. CDK4/6 are rendered hyperactive in the majority of human cancers through a multitude of genomic alterations. Sustained activation of these protein kinases provides cancer cells with the power to enter the cell cycle continuously by triggering G1-S-phase transitions and dramatically shortening the duration of the G1 phase. It has also become clear, however, that CDK4/6 effectively counter cancer cell-intrinsic tumor suppression mechanisms, senescence and apoptosis, which must be overcome during cell transformation and kept at bay throughout all stages of tumorigenesis. As a central 'node' in cellular signaling networks, cyclin D-dependent kinases sense a plethora of mitogenic signals to orchestrate specific transcriptional programs. As the complexity of the cellular signaling network regulated by these oncogenic kinases unfolds, much remains to be learned about its architecture, its dynamics and the consequences of its perturbation.

A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide new and important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods provide a powerful approach to enhance understanding of therapeutic action and characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.

Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTP is cleaved by three proteases: furin, ADAM 10, and ␥-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTP. This is followed by ␥-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates ␤-catenin, an RPTP substrate. The expression of RPTP suppresses ␤-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of ␤-catenin signaling. We also found that RPTP, another MAM family member, undergoes ␥-secretasedependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTP signaling and implicate PIC in the activation of ␤-catenin-mediated transcription.The phosphorylation of cellular proteins on tyrosine residues is reversible and regulated by the coordinated and competing actions of two enzyme families: protein tyrosine kinases and protein tyrosine phosphatases (PTPs). The PTP family is structurally diverse and includes both receptor-like and cytoplasmic enzymes. The majority of the receptor PTPs (RPTPs) contain two catalytic domains: a membrane-proximal domain (D1), which is responsible for mainly catalysis, and a membrane-distal domain (D2), which contains little or no phosphatase activity (45). The extracellular (E) portion exhibits broad structural variation. The MAM (meprin/A5 glycoprotein/PTPmu) family of RPTPs, including RPTP, -, -, -, and PCP-2, are characterized by the presence of the MAM domain at their N termini (3,6,18,29,46). Additional structural features of the extracellular portions involve one immunoglobulin-like domain and four fibronectin type III domains which are commonly found in cell adhesion molecules.Functionally, MAM family RPTPs have been recognized as homophilic cell adhesion receptors. In fact, when expressed at the cell surface, the extracellular fragments of the enzymes can mediate cell aggregation (3, 39). These trans-interactions are highly specific, i.e., RPTP proteins interact with only each other and there is no heterophilic binding between, for instance, RPTP and RPTP. Moreover, the expression of MAM family RPTPs is increased in high-cell density cultures (11, 12). These observations have led to the proposal that these enzymes could directly sense cell-cell contact (4, 45) and thereby mediate contact inhibit...

SUMMARY Cyclins and cyclin-dependent kinases (CDKs) are hyperactivated in numerous tumors. To identify means of interfering with cyclins/CDKs, we performed nine genome-wide screens for human microRNAs (miRNAs) directly regulating cell cycle proteins. We uncovered a distinct class of miRNAs that target nearly all cyclins/CDKs, which are very effective in inhibiting cancer cell proliferation. By profiling the response of over 120 human cancer cell lines, we derived an expression-based algorithm that can predict the response of primary tumors to cell cycle-targeting miRNAs. Using systemic administration of nanoparticle-formulated miRNAs, we inhibited tumor progression in seven mouse xenograft models, including three treatment-refractory patient-derived tumors, without affecting normal tissues. Our results highlight the utility of using cell cycle-targeting miRNAs for treatment of refractory cancer types.