A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

Light-Controllable Binary Switch Activation of CAR T Cells.

Major challenges to chimeric antigen receptor (CAR) T cell therapies include uncontrolled immune activity, off-tumor toxicities and tumor heterogeneity. To overcome these challenges, we engineered CARs directed against small molecules. By conjugating the same small molecule to distinct tumor-targeting antibodies, we show that small molecule specific-CAR T cells can be redirected to different tumor antigens. Such binary switches allow control over the degree of CAR T cell activity and enables simultaneous targeting of multiple tumor-associated antigens. We also demonstrate that ultraviolet light-sensitive caging of small molecules blocks CAR T cell activation. Exposure to ultraviolet light, uncaged small molecules and restored CAR T cell-mediated killing. Together, our data demonstrate that a light-sensitive caging system enables an additional level of control over tumor cell killing, which could improve the therapeutic index of CAR T cell therapies.

Genome-wide functional screen of 3'UTR variants uncovers causal variants for human disease and evolution.

3' untranslated region (3'UTR) variants are strongly associated with human traits and diseases, yet few have been causally identified. We developed the massively parallel reporter assay for 3'UTRs (MPRAu) to sensitively assay 12,173 3'UTR variants. We applied MPRAu to six human cell lines, focusing on genetic variants associated with genome-wide association studies (GWAS) and human evolutionary adaptation. MPRAu expands our understanding of 3'UTR function, suggesting that simple sequences predominately explain 3'UTR regulatory activity. We adapt MPRAu to uncover diverse molecular mechanisms at base pair resolution, including an adenylate-uridylate (AU)-rich element of LEPR linked to potential metabolic evolutionary adaptations in East Asians. We nominate hundreds of 3'UTR causal variants with genetically fine-mapped phenotype associations. Using endogenous allelic replacements, we characterize one variant that disrupts a miRNA site regulating the viral defense gene TRIM14 and one that alters PILRB abundance, nominating a causal variant underlying transcriptional changes in age-related macular degeneration.

Arhgef2 regulates mitotic spindle orientation in hematopoietic stem cells and is essential for productive hematopoiesis.

How hematopoietic stem cells (HSCs) coordinate their divisional axis and whether this orientation is important for stem cell-driven hematopoiesis is poorly understood. Single-cell RNA sequencing data from patients with Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome, show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor and determinant of mitotic spindle orientation, is specifically downregulated in SDS hematopoietic stem and progenitor cells (HSPCs). We demonstrate that transplanted Arhgef2-/- fetal liver and bone marrow cells yield impaired hematopoietic recovery and a production deficit from long-term HSCs, phenotypes that are not the result of differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output, or homing ability. Notably, these defects are functionally restored in vivo by overexpression of ARHGEF2 or its downstream activated RHOA GTPase. By using live imaging of dividing HSPCs, we show an increased frequency of misoriented divisions in the absence of Arhgef2. ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller xenografts. Together, these data demonstrate a conserved role for Arhgef2 in orienting HSPC division and suggest that HSCs may divide in certain orientations to establish hematopoiesis, the loss of which could contribute to HSC dysfunction in bone marrow failure.

Author Correction: On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

On-chip recapitulation of clinical bone marrow toxicities and patient-specific pathophysiology.

The inaccessibility of living bone marrow (BM) hampers the study of its pathophysiology under myelotoxic stress induced by drugs, radiation or genetic mutations. Here, we show that a vascularized human BM-on-a-chip (BM chip) supports the differentiation and maturation of multiple blood cell lineages over 4 weeks while improving CD34+ cell maintenance, and that it recapitulates aspects of BM injury, including myeloerythroid toxicity after clinically relevant exposures to chemotherapeutic drugs and ionizing radiation, as well as BM recovery after drug-induced myelosuppression. The chip comprises a fluidic channel filled with a fibrin gel in which CD34+ cells and BM-derived stromal cells are co-cultured, a parallel channel lined by human vascular endothelium and perfused with culture medium, and a porous membrane separating the two channels. We also show that BM chips containing cells from patients with the rare genetic disorder Shwachman-Diamond syndrome reproduced key haematopoietic defects and led to the discovery of a neutrophil maturation abnormality. As an in vitro model of haematopoietic dysfunction, the BM chip may serve as a human-specific alternative to animal testing for the study of BM pathophysiology.

Targeting the Oncogenic Long Non-coding RNA SLNCR1 by Blocking Its Sequence-Specific Binding to the Androgen Receptor.

Long non-coding RNAs (lncRNAs) are critical regulators of numerous physiological processes and diseases, especially cancers. However, development of lncRNA-based therapies is limited because the mechanisms of many lncRNAs are obscure, and interactions with functional partners, including proteins, remain uncharacterized. The lncRNA SLNCR1 binds to and regulates the androgen receptor (AR) to mediate melanoma invasion and proliferation in an androgen-independent manner. Here, we use biochemical analyses coupled with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing to show that the N-terminal domain of AR binds a pyrimidine-rich motif in an unstructured region of SLNCR1. This motif is predictive of AR binding, as we identify an AR-binding motif in lncRNA HOXA11-AS-203. Oligonucleotides that bind either the AR N-terminal domain or the AR RNA motif block the SLNCR1-AR interaction and reduce SLNCR1-mediated melanoma invasion. Delivery of oligos that block SLNCR1-AR interaction thus represent a plausible therapeutic strategy.

TGFß signaling underlies hematopoietic dysfunction and bone marrow failure in Shwachman-Diamond Syndrome.

Shwachman-Diamond Syndrome (SDS) is a rare and clinically-heterogeneous bone marrow (BM) failure syndrome caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene. Although SDS was described over 50 years ago, the molecular pathogenesis is poorly understood due, in part, to the rarity and heterogeneity of the affected hematopoietic progenitors. To address this, we used single cell RNA sequencing to profile scant hematopoietic stem and progenitor cells from SDS patients. We generated a single cell map of early lineage commitment and found that SDS hematopoiesis was left-shifted with selective loss of granulocyte-monocyte progenitors. Transcriptional targets of transforming growth factor-beta (TGFß) were dysregulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed progenitors. TGFß inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, TGFß3 and other TGFß pathway members were elevated in SDS patient blood plasma. These data establish the TGFß pathway as a novel candidate biomarker and therapeutic target in SDS and translate insights from single cell biology into a potential therapy.

Therapeutic discovery for marrow failure with MDS predisposition using pluripotent stem cells.

Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFß pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.

The lncRNA SLNCR Recruits the Androgen Receptor to EGR1-Bound Genes in Melanoma and Inhibits Expression of Tumor Suppressor p21.

Melanoma is the deadliest form of skin cancer, affecting men more frequently and severely than women. Although recent studies suggest that differences in activity of the androgen receptor (AR) underlie the observed sex bias, little is known about AR activity in melanoma. Here we show that AR and EGR1 bind to the long non-coding RNA SLNCR and increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes. ChIP-seq reveals that ligand-free AR is enriched on SLNCR-regulated melanoma genes and that AR genomic occupancy significantly overlaps with EGR1 at consensus EGR1 binding sites. We present a model in which SLNCR recruits AR to EGR1-bound genomic loci and switches EGR1-mediated transcriptional activation to repression of the tumor suppressor p21Waf1/Cip1. Our data implicate the regulatory triad of SLNCR, AR, and EGR1 in promoting oncogenesis and may help explain why men have a higher incidence of and more rapidly progressive melanomas compared with women.

Protein engineering strategies for improving the selective methylation of target CpG sites by a dCas9-directed cytosine methyltransferase in bacteria.

Mammalian gene expression is a complex process regulated in part by CpG methylation. The ability to target methylation for de novo gene regulation could have therapeutic and research applications. We have previously developed a dCas9-MC/MN protein for targeting CpG methylation. dCas9-MC/MN is composed of an artificially split M.SssI methyltransferase (MC/MN), with the MC fragment fused to a nuclease-null CRISPR/Cas9 (dCas9). Guide RNAs directed dCas9-MC/MN to methylate target sites in E. coli and human cells but also caused some low-level off-target methylation. Here, in E. coli, we show that shortening the dCas9-MC linker increases methylation of CpG sites located at select distances from the dCas9 binding site. Although a shortened linker decreased methylation of other CpGs proximal to the target site, it did not reduce off-target methylation of more distant CpG sites. Instead, targeted mutagenesis of the methyltransferase's DNA binding domain, designed to reduce DNA affinity, significantly and preferentially reduced methylation of such sites.

RATA: A method for high-throughput identification of RNA bound transcription factors.

Long non-coding RNAs (lncRNAs) regulate critical cellular processes and their dysregulation contributes to multiple diseases. Although only a few lncRNAs have defined mechanisms, many of these characterized lncRNAs interact with transcription factors to regulate gene expression, suggesting a common mechanism of action. Identifying RNA-bound transcription factors is especially challenging due to inefficient RNA immunoprecipitation and low abundance of many transcription factors. Here we describe a highly sensitive, user-friendly, and inexpensive technique called RATA (RNA-associated transcription factor array), which utilizes a MS2-aptamer pulldown strategy coupled with transcription factor activation arrays for identification of transcription factors associated with a nuclear RNA of interest. RATA requires only ~5 million cells and standard molecular biology reagents for multiplexed identification of up to 96 transcription factors in 2-3 d. Thus, RATA offers significant advantages over other technologies for analysis of RNA-transcription factor interactions.

Targeted DNA methylation in human cells using engineered dCas9-methyltransferases.

Mammalian genomes exhibit complex patterns of gene expression regulated, in part, by DNA methylation. The advent of engineered DNA methyltransferases (MTases) to target DNA methylation to specific sites in the genome will accelerate many areas of biological research. However, targeted MTases require clear design rules to direct site-specific DNA methylation and minimize the unintended effects of off-target DNA methylation. Here we report a targeted MTase composed of an artificially split CpG MTase (sMTase) with one fragment fused to a catalytically-inactive Cas9 (dCas9) that directs the functional assembly of sMTase fragments at the targeted CpG site. We precisely map RNA-programmed DNA methylation to targeted CpG sites as a function of distance and orientation from the protospacer adjacent motif (PAM). Expression of the dCas9-sMTase in mammalian cells led to predictable and efficient (up to ~70%) DNA methylation at targeted sites. Multiplexing sgRNAs enabled targeting methylation to multiple sites in a single promoter and to multiple sites in multiple promoters. This programmable de novo MTase tool might be used for studying mechanisms of initiation, spreading and inheritance of DNA methylation, and for therapeutic gene silencing.

PRMT1-Mediated Translation Regulation Is a Crucial Vulnerability of Cancer.

Through an shRNA screen, we identified the protein arginine methyltransferase Prmt1 as a vulnerable intervention point in murine p53/Rb-null osteosarcomas, the human counterpart of which lacks effective therapeutic options. Depletion of Prmt1 in p53-deficient cells impaired tumor initiation and maintenance in vitro and in vivo Mechanistic studies reveal that translation-associated pathways were enriched for Prmt1 downstream targets, implicating Prmt1 in translation control. In particular, loss of Prmt1 led to a decrease in arginine methylation of the translation initiation complex, thereby disrupting its assembly and inhibiting translation. p53/Rb-null cells were sensitive to p53-induced translation stress, and analysis of human cancer cell line data from Project Achilles further revealed that Prmt1 and translation-associated pathways converged on the same functional networks. We propose that targeted therapy against Prmt1 and its associated translation-related pathways offer a mechanistic rationale for treatment of osteosarcomas and other cancers that exhibit dependencies on translation stress response. Cancer Res; 77(17); 4613-25. ©2017 AACR.

The microRNA miR-31 inhibits CD8+ T cell function in chronic viral infection.

During infection, antigen-specific T cells undergo tightly regulated developmental transitions controlled by transcriptional and post-transcriptional regulation of gene expression. We found that the microRNA miR-31 was strongly induced by activation of the T cell antigen receptor (TCR) in a pathway involving calcium and activation of the transcription factor NFAT. During chronic infection with lymphocytic choriomeningitis virus (LCMV) clone 13, miR-31-deficent mice recovered from clinical disease, while wild-type mice continued to show signs of disease. This disease phenotype was explained by the presence of larger numbers of cytokine-secreting LCMV-specific CD8+ T cells in miR-31-deficent mice than in wild-type mice. Mechanistically, miR-31 increased the sensitivity of T cells to type I interferons, which interfered with effector T cell function and increased the expression of several proteins related to T cell dysfunction during chronic infection. These studies identify miR-31 as an important regulator of T cell exhaustion in chronic infection.

Differential Regulation of the Melanoma Proteome by eIF4A1 and eIF4E.

Small molecules and antisense oligonucleotides that inhibit the translation initiation factors eIF4A1 and eIF4E have been explored as broad-based therapeutic agents for cancer treatment, based on the frequent upregulation of these two subunits of the eIF4F cap-binding complex in many cancer cells. Here, we provide support for these therapeutic approaches with mechanistic studies of eIF4F-driven tumor progression in a preclinical model of melanoma. Silencing eIF4A1 or eIF4E decreases melanoma proliferation and invasion. There were common effects on the level of cell-cycle proteins that could explain the antiproliferative effects in vitro Using clinical specimens, we correlate the common cell-cycle targets of eIF4A1 and eIF4E with patient survival. Finally, comparative proteomic and transcriptomic analyses reveal extensive mechanistic divergence in response to eIF4A1 or eIF4E silencing. Current models indicate that eIF4A1 and eIF4E function together through the 5'UTR to increase translation of oncogenes. In contrast, our data demonstrate that the common effects of eIF4A1 and eIF4E on translation are mediated by the coding region and 3'UTR. Moreover, their divergent effects occur through the 5'UTR. Overall, our work shows that it will be important to evaluate subunit-specific inhibitors of eIF4F in different disease contexts to fully understand their anticancer actions. Cancer Res; 77(3); 613-22. ©2016 AACR.

Bidirectional cross talk between patient-derived melanoma and cancer-associated fibroblasts promotes invasion and proliferation.

Tumor-stroma interactions are critical for epithelial-derived tumors, and among the stromal cell types, cancer-associated fibroblasts (CAFs) exhibit multiple functions that fuel growth, dissemination, and drug resistance. However, these interactions remain insufficiently characterized in non-epithelial tumors such as malignant melanoma. We generated monocultures of melanoma cells and matching CAFs from patients' metastatic lesions, distinguished by oncogenic drivers and immunoblotting of characteristic markers. RNA sequencing of CAFs revealed a homogenous epigenetic program that strongly resembled the signatures from epithelial cancers, including enrichment for an epithelial-to-mesenchymal transition (EMT). Melanoma CAFs in monoculture displayed robust invasive behavior while patient-derived melanoma monocultures showed very little invasiveness. Instead, melanoma cells showed increased invasion when co-cultured with CAFs. In turn, CAFs showed increased proliferation when exposed to melanoma conditioned media (CM), mediated in part by melanoma-secreted transforming growth factor-alpha that acted on CAFs via the epidermal growth factor receptor. This study provides evidence that bidirectional interactions between melanoma and CAFs regulate progression of metastatic melanoma.

The lncRNA SLNCR1 Mediates Melanoma Invasion through a Conserved SRA1-like Region.

Long non-coding RNAs (lncRNAs) have been implicated in numerous physiological processes and diseases, most notably cancers. However, little is known about the mechanism of many functional lncRNAs. We identified an abundantly expressed lncRNA associated with decreased melanoma patient survival. Increased expression of this lncRNA, SLNCR1, mediates melanoma invasion through a highly conserved sequence similar to that of the lncRNA SRA1. Using a sensitive technique we term RATA (RNA-associated transcription factor array), we show that the brain-specific homeobox protein 3a (Brn3a) and the androgen receptor (AR) bind within and adjacent to SLNCR1's conserved region, respectively. SLNCR1, AR, and Brn3a are specifically required for transcriptional activation of matrix metalloproteinase 9 (MMP9) and increased melanoma invasion. Our observations directly link AR to melanoma invasion, possibly explaining why males experience more melanoma metastases and have an overall lower survival in comparison to females.