HIGHLIGHTS A super-enhancer drives the expression of lncRNA UCA1 in EOC Inactivation of UCA1 impairs tumor growth in vivo UCA1 activates transcription coactivator YAP and its target genes UCA1 promotes YAP dephosphorylation and nuclear translocation via AMOTp130 Lin et al., iScience 17, 242-255 SUMMARYLong noncoding RNAs (lncRNAs) have emerged as critical regulators of tumorigenesis, and yet their mechanistic roles remain challenging to characterize. Here, we integrate functional proteomics with lncRNA-interactome profiling to characterize Urothelial Cancer Associated 1 (UCA1), a candidate driver of ovarian cancer development. Reverse phase protein array (RPPA) analysis indicates that UCA1 activates transcription coactivator YAP and its target genes. In vivo RNA antisense purification (iRAP) of UCA1 interacting proteins identified angiomotin (AMOT), a known YAP regulator, as a direct binding partner. Loss-of-function experiments show that AMOT mediates YAP activation by UCA1, as UCA1 enhances the AMOT-YAP interaction to promote YAP dephosphorylation and nuclear translocation. Together, we characterize UCA1 as a lncRNA regulator of Hippo-YAP signaling and highlight the UCA1-AMOT-YAP signaling axis in ovarian cancer development.We used RPPAs to profile changes in protein abundance and phosphorylation following UCA1 KO. The most differentially expressed proteins between WT and UCA1 KO cells included phosphorylated YAP at iScience 17, 242-255, July 26, 2019 243 A B C E D 244 iScience 17, 242-255,

Genome-wide association studies have reported eleven regions conferring risk of high-grade serous epithelial ovarian cancer (HGSOC). Expression quantitative trait locus (eQTL) analyses can identify candidate susceptibility genes at risk loci. Here we evaluate cis-eQTL associations at 47 regions associated with HGSOC risk (P≤10−5). For three cis-eQTL associations (P<1.4×10−3, FDR<0.05) at 1p36 (CDC42), 1p34 (CDCA8) and 2q31 (HOXD9), we evaluate the functional role of each candidate by perturbing expression of each gene in HGSOC precursor cells. Overexpression of HOXD9 increases anchorage-independent growth, shortens population-doubling time and reduces contact inhibition. Chromosome conformation capture identifies an interaction between rs2857532 and the HOXD9 promoter, suggesting this SNP is a leading causal variant. Transcriptomic profiling after HOXD9 overexpression reveals enrichment of HGSOC risk variants within HOXD9 target genes (P=6×10−10 for risk variants (P<10−4) within 10kb of a HOXD9 target gene in ovarian cells), suggesting a broader role for this network in genetic susceptibility to HGSOC.

Genome-wide association studies have identified several risk associations for ovarian carcinomas (OC) but not for mucinous ovarian carcinomas (MOC). Genotypes from OC cases and controls were imputed into the 1000 Genomes Project reference panel. Analysis of 1,644 MOC cases and 21,693 controls identified three novel risk associations: rs752590 at 2q13 (P = 3.3 × 10−8), rs711830 at 2q31.1 (P = 7.5 × 10−12) and rs688187 at 19q13.2 (P = 6.8 × 10−13). Expression Quantitative Trait Locus (eQTL) analysis in ovarian and colorectal tumors (which are histologically similar to MOC) identified significant eQTL associations for HOXD9 at 2q31.1 in ovarian (P = 4.95 × 10−4, FDR = 0.003) and colorectal (P = 0.01, FDR = 0.09) tumors, and for PAX8 at 2q13 in colorectal tumors (P = 0.03, FDR = 0.09). Chromosome conformation capture analysis identified interactions between the HOXD9 promoter and risk SNPs at 2q31.1. Overexpressing HOXD9 in MOC cells augmented the neoplastic phenotype. These findings provide the first evidence for MOC susceptibility variants and insights into the underlying biology of the disease.

Mesenchymal stem cells (MSCs), as well as osteoblastic cells derived from these MSCs, have been shown to be key components of the hematopoietic stem cell (HSC) niche. In this study, we wished to examine whether other cell types that are known to differentiate from MSCs similarly regulate the stem cell niche, namely cells of the adipocyte lineage. Recent studies have examined the role that adipocytes play in the biology of the HSCs in different bone locations and in transplantation settings; however, none have examined their role under homeostatic conditions. We compared the ability of adipocytic and nonadipocytic cell lines to support primitive hematopoietic cells in vitro. Preadipocytic cell lines demonstrated enhanced support of hematopoietic cells. Similarly, primary bone marrow (BM) cells treated with troglitazone, a drug that enhances adipogenesis, also demonstrated augmented support over control-treated stromal cells. We further examined the effects of increased adipocyte number in vivo under homeostatic conditions using troglitazone treatment and found that these alterations had no effect on HSC frequency. Taken together, we demonstrate that cells of the adipocyte lineage promote the ability of stromal cells to support primitive hematopoietic cells in vitro, yet alterations of adipocyte number and volume in vivo have no effect. These data suggest that adipocytes are not a component of the adult BM HSC niche under homeostatic conditions.

SummaryPrior efforts to identify regulators of hematopoietic stem cell physiology have relied mainly on candidate gene approaches with genetically modified mice. Here we used a genome-wide association study (GWAS) strategy with the hybrid mouse diversity panel to identify the genetic determinants of hematopoietic stem/progenitor cell (HSPC) frequency. Among 108 strains, we observed ∼120- to 300-fold variation in three HSPC populations. A GWAS analysis identified several loci that were significantly associated with HSPC frequency, including a locus on chromosome 5 harboring the homeodomain-only protein gene (Hopx). Hopx previously had been implicated in cardiac development but was not known to influence HSPC biology. Analysis of the HSPC pool in Hopx−/− mice demonstrated significantly reduced cell frequencies and impaired engraftment in competitive repopulation assays, thus providing functional validation of this positional candidate gene. These results demonstrate the power of GWAS in mice to identify genetic determinants of the hematopoietic system.

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three-dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early-stage EOC, generated by expressing CMYC and KRAS G12V in TERT-immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography-tandem mass spectrometry. Thirty-seven and 55 proteins were differentially expressed by CMYC and CMYC1KRAS G12V expressing cells respectively (p < 0.05; >2-fold). We evaluated expression of the top candidate biomarkers in~210 primary EOCs: CHI3L1 and FKBP4 are both expressed by >96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p 5 0.042 and p 5 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p 5 0.0001) and suboptimal debulking (p 5 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p 5 3 3 10 24 , p 5 0.016, p 5 0.010, respectively). We show in vitro cell biology models of early-stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease.Despite recent advances in surgery and chemotherapy, epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy, mainly due to the absence of specific symptoms and a lack of effective screening tools. The majority of patients present with advanced stage disease where the 5-year survival rate is only 27%. For advanced stage tumours, recurrence rates are over 60%, and 20% of these patients respond poorly to platinum-based chemotherapy. Therefore, detecting patients with early EOC continues to be an urgent clinical need and it remains a clinical priority to detect highgrade serous EOC early during disease development.Given the difficulties accessing the ovary for biopsy and the rapid rate at which the most aggressive EOC subtypes progress, the identification of clinical biomarkers detectable in the blood would represent a significant advance for the identification of patients with EOC. Currently the most widely used ovarian cancer biomarker is serum CA125, and this marker is particularly good at detecting disease recurrence. A second marker, HE4, was approved in 2009 for monitoring ovarian cancer progression, and can be elevated

Background: Multiple myeloma (MM) is a usually fatal malignancy of plasma cells, with no current therapy considered curative. About 15% of patients diagnosed with MM are stratified as high risk with poor treatment outcomes and short (2-3 years) survival from diagnosis. Standard risk patients tend to live longer but undergo chronic and/or high intensity therapy and likely experience a relapsing and remitting disease pattern. Therefore, there is still a considerable unmet need for innovative therapies that improve outcomes in MM. One such approach is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against malignant cells. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. KITE-585 was developed as a CAR T cell immunotherapy product candidate directed against B cell maturation antigen (BCMA). BCMA is nearly ubiquitously expressed on MM cells, plasma cells and subsets of mature B cells, but with limited or absent expression on other tissues. Methods: We generated &gt;50 fully human IgGs directed against BCMA using the BCMA protein as antigen and selection criteria including affinity, cross-reactivity and poly-specificity. Following assessment of the binding of the IgGs to a MM cell line known to express BCMA, &gt;10 IgGs were identified that met the criteria for affinity and selectivity and had a &gt;50-fold binding over background. The 8 IgGs that demonstrated the highest specific binding were then sequence-converted to single-chain variable fragments (scFvs) and incorporated into CARs. Results: In all but one case, human T cells engineered to express these CAR constructs exhibited specific cytolytic activity against MM cell lines (NCI-H929 and MM.1s). These CAR T cells demonstrated killing efficiencies of &gt;95% at effector:target ratios of 1:1 over a 24-hour period. Similarly antigen-specific production of inflammatory cytokines was observed in response to target cell lines in vitro. Assessment of antigen-dependent proliferation over a 5 day period revealed &gt;80% proliferation in the 7 constructs that showed cytolytic activity in vitro. Multiple different anti-BCMA CAR constructs representing distinct epitope binding bins of BCMA were then selected for in vivo evaluation. In two disseminated tumor models of luciferase labeled NCI-H929 or MM.1s cells injected intravenously (i.v.), a single i.v. injection of anti-BCMA CAR T-cells delayed the progression of disease and significantly increased survival when compared to control treatment. Conclusions: The results of these studies highlight the potential of targeting BCMA with adoptive transfer of engineered T cells for the treatment of MM. Given these positive findings, progress towards Phase 1 clinical studies in MM patients with KITE-585 is continuing. Citation Format: Gregor B. Adams, Jun Feng, Atefah Ghogha, Armen Mardiros, Jodi Murakami, Tammy Phung, Ruben Rodriguez, Stuart Sievers, Tassja J. Spindler, Jed Wiltzius, Clare Yarka, Sean C. Yoder, Tony Polverino. Development of KITE-585: A fully human BCMA CAR T-cell therapy for the treatment of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4979. doi:10.1158/1538-7445.AM2017-4979

Background: Immunotherapy has provided treatment options for cancers that are otherwise refractory to standard approaches. One such technique is to use adoptive transfer of engineered autologous T cells expressing a chimeric antigen receptor (CAR) directed against a tumor antigen. The efficacy of CAR T cells directed against hematological malignancies, particularly CD19-expressing B cell leukemia and lymphomas, has been demonstrated in multiple clinical studies. The success of this approach has prompted development of CAR T cells directed to different tumor antigens for other tumor types. To ensure the selectivity and specificity of the CAR T cells against their intended target, screening methods need to be employed. Multiple myeloma is an incurable malignancy of plasma cells. B-cell maturation antigen (BCMA), also known as tumor necrosis factor superfamily member 17 (TNFRSF17) is nearly ubiquitously expressed on multiple myeloma cells, plasma cells and subsets of mature B cells. Methods: In order to screen for the specificity of novel CAR T cells directed against BCMA, we utilized a cell microarray platform developed by Retrogenix. In this screen, approximately 4500 human plasma membrane proteins (representing up to 75% of the human plasma membrane proteome) are individually expressed in human HEK293 cells. Fluorescently labeled CAR T cells, which showed cytolytic activity against MM cell lines expressing BCMA, were applied to the cell microarray and specific binding of the CAR T cells to target cells was determined. Results: Primary hits were sequenced to confirm identity and secondary specificity screens were performed on the identified hits. Specific binding of both mock transduced and BCMA CAR transduced T cells were confirmed for different plasma membrane proteins expressed from the HEK293 cells. These included known T cell interactors, such as ICOSLG, CD244 and CD86, where binding is proposed to be independent of CAR expression. Subtracting the hits of the mock transduced T cells from the BCMA CAR T cells demonstrated specific binding of the CAR T cells to BCMA. Utilizing the fully human IgGs directed against BCMA from which the single-chain variable fragments (scFvs) of the CARs were derived, we further confirmed specific binding to BCMA in additional secondary screens. Additionally, a lack of off-target binding of the fully human IgGs to normal tissue was demonstrated in a tissue cross reactivity screen. Conclusions: These studies highlight the tractability of this cell microarray approach for determining the specificity of novel CAR constructs expressed in T cell. Demonstrating the selectivity and specificity of anti-BMCA CAR T cells further supports the progression of KITE-585 towards Phase 1 clinical studies in MM patients. Citation Format: Gregor B. Adams, Jun Feng, Atefeh Ghogha, Armen Mardiros, Ruben Rodriguez, Tassja J. Spindler, Jed Wiltzius, Tony Polverino. Selectivity and specificity of engineered T cells expressing KITE-585, a chimeric antigen receptor targeting B-cell maturation antigen (BCMA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2135. doi:10.1158/1538-7445.AM2017-2135