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Somatic copy-number alterations (CNA) promote cancer, but the underlying driver genes may not be comprehensively identified if only the functions of the encoded proteins are considered. mRNAs can act as competitive endogenous RNAs (ceRNA), which sponge miRNAs to posttranscriptionally regulate gene expression in a protein coding–independent manner. We investigated the contribution of ceRNAs to the oncogenic effects of CNAs. Chromosome 1q gains promoted melanoma progression and metastasis at least in part through overexpression of three mRNAs with ceRNA activity: CEP170, NUCKS1, and ZC3H11A. These ceRNAs enhanced melanoma metastasis by sequestering tumor suppressor miRNAs. Orthogonal genetic assays with miRNA inhibitors and target site blockers, along with rescue experiments, demonstrated that miRNA sequestration is critical for the oncogenic effects of CEP170, NUCKS1, and ZC3H11A mRNAs. Furthermore, chromosome 1q ceRNA-mediated miRNA sequestration alleviated the repression of several prometastatic target genes. This regulatory RNA network was evident in other cancer types, suggesting chromosome 1q ceRNA deregulation as a common driver of cancer progression. Taken together, this work demonstrates that ceRNAs mediate the oncogenicity of somatic CNAs. Significance: The function of CEP170, NUCKS1, and ZC3H11A mRNAs as competitive endogenous RNAs that sequester tumor suppressor microRNAs underlies the oncogenic activity of chromosome 1q gains.

Obesity is a risk factor for the development of post-menopausal breast cancer. Breast white adipose tissue (WAT) inflammation, which is commonly found in women with excess body fat, is also associated with increased breast cancer risk. Both local and systemic effects are probably important for explaining the link between excess body fat, adipose inflammation and breast cancer. The first goal of this cross-sectional study of 196 women was to carry out transcriptome profiling to define the molecular changes that occur in the breast related to excess body fat and WAT inflammation. A second objective was to determine if commonly measured blood biomarkers of risk and prognosis reflect molecular changes in the breast. Breast WAT inflammation was assessed by immunohistochemistry. Bulk RNA-sequencing was carried out to assess gene expression in non-tumorous breast. Obesity and WAT inflammation were associated with a large number of differentially expressed genes and changes in multiple pathways linked to the development and progression of breast cancer. Altered pathways included inflammatory response, complement, KRAS signaling, tumor necrosis factor α signaling via NFkB, interleukin (IL)6-JAK-STAT3 signaling, epithelial mesenchymal transition, angiogenesis, interferon γ response and transforming growth factor (TGF)-β signaling. Increased expression of several drug targets such as aromatase, TGF-β1, IDO-1 and PD-1 were observed. Levels of various blood biomarkers including high sensitivity C-reactive protein, IL6, leptin, adiponectin, triglycerides, high-density lipoprotein cholesterol and insulin were altered and correlated with molecular changes in the breast. Collectively, this study helps to explain both the link between obesity and breast cancer and the utility of blood biomarkers for determining risk and prognosis.

Heat shock proteins (HSPs) are evolutionary conserved 'stress-response' proteins that facilitate cell survival against various adverse conditions. HSP-mediated cytoprotection was hitherto reported to occur principally in two ways. Firstly, HSPs interact directly or indirectly with apoptosis signaling components and suppress apoptosis. Secondly, through chaperon activity, HSPs suppress proteotoxicity and maintain protein-homeostasis. Recent studies highlight the interaction of HSPs with cytoplasmic stress granules (SGs). SGs are conserved cytoplasmic mRNPs granules that aid in cell survival under stressful conditions. We primarily aim to describe the distinct cell survival strategy mediated by HSPs as the crucial regulators of SGs assembly and disassembly. Based on the growing evidence, HSPs and associated co-chaperones act as important determinants of SG assembly, composition and dissolution. Under cellular stress, as a 'stress-coping mechanism', the formation of SGs reprograms protein translation machinery and modulates signaling pathways indispensable for cell survival. Besides their role in suppressing apoptosis, HSPs also regulate protein-homeostasis by their chaperone activity as well as by their tight regulation of SG dynamics. The intricate molecular signaling in and around the nexus of HSPs-SGs and its importance in diseases has to be unearthed. These studies have significant implications in the management of chronic diseases such as cancer and neurodegenerative diseases where SGs possess pathological functions.

Chromosome instability (CIN) is an attribute of cancer cells that has yet to be fully exploited therapeutically, despite its prevalence across many tumor types and lack of occurrence in normal cells. To identify genes uniquely essential to CINhigh cells, we mined the Cancer Dependency map (DepMap) for genes required for proliferation of tumor cell lines with high levels of copy number aberrations, an indicator of CIN. This analysis identified KIF18A, a mitotic kinesin also identified by others as a vulnerability of aneuploid and whole genome doubled cells. Genetic disruption of KIF18A with RNAi confirmed its necessity in CINhigh cells, and these findings translated to in vivo reduction in tumor growth, illustrating the potential of KIF18A as a therapeutic target. High-throughput screening of a chemical library for inhibitors of KIF18A motor domain’s ATPase activity identified an ATP-noncompetitive hit that was optimized for potency, selectivity, and pharmacokinetic properties. These optimized inhibitors are orally bioavailable, exhibit sub-nM potency, and are highly selective for KIF18A vs. other kinesins. They also display a long drug-target residence time, a unique property that allows cells to be trapped in mitosis long enough to result in cell death. Mechanistically, inhibition of KIF18A’s ATPase activity renders KIF18A unable to localize to the metaphase plate, instead becoming trapped at the spindle poles. This inability to translocate results in defects in chromosome congression, evidenced by a less compact metaphase plate, and an accumulation of cells in G2/M. Treatment of CIN cancer cells with these inhibitors results in a proliferative defect due in part to induction of programmed cell death. In contrast to the effect in cancer cells, KIF18A inhibition has no effect on rapidly proliferating non-transformed epithelial cells and primary T cells. The differential impact between normal and cancer cells sets KIF18A inhibitors apart from other anti-mitotic therapies, which have had limited clinical benefit due to their detrimental impact on both cancer and highly proliferative normal cells. The therapeutic potential of KIF18A inhibitors was assessed in human tumor xenograft models. Treatment of multiple tumor models resulted in substantial, dose-dependent inhibition of tumor growth. KIF18A inhibition in the tumor was confirmed through evaluation of KIF18A localization changes, chromosome congression and an increase in mitotic index. The strong biological rationale, robust preclinical data, and superior compound properties warrant clinical development of Volastra’s KIF18A inhibitor which is planned for development for the treatment of chromosomally unstable cancers. Citation Format: Aaron F. Phillips, Rumin Zhang, Akanksha Verma, Tamar Feinberg, Mia Jaffe, Marysol Chu Carty, Ryan Schulz, Sarah Bettigole, Celia Andreu, Scott Drutman, Michael Su, Derek Cogan, Christina H. Eng. Targeting chromosomally unstable tumors with a selective KIF18A inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 517.

Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive subtype of lymphoma usually associated with inferior outcomes. ABC-DLBCL exhibits plasmablastic features and is characterized by aberrancies in the molecular networks controlled by IRF4. The signaling pathways that are dysregulated in ABC-DLBCL are, however, not fully understood. ROCK2 is a serine-threonine kinase whose role in lymphomagenesis is unknown. Here we show that ROCK2 activity is constitutively dysregulated in ABC-DLBCL but not in GCB-DLBCL and BL. We furthermore show that ROCK2 phosphorylates IRF4 and that the ROCK2-mediated phosphorylation of IRF4 modulates its ability to regulate a subset of target genes. In addition to its effects on IRF4, ROCK2 also controls the expression of MYC in ABC-DLBCL by regulating MYC protein levels. ROCK inhibition furthermore selectively decreases the proliferation and survival of ABC-DLBCL in vitro and inhibits ABC-DLBCL growth in xenograft models. Thus, dysregulated ROCK2 activity contributes to the aberrant molecular program of ABC-DLBCL via its dual ability to modulate both IRF4- and MYC-controlled gene networks and ROCK inhibition could represent an attractive therapeutic target for the treatment of ABC-DLBCL.

Deregulated gene expression through epigenetic, transcriptional, and copy number alterations is a major driver of melanoma progression and metastasis. In addition to serving as blueprints for translation, some mRNAs post-transcriptionally regulate gene expression by competitively sequestering miRNAs they share with other targets. Here we report that such mRNAs, termed competitive endogenous RNAs (ceRNAs), contribute to melanoma progression and metastasis. ceRNA predictions identified multiple candidate genes on chromosome 1q, which is recurrently amplified in melanoma. Genetic studies reveal that three of these mRNAs, CEP170, NUCKS1, and ZC3H11A, promote melanoma migration, invasion, and metastasis in a protein coding-independent and miRNA binding site-dependent manner. Interestingly, CEP170, NUCKS1, and ZC3H11A cooperate to elicit oncogenic effects by collectively impairing the tumor suppressor activity of 8 miRNAs on several pro-metastatic target genes. Finally, this complex chromosome 1q ceRNA network is evident in other cancer types, suggesting ceRNA network deregulation is a common driver of cancer progression.

Androgen receptor signaling inhibitors (ARSi) are a mainstay for patients with metastatic castration-resistant prostate cancer (mCRPC). However, patient response is heterogeneous and the molecular underpinnings of ARSi resistance are not well elucidated. Here we performed transcriptome analysis of circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMC) in the context of a prospective clinical trial of men with mCRPC treated with abiraterone (Abi) or enzalutamide (Enza). CTC RNA-sequencing identified that RB loss and enhanced E2F signaling along with BRCA loss transcriptional networks were associated with intrinsic ARSi resistance, while an inflammatory response signature was significantly associated with acquired resistance. Transcriptomic analysis of matching PBMCs identified enrichment of inflammasome gene signatures indicative of activated innate immunity at progression, with concurrent downregulation of T and NK cells. Importantly, CTC gene signatures had a significant positive association with circulating immune macroenvironment (CIME) signatures. Taken together, these data demonstrate that liquid biopsy transcriptomics can identify molecular pathways associated with clinical ARSi resistance paving the way for treatment optimization in patients with mCRPC.

There is a need for better classification and understanding of tumor infiltrating lymphocytes (TILs). Here we applied advanced functional genomics to interrogate 9000 human tumors, and multiple single-cell sequencing sets using benchmarked T cell states, comprehensive T cell differentiation trajectories, human and mouse vaccine responses, and other human TILs. Compared to other T cell states, enrichment of T memory/resident memory programs was observed across solid tumors. Trajectory analysis of single-cell melanoma CD8(+) TILs also identified a high fraction of memory/resident memory-scoring TILs in anti-PD-1 responders, which expanded post therapy. In contrast, TILs scoring highly for early T cell activation, but not exhaustion, associated with non-response. Late/persistent, but not early activation signatures, prognosticate melanoma survival, and co-express with dendritic cell and IFN-γ response programs. These data identify an activation-like state associated to poor response and suggest successful memory conversion, above resuscitation of exhaustion, is an under-appreciated aspect of successful anti-tumoral immunity.

Loss of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) expression by CpG promoter hypermethylation is associated with metastasis in gastroenteropancreatic neuroendocrine tumors; however, the mechanism of how UCHL1 loss contributes to metastatic potential remains unclear. In this study, we first confirmed that the loss of UCHL1 expression on immunohistochemistry was significantly associated with metastatic tumors in a translational pancreatic neuroendocrine tumor (PNET) cohort, with a sensitivity and specificity of 78% and 89%, respectively. To study the mechanism driving this aggressive phenotype, BON and QGP-1 metastatic PNET cell lines, which do not produce UCHL1, were stably transfected to re-express UCHL1. In vitro assays, RNA sequencing and reverse phase protein array (RPPA) analyses were performed comparing empty-vector negative controls and UCHL1-expressing cell lines. UCHL1 re-expression is associated with lower anchorage-independent colony growth in BON cells, lower colony formation in QGP cells and a higher percentage of cells in the G0/G1 cell-cycle phase in BON and QGP cells. On RPPA proteomic analysis, there was an upregulation of cell-cycle regulatory proteins CHK2 (1.2-fold change, P = 0.004) and P21 (1.2-fold change, P = 0.023) in BON cells expressing UCHL1; western blot confirmed upregulation of phosphorylated CHK2 and P21. There were no transcriptomic differences detected on RNA sequencing between empty-vector negative controls and UCHL1-expressing cell lines. In conclusion, UCHL1 loss correlates with metastatic potential in PNETs and its re-expression induces a less aggressive phenotype in vitro, in part by inducing cell-cycle arrest through posttranslational regulation of phosphorylated CHK2. UCHL1 expression should be considered as a functional biomarker in detecting PNETs capable of metastasis.

Androgen receptor signaling inhibitors (ARSi) and taxanes are mainstays for patients with metastatic castration-resistant prostate cancer (mCRPC). However, patient response is heterogeneous, and the molecular underpinnings of treatment resistance are not well elucidated. To identify clinically meaningful mechanisms of treatment resistance, we performed transcriptome analysis of circulating tumor cells (CTCs) isolated from mCRPC patients enrolled in two independent prospective clinical trials: PROPHECY, a clinical study of patients (n=118) treated with abiraterone or enzalutamide followed by docetaxel; and TAXYNERGY where patients were randomized to docetaxel or cabazitaxel treatment. CTCs were obtained at baseline (before treatment), on treatment and at progression and their comprehensive transcriptomic analysis was correlated with clinical outcomes. To uncover potential involvement of the circulating immune macroenvironment (CIME) in treatment resistance, we performed transcriptomic analysis of matching peripheral blood mononuclear cells (PBMCs) using an established, rigorous, blood-derived transcriptional modular framework. In PROPHECY, CTC RNA-seq identified that RB loss concurrently with enhanced E2F signaling networks were associated with intrinsic ARSi resistance. Using single sample GSEA (ssGSEA) score, we identified that the RB/E2F common signature at baseline was associated with short PFS (median PFS=6.5 months) and OS (median OS=24.5 months) (hazard ratio (HR) = 3.5; 95% CI 1.5-8.2) in men with mCRPC. We further developed a BRCA-loss transcriptional signature, and validated it in the SU2C mCRPC patient cohort, expanding the identification of patients with BRCA-loss phenotypes beyond genomic loss. Applying this signature to PROPHECY baseline samples, we showed that men with high BRCA-loss scores experienced shorter OS (HR=2.42; 95% CI=1-5.9). Through the comparison of CTC transcriptomic profiles at progression with baseline, we identified an inflammatory response signature in CTCs which was significantly associated with acquired ARSi resistance. Transcriptomic PBMC analysis further identified enrichment of inflammasome gene signatures at progression, with concurrent downregulation of CD8+ T and NK cells. Furthermore, preliminary data from both clinical trials, showed a significant upregulation of TGF-β1 and corresponding TGFβ-Receptor signaling pathway in CTCs from patients at progression following taxane treatment, suggesting a role for TGFβ pathway in clinical response to taxane chemotherapy. Taken together, these data demonstrate that liquid biopsy transcriptomics of both tumor cells and immune cells can identify molecular pathways associated with treatment resistance paving the way for treatment optimization and the development of novel precision therapies in patients with mCRPC. Citation Format: Jiaren Zhang, Bob Zimmermann, Giuseppe Galletti, Susan Halabi, Ada Gjyrezi, Qian Yang, Santosh Gupta, Akanksha Verma, Andrea Sboner, Monika Anand, Daniel J. George, Simon G. Gregory, Prerna Mahtani, Seunghee Hong, Virginia Pascual, Clio P. Mavragani, Emmanuel S. Antonarakis, David M. Nanus, Scott T. Tagawa, Olivier Elemento, Andrew J. Armstrong, Paraskevi Giannakakou. Liquid biopsy transcriptomics identify pathways associated with poor outcomes and immune phenotypes in men with mCRPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 646.

Pancreatic neuroendocrine tumors (PNETS) represent the second most common pancreatic neoplasm with surgical resection serving as the mainstay of treatment for localized PNETs (lPNETs) > 2 cm in size; however, therapeutic options for managing metastatic PNETs (mPNETs) remain limited. Recently, immunotherapy with checkpoint inhibitors has emerged as effective therapy across a range of malignancies but has shown limited efficacy in PNET patients partly due to what has been reported as a “cold” tumor microenvironment (TME). Here, we aimed to characterize the TME among both localized and mPNETs and identify mechanisms to increase tumor immunogenicity and T cell recruitment among these patients. We analyzed tumor-infiltrating and peripheral T cells from patients with lPNETs and mPNETs by flow cytometry and found higher expression of CD69+ (activation), CCR5+ (recruitment), and PD1+ (exhaustion) in T cells isolated from TME compared to peripheral blood. Further, we identified a more profound degree of CD3+ and CD8+ T cell infiltration among mPNETs compared to lPNETs by immunohistochemistry analysis, suggesting that mPNETs are at least moderately enriched with activated T cells. RNA-sequencing performed on 22 sporadic, well-differentiated primary PNETs revealed distinct transcriptional signatures dependent upon metastatic potential. These divergent transcriptomes were analyzed by an L1000 computational algorithm, which classifies landmark gene signatures perturbed by drug treatments. We identified the histone deacetylase inhibitor vorinostat as the drug most likely to perturb the PNET gene signature. A KEGG pathway analysis revealed that these vorinostat-target genes were commonly involved in both Th1, Th2, and Th17 cell differentiation and in the NF-κB pathway. Vorinostat treatment of patient-derived mPNET resulted in increased T cell migration towards metastatic tumors across a semi-permeable membrane; cotreatment with a competitive CCR5 antagonist abrogated this effect. In addition, vorinostat treatment of BON-1 and QGP-1, two PNET cell lines, elevated both NF-κB activity and CCR5 expression, with induction reduced following inhibition of NF-κB nuclear localization. Our study demonstrates that T cell recruitment toward mPNET tissue is dependent upon CCR5 expression. These results reveal that PNET TME is regulatable by vorinostat and opens an avenue of potential sensitization to immune checkpoint blockade among patients with PNETs. Citation Format: Jacques A. Greenberg, Jessica N. Limberg, Akanksha Verma, David Kim, Maureen D. Moore, Timothy M. Ullmann, Jessica W. Thiesmeyer, Zachary Loewenstein, Kevin J. Chen, Caitlin E. Egan, Dessislava I. Stefanova, Rohan Bareja, Rasa Zarnegar, Brendan M. Finnerty, Theresa Scognamiglio, Olivier Elemento, Thomas J. Fahey, Irene M. Min. Histone deacetylase inhibitor modulates T cell recruitment to metastatic pancreatic neuroendocrine tumors by CCR5 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6162.

e15046 Background: KIF18A is a kinesin assisting chromosome congression and alignment during mitosis. It is nonessential in normal, euploid cell division but essential for a subset of aneuploid cancer cells with high chromosomal instability (CIN-high). KIF18A knockdown in these cells leads to mitotic arrest, cytostasis and cell death. As a result, KIF18A is an attractive anticancer target. Methods: Volastra drug discovery efforts identified and validated KIF18A as a promising target using genetic knockdown and pharmacological inhibition with a tool compound. High-throughput screening identified an ATP-noncompetitive hit that was optimized for potency, selectivity, and pharmacokinetic properties. Breadth of efficacy studies against a panel of cancer cell lines were used to formulate responder hypotheses and aid in patient selection. Mechanistic profiling and cell fate determination upon KIF18A inhibition was used to identify target engagement and clinical response biomarkers. Results: Our lead candidates bind to KIF18A with sub-nM potency and exhibit long (̃2 days) drug-target residence time. They are ATP and microtubule uncompetitive. They are highly selective for KIF18A and have no known off-target effects on related kinesins and in an in vitro safety screen. They are orally bioavailable in preclinical species, well tolerated and demonstrate tumor regression in mouse xenograft models. In contrast to many agents that disrupt the mitotic machinery, KIF18A inhibition does not impact growth of proliferating normal cells. Breadth of efficacy studies performed on a large panel of human cancer cell lines reveal sensitivity to KIF18A inhibitors across multiple tumor types including high-grade serous ovarian, as well as subtypes of breast and lung cancer. Conclusions: Volastra’s research efforts have culminated in the discovery of orally active, highly potent, selective, and efficacious KIF18A inhibitors as development candidates.

Triple negative breast cancers (TNBCs) are molecularly heterogeneous, and the link between their aggressiveness with African ancestry is not established. We investigated primary TNBCs for gene expression among self-reported race (SRR) groups of African American (AA, n = 42) and European American (EA, n = 33) women. RNA sequencing data were analyzed to measure changes in genome-wide expression, and we utilized logistic regressions to identify ancestry-associated gene expression signatures. Using SNVs identified from our RNA sequencing data, global ancestry was estimated. We identified 156 African ancestry-associated genes and found that, compared to SRR, quantitative genetic analysis was a more robust method to identify racial/ethnic-specific genes that were differentially expressed. A subset of African ancestry-specific genes that were upregulated in TNBCs of our AA patients were validated in TCGA data. In AA patients, there was a higher incidence of basal-like two tumors and altered TP53, NFB1, and AKT pathways. The distinct distribution of TNBC subtypes and altered oncologic pathways show that the ethnic variations in TNBCs are driven by shared genetic ancestry. Thus, to appreciate the molecular diversity of TNBCs, tumors from patients of various ancestral origins should be evaluated.

Serine is a nonessential amino acid generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. In addition, cancer cells can harness exogenous serine to enhance their metabolism and proliferation. Here we tested the relative contributions of exogenous and endogenous sources of serine on the biology of colorectal cancer. In murine tumors, Apc status was identified as a determinant of the expression of genes controlling serine synthesis. In patient samples, PSAT1 was overexpressed in both colorectal adenomas and adenocarcinomas. Combining genetic deletion of PSAT1 with exogenous serine deprivation maximally suppressed the proliferation of colorectal cancer cells and induced profound metabolic defects including diminished nucleotide production. Inhibition of serine synthesis enhanced the transcriptional changes following exogenous serine removal as well as alterations associated with DNA damage. Both loss of PSAT1 and removal of serine from the diet were necessary to suppress colorectal cancer xenograft growth and enhance the antitumor activity of 5-fluorouracil (5-FU). Restricting endogenous and exogenous serine in vitro augmented 5-FU–induced cell death, DNA damage, and metabolic perturbations, likely accounting for the observed antitumor effect. Collectively, our results suggest that both endogenous and exogenous sources of serine contribute to colorectal cancer growth and resistance to 5-FU. Significance: These findings provide insights into the metabolic requirements of colorectal cancer and reveal a novel approach for its treatment.

The molecular mechanisms leading to the transformation of anaplastic lymphoma kinase negative (ALK−) anaplastic large cell lymphoma (ALCL) have been only in part elucidated. To identify new culprits which promote and drive ALCL, we performed a total transcriptome sequencing and discovered 1208 previously unknown intergenic long noncoding RNAs (lncRNAs), including 18 lncRNAs preferentially expressed in ALCL. We selected an unknown lncRNA, BlackMamba, with an ALK− ALCL preferential expression, for molecular and functional studies. BlackMamba is a chromatin-associated lncRNA regulated by STAT3 via a canonical transcriptional signaling pathway. Knockdown experiments demonstrated that BlackMamba contributes to the pathogenesis of ALCL regulating cell growth and cell morphology. Mechanistically, BlackMamba interacts with the DNA helicase HELLS controlling its recruitment to the promoter regions of cell-architecture-related genes, fostering their expression. Collectively, these findings provide evidence of a previously unknown tumorigenic role of STAT3 via a lncRNA-DNA helicase axis and reveal an undiscovered role for lncRNA in the maintenance of the neoplastic phenotype of ALK−ALCL.

Background and objectives Hurthle cell carcinoma (HCC) is an unusual and relatively rare type of differentiated thyroid cancer. Currently, cytologic analysis of fine-needle aspiration biopsy is limited in distinguishing benign Hurthle cell neoplasms from malignant ones. The aim of this study was to determine whether differences in the expression of specific genes could differentiate HCC from benign Hurthle cell nodules by evaluating differential gene expression in Hurthle cell disease. Methods Eighteen benign Hurthle cell nodules and seven HCC samples were analyzed by whole-transcriptome sequencing. Bioinformatics analysis was carried out to identify candidate differentially expressed genes. Expression of these candidate genes was re-examined by quantitative real-time polymerase chain reaction (qRT-PCR). Protein expression was quantified by immunohistochemistry. Results Close homolog of L1 (CHL1) was identified as overexpressed in HCC. CHL1 was found to have greater than 15-fold higher expression in fragments per kilobase million in HCC compared with benign Hurthle cell tumors. This was confirmed by qRT-PCR. Moreover, the immunoreactivity score of the CHL1 protein was significantly higher in HCC compared with benign Hurthle cell nodules. Conclusions CHL1 expression may represent a novel and useful prognostic biomarker to distinguish HCC from benign Hurthle cell disease.

Summary Here, we show that HEMATOLOGICAL AND NEUROLOGICAL EXPRESSED 1-LIKE (HN1L) is a targetable breast cancer stem cell (BCSC) gene that is altered in 25% of whole breast cancer and significantly correlated with shorter overall or relapse-free survival in triple-negative breast cancer (TNBC) patients. HN1L silencing reduced the population of BCSCs, inhibited tumor initiation, resensitized chemoresistant tumors to docetaxel, and hindered cancer progression in multiple TNBC cell line-derived xenografts. Additionally, gene signatures associated with HN1L correlated with shorter disease-free survival of TNBC patients. We defined HN1L as a BCSC transcription regulator for genes involved in the LEPR-STAT3 signaling axis as HN1L binds to a putative consensus upstream sequence of STAT3, LEPTIN RECEPTOR, and MIR-150. Our data reveal that BCSCs in TNBC depend on the transcription regulator HN1L for the sustained activation of the LEPR-STAT3 pathway, which makes it a potentially important target for both prognosis and BCSC therapy., Graphical Abstract, Highlights • HN1L expression is correlated with shorter survival of TNBC patients • HN1L regulates BCSCs by promoting the STAT3 signaling pathway • HN1L: novel transcription regulator of LEPR and miR-150, upstream regulators of STAT3 • HN1L-regulated gene signatures can predict clinical outcomes in TNBC patients, Yi et al. describe HN1L as a novel transcription regulator for breast cancer stem cells (BCSCs) in triple-negative breast cancer (TNBC), promoting LEPR and miR-150 expression and activating the STAT3 pathway. Since BCSCs contribute to chemoresistance and metastasis in TNBC, further investigation of HN1L will offer new therapeutic strategies.

// Michelle Faria 1 , Samaneh Karami 1 , Sergio Granados-Principal 2 , Prasenjit Dey 3, 4 , Akanksha Verma 5 , Dong S. Choi 6 , Olivier Elemento 5 , Tasneem Bawa-Khalfe 1 , Jenny C. Chang 6 , Anders M. Strom 1 and Jan-Ake Gustafsson 1, 7 1 University of Houston, Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, Science & Engineering Research Center, Houston, Texas, USA 2 Department of Medical Oncology, Hospital of Jaen, Jaen, Spain 3 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 5 Institute for Computational Biomedicine, Department of Physiology and Biophysics Weill Cornell Medicine, New York, NY, USA 6 Methodist Cancer Center, Houston Methodist Hospital, Houston, Texas, USA 7 Department of BioSciences and Nutrition, Karolinska Institutet, Huddinge, Sweden Correspondence to: Anders M. Strom, email: amstrom@uh.edu Keywords: slug; twist1; SOX2; CD133; c-Myc Received: July 22, 2017 Accepted: November 05, 2017 Published: January 10, 2018 ABSTRACT Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that two of the variants of ERβ, namely ERβ2 and ERβ5, control aggressiveness of TNBC by regulating hypoxic signaling through stabilization of HIF-1α. RNA-seq of patient derived xenografts (PDX) from TNBC shows expression of ERβ2, ERβ4 and ERβ5 variants in more than half of the samples. Furthermore, expression of ERβ4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to tumorsphere formation caused transformation and development of tumorspheres. By contrast, ERβ1, ERβ2 or ERβ5 were unable to support tumorsphere formation. We have previously shown that all variants except ERβ1 stabilize HIF-1α but only ERβ4 appears to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERβ4. We propose that ERβ variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer.

Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Obesity is associated with an increased incidence of high-grade prostate cancer (PC) and worse prognosis for PC patients. Recently, we showed in men that obesity-related periprostatic white adipose tissue (WAT) inflammation, characterized by macrophages surrounding dead or dying adipocytes forming crown-like structures, was associated with high-grade PC. Possibly, interventions that suppress periprostatic WAT inflammation will improve outcomes for men with PC. Here, we tested the hypothesis that supplemental 17β-estradiol (E2) could decrease periprostatic WAT inflammation in obese male mice. Mice were fed a high-fat diet to induce periprostatic WAT inflammation before being treated with supplemental E2. E2 supplementation suppressed caloric intake, induced weight loss, decreased periprostatic WAT inflammation and downregulated the expression of genes linked to inflammation including Cd68, Mcp1 and Tnf. Similar to the effects of E2 supplementation, treatment with diethylstilbestrol, a synthetic estrogen, also suppressed caloric intake and reduced periprostatic WAT inflammation. To determine whether the observed effects of supplemental estrogen could be reproduced by caloric restriction (CR) alone, obese mice were put on a 30% CR diet. Like estrogen treatment, CR was effective in reducing body weight, periprostatic WAT inflammation and the expression of pro-inflammatory genes. Transcriptomic analyses of periprostatic fat showed that obesity was associated with enrichment in inflammatory response pathways, which were normalized by both supplemental E2 and CR. Taken together, these findings strengthen the rationale for future efforts to determine whether either CR or supplemental estrogen will decrease periprostatic WAT inflammation and thereby improve outcomes for men with PC.

The receptor for hyaluronic acid-mediated motility (RHAMM) is upregulated in various cancers. We previously screened genes upregulated in human hepatocellular carcinomas for their metastatic function in a mouse model of pancreatic neuroendocrine tumor (PNET) and identified that humanRHAMMBpromoted liver metastasis. It was unknown whetherRHAMMBis upregulated in pancreatic cancer or contributes to its progression. In this study, we found that RHAMM protein was frequently upregulated in human PNETs. We investigated alternative splicing isoforms,RHAMMAandRHAMMB, by RNA-Seq analysis of primary PNETs and liver metastases.RHAMMB, but notRHAMMA, was significantly upregulated in liver metastases. RHAMMBwas crucial forin vivometastatic capacity of mouse and human PNETs. RHAMMA, carrying an extra 15-amino acid-stretch, did not promote metastasis in spontaneous and experimental metastasis mouse models. Moreover,RHAMMBwas substantially higher thanRHAMMAin pancreatic ductal adenocarcinoma (PDAC).RHAMMB, but notRHAMMA, correlated with both higherEGFRexpression and poorer survival of PDAC patients. Knockdown of EGFR abolished RHAMMB-driven PNET metastasis. Altogether, our findings suggest a clinically relevant function ofRHAMMB, but notRHAMMA, in promoting PNET metastasis in part through EGFR signaling.RHAMMBcan thus serve as a prognostic factor for pancreatic cancer.

Epigenetic silencing of promoter and enhancer regions is a common phenomenon in malignant cells. The transcription factor STAT3 is aberrantly activated in several tumors, where its constitutive acetylation accounts for the transcriptional repression of a number of tumor suppressor genes (TSG) via molecular mechanisms that remain to be understood. Using nucleophosmin-anaplastic lymphoma kinase–positive (NPM-ALK+) anaplastic large-cell lymphoma (ALCL) as model system, we found in cells and patient-derived tumor xenografts that STAT3 is constitutively acetylated as a result of ALK activity. STAT3 acetylation relied on intact ALK-induced PI3K- and mTORC1-dependent signaling and was sensitive to resveratrol. Resveratrol lowered STAT3 acetylation, rescued TSG expression, and induced ALCL apoptotic cell death. STAT3 constitutively bound the Sin3A transcriptional repressor complex, and both STAT3 and Sin3A bound the promoter region of silenced TSG via a resveratrol-sensitive mechanism. Silencing SIN3A caused reexpression of TSG, induced ALCL apoptotic cell death in vitro, and hindered ALCL tumorigenic potential in vivo. A constitutive STAT3–Sin3A interaction was also found in breast adenocarcinoma cells and proved critical for TSG silencing and cell survival. Collectively, these results suggest that oncogene-driven STAT3 acetylation and its constitutive association with Sin3A represent novel and concomitant events contributing to STAT3 oncogenic potential. Significance: This study delineates the transcriptional regulatory complex Sin3A as a mediator of STAT3 transcriptional repressor activity and identifies the STAT3/Sin3A axis as a druggable target to antagonize STAT3-addicted tumors.

Purpose: Both gain-of-function enhancer of zeste homolog 2 (EZH2) mutations and inactivating histone acetyltransferases mutations, such as CREBBP and EP300, have been implicated in the pathogenesis of germinal center (GC)–derived lymphomas. We hypothesized that direct inhibition of EZH2 and histone deacetyltransferase (HDAC) would be synergistic in GC-derived lymphomas. Experimental Design: Lymphoma cell lines (n = 21) were exposed to GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor. Synergy was assessed by excess over bliss. Western blot, mass spectrometry, and coimmunoprecipitation were performed. A SU-DHL-10 xenograft model was utilized to validate in vitro findings. Pretreatment RNA-sequencing of cell lines was performed. MetaVIPER analysis was used to infer protein activity. Results: Exposure to GSK126 and romidepsin demonstrated potent synergy in lymphoma cell lines with EZH2 dysregulation. Combination of romidepsin with other EZH2 inhibitors also demonstrated synergy suggesting a class effect of EZH2 inhibition with romidepsin. Dual inhibition of EZH2 and HDAC led to modulation of acetylation and methylation of H3K27. The synergistic effects of the combination were due to disruption of the PRC2 complex secondary to acetylation of RbAP 46/48. A common basal gene signature was shared among synergistic lymphoma cell lines and was characterized by upregulation in chromatin remodeling genes and transcriptional regulators. This finding was supported by metaVIPER analysis which also revealed that HDAC 1/2 and DNA methyltransferase were associated with EZH2 activation. Conclusions: Inhibition of EZH2 and HDAC is synergistic and leads to the dissociation of PRC2 complex. Our findings support the clinical translation of the combination of EZH2 and HDAC inhibition in EZH2 dysregulated lymphomas.

Serine is a non-essential amino acid that is generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1) and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. In addition to serine synthesis, exogenous serine is taken up by cells and can also fuel tumor growth. Interestingly, colon cancer cells increase expression of serine biosynthesis enzymes in the absence of exogenous serine, suggesting a compensatory adaptive response to reduced availability of serine. This study explored the relative contributions of exogenous and synthesized serine to colon cancer cell growth, metabolism and response to anti-cancer therapy. We found that PSAT1 expression was markedly increased in human colonic adenomas and colorectal cancer (CRC) compared to normal adjacent tissue. Additionally, high levels of PSAT1 in CRC were associated with reduced patient survival. Deletion of PSAT1 only modestly reduced colon cancer cell proliferation in vitro. Removal of serine from the medium strongly suppressed cell proliferation whereas the combination of removing exogenous serine and deleting PSAT1 caused even greater inhibition of cell proliferation. Metabolite profiling revealed altered nucleotide levels upon exogenous serine depletion, an effect that was enhanced when PSAT1 was deleted. Global gene expression profiling showed approximately 3,000 differentially expressed genes when only exogenous serine was removed, with an additional ~800 changes in PSAT1 knockout (KO) cells grown in serine deficient medium. A marked DNA damage response was induced selectively in PSAT1KO cells in serine deficient medium, which was prevented by the addition of formate. Xenografts derived from PSAT1 sufficient and PSAT1KO cells showed that neither PSAT1 deletion alone nor removal of dietary serine alone affected tumor growth, but the combination resulted in profound growth suppression. Additionally, treatment with 5-fluorouracil (5-FU) in the xenograft model induced a strong regression of established tumors derived from mice carrying PSAT1KO cells fed a serine deficient diet. Complementary in vitro studies showed enhanced sensitivity of PSAT1KO cells grown in serine deficient medium to 5-FU induced death, which was preceded by specific metabolite changes including markedly reduced levels of TMP and TTP. Taken together, our results suggest that both exogenous and endogenous sources of serine are important contributors to colon cancer cell proliferation and metabolism. Limiting exogenously available and synthesized serine may be an effective strategy to limit colon cancer growth and enhance the efficacy of treatment. Citation Format: David C. Montrose, Miguel Foronda, Suchandrima Saha, Erin M. McNally, Xi Kathy Zhou, Jan Krumsiek, Akanksha Verma, Olivier Elemento, Rhonda K. Yantiss, Qiuying Chen, Steven S. Gross, Lorenzo Galluzzi, Lukas E. Dow, Andrew J. Dannenberg. Exogenous and endogenous sources of serine contribute to colon cancer metabolism and growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3745.

1547 Background: Due to persistent disparities in breast cancer mortality, there has been a renewed focus on investigating tumor biology. Deeper exploration has exposed distinctions in tumor biology based upon self-reported race and ancestry. The disparities associated with Triple Negative Breast Cancer (TNBC) across the modern African Diaspora suggests that there is a genetic ancestry connection between its aggressive tumor biology and clinical outcomes. Understanding this connection could hold the key to improving clinical outcomes in this group. Methods: We investigated 75 TNBC primary tumors using Self-Reported Race (SRR) groups: African American (AA, n = 42) and European American (EA, n = 33). Using best practices established by TCGA, we analyzed bulk RNA sequencing to measure changes in genome-wide expression levels. We next quantified global ancestry in a novel manner using RNAseq variants using 1000 Genomes as the reference data. We then identified African and European ancestry-associated genes using a logistic regression (adjusted FDR p < 0.05) between quantified ancestry and gene expression levels. Results: We identified > 150 genes associated with quantified African ancestry. We also found using quantified ancestry was a more robust method to screen for differentially expressed genes than SRR. Using an updated TNBC subtyping method, we noted higher incidences of Basal-like 2 tumors in AAs. Pathway analyses indicated several canonical cancer pathways; including, TP53, NFKB1 and AKT, have altered functionality in patients of African descent. For example, TP53-associated genes were activated in TNBC tumors of AA versus EA. This upregulation, rather than loss of function, is suggestive of polymorphic and/or ancestry-specific expression regulation, likely driven by population-private genetic variants. Lastly, we used TCGA data to validate a subset of African ancestry-specific genes that were upregulated in AA patients in our cohort. Specifically, PIM3, ZBTB22 and PPP2R4 each retained significant upregulation, in our cohort, but also TNBC tumors from TCGA (p = 0.0018, 0.023 and 0.022, respectively). Conclusions: Our study has uncovered ancestry-specific gene expression profiles in TNBC tumors. The distinct distribution of TNBC subtypes and altered functional oncologic pathways are evidence that biological underpinnings in TNBC can be driven by shared genetic ancestry. These findings emphasize the need to investigate patient populations of various ancestral origins in order to fully appreciate the molecular diversity in tumor biology for precision of disease management.

Summary: Epigenetic changes are increasingly being appreciated as key events in breast cancer progression. However, breast cancer subtype-specific epigenetic regulation remains poorly investigated. Here we report that EZH2 is a leading candidate of epigenetic modulators associated with the TNBC subtype and that it predicts poor overall survival in TNBC patients. We demonstrate that specific pharmacological or genetic inhibition of EZH2 catalytic activity impairs distant metastasis. We further define a specific EZH2high population with enhanced invasion, mammosphere formation, and metastatic potential that exhibits marked sensitivity to EZH2 inhibition. Mechanistically, EZH2 inhibition differentiates EZH2high basal cells to a luminal-like phenotype by derepressing GATA3 and renders them sensitive to endocrine therapy. Furthermore, dissection of human TNBC heterogeneity shows that EZH2high basal-like 1 and mesenchymal subtypes have exquisite sensitivity to EZH2 inhibition compared with the EZH2low luminal androgen receptor subtype. These preclinical findings provide a rationale for clinical development of EZH2 as a targeted therapy against TNBC metastasis. : TNBC presents a clinical challenge because it exhibits a high incidence of metastatic recurrence. Yomtoubian et al. identify a highly metastatic basal-like subpopulation in the primary tumor that expresses elevated EZH2 levels. EZH2 inhibition induces luminal differentiation by derepressing GATA3, rendering tumors sensitive to endocrine therapy, and inhibiting metastasis. Keywords: breast cancer, metastasis, circulating tumor cells, epigenetics, EZH2, GATA3, differentiation

Anaplastic Large Cell Lymphoma (ALCL) is an aggressive subtype of T-cell lymphoma. ALCLs are stratified based on the presence of anaplastic lymphoma kinase (ALK) translocations. ALK negative (ALK-ALCLs) are heterogeneous subtypes characterized by higher aggressiveness and poorer outcome than ALK+ALCL. The molecular and genetic asset of ALK-ALCLs has recently begun to emerge (i.e. JAK/STAT3 activating mutations, DUSP22, TP63, TP53 and IRF4 rearrangements), but an exhaustive picture of the molecular drivers leading to ALK-ALCLs transformation, progression, and immune evasion is still lacking. Long non coding RNAs (LncRNAs) are transcripts longer than 200 nucleotides with different regulatory functions ranging from transcriptional regulation to structural functions, which are emerging as relevant players in many cellular processes including cancer. Using deep RNA-sequencing profiling combined with de novo transcriptome assembly, we explored and validated the potential contribution of non-coding RNAs in a large series of ALCLs. 24 lncRNAs were found specifically enriched in ALCL samples. Among these, a 70Kb chromatin associated lncRNA (BlackMamba) was identified as preferentially associated with the ALK-ALCLs subtypes and was shown to be a target of the JAK/STAT3 signaling. BlackMamba was overexpressed in ALK-ALCL patient samples as well as in patient-derived tumor xenograft (PDTX) models. Its shRNA mediated knockdown (KD) in ALK-ALCL cells reduces cell proliferation and clonogenicity. BlackMamba KD cells also showed a remarkable increase in the number of multinucleated cells (without ploidy alteration) providing evidence that this lncRNA may be required for correct cytokinesis. To better characterize the role of BlackMamba we performed RNA-sequencing profiling in BlackMamba KD ALK-ALCL cells showing that this lncRNA affects primarily the expression of genes involved in cytoskeleton organization and remodeling. Noticeably, the DNA-helicase HELLS emerged among the most relevant BlackMamba target gene in ALK-ALCL cells and patients. Loss of BlackMamba causes profound reduction of HELLS concomitant with a reorganization of chromatin markers (reduction of K4me3 and increase of K27me3) in the HELLS locus. To test whether HELLS enforces BlackMamba-mediated transcription, we silenced HELLS by shRNA approach in ALK-ALCLs. Noticeably, loss of HELLS led to a reduction in cell growth, a delay in the duplication rate and a dramatic drop of clonogenicity potential of ALK-ALCL cells. This phenotype was also associated with an increased number of multinucleated cells, phenocopying the BlackMamba KD cells. We also showed that, HELLS KD causes expression changes in a subset of cytoskeleton-related genes previously identified as BlackMamba targets confirming that HELLS is a crucial mediator of BlackMamba function. Indeed, ectopic over-expression of HELLS in BlackMamba KD cells rescued the cell growth defects induced by the loss of lncRNA mitigated the polynucleation phenotype and restored the baseline expression of BlackMamba target genes. Being established that lncRNAs affect gene expression by recruiting chromatin remodeling complexes to target promoters or enhancers we also investigated whether BlackMamba may associates with HELLS and dictates its chromatin positioning in ALK-ALCL cells. By RNA-immunoprecipitation (RIP) we showed that HELLS binds to BlackMamba at in two distinct regions the 3'-end of the lncRNA. Next, by Chromatin Immunoprecipitation (ChIP) we demonstrated that HELLS is associated to BlackMamba target gene promoters exclusively in ALK-ALCLs in which BlackMamba is expressed. Collectively, these data demonstrate the existence of a new lncRNA-dependent mechanism controlling the recruitment of HELLS on chromatin sites and its expression in lymphomas. The axis BlackMamba-HELLS sustains the neoplastic phenotype of ALK-ALCL representing a potential vulnerability of ALCL cells. Disclosures Merli: Mundipharma: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Sandoz: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Janssen: Honoraria; Takeda: Honoraria, Other: Travel Expenses; Gilead: Honoraria.

Obesity is associated with adipose inflammation, defined by macrophages encircling dead adipocytes, as well as extracellular matrix (ECM) remodeling and increased risk of breast cancer. Whether ECM affects macrophage phenotype in obesity is uncertain. A better understanding of this relationship could be strategically important to reduce cancer risk or improve outcomes in the obese. Using clinical samples, computational approaches, and in vitro decellularized ECM models, this study quantified the relative abundance of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in human breast adipose tissue, determined molecular similarities between obesity and tumor-associated macrophages, and assessed the regulatory effect of obese versus lean ECM on macrophage phenotype. Our results suggest that breast adipose tissue contains more M2- than M1-biased macrophages across all body mass index categories. Obesity further increased M2-biased macrophages but did not affect M1-biased macrophage density. Gene Set Enrichment Analysis suggested that breast tissue macrophages from obese versus lean women are more similar to tumor-associated macrophages. These changes positively correlated with adipose tissue interstitial fibrosis, and in vitro experiments indicated that obese ECM directly stimulates M2-biased macrophage functions. However, mammographic density cannot be used as a clinical indicator of these changes. Collectively, these data suggest that obesity-associated interstitial fibrosis promotes a macrophage phenotype similar to tumor-associated macrophages, which may contribute to the link between obesity and breast cancer.

Obesity, a cause of subclinical inflammation, is a risk factor for the development of postmenopausal breast cancer and is associated with poorer cancer outcomes. Docosahexaenoic acid (DHA), an omega-3 fatty acid, possesses anti-inflammatory properties. We hypothesized that treatment with DHA would reduce the expression of proinflammatory genes and aromatase, the rate-limiting enzyme for estrogen biosynthesis, in benign breast tissue of overweight/obese women. A randomized, placebo-controlled, double-blind phase II study of DHA given for 12 weeks to overweight/obese women with a history of stage I–III breast cancer, DCIS/LCIS, Paget's disease, or proliferative benign breast disease was carried out. In this placebo controlled trial, the primary objective was to determine whether DHA (1,000 mg by mouth twice daily) reduced breast tissue levels of TNFα. Secondary objectives included evaluation of the effect of DHA on breast tissue levels of COX-2, IL1β, aromatase, white adipose tissue inflammation, and gene expression by RNA-seq. Red blood cell fatty acid levels were measured to assess compliance. From July 2013 to November 2015, 64 participants were randomized and treated on trial (32 women per arm). Increased levels of omega-3 fatty acids in red blood cells were detected following treatment with DHA (P < 0.001) but not placebo. Treatment with DHA did not alter levels of TNFα (P = 0.71), or other biomarkers including the transcriptome in breast samples. Treatment with DHA was overall well-tolerated. Although compliance was confirmed, we did not observe changes in the levels of prespecified biomarkers in the breast after treatment with DHA when compared with placebo. Cancer Prev Res; 11(4); 203–14. ©2018 AACR. See related editorial by Fabian and Kimler, p. 187

Purpose: On the basis of the identified stress-independent cellular functions of activating transcription factor 4 (ATF4), we reported enhanced ATF4 levels in MCF10A cells treated with TGFβ1. ATF4 is overexpressed in patients with triple-negative breast cancer (TNBC), but its impact on patient survival and the underlying mechanisms remain unknown. We aimed to determine ATF4 effects on patients with breast cancer survival and TNBC aggressiveness, and the relationships between TGFβ and ATF4. Defining the signaling pathways may help us identify a cell signaling–tailored gene signature. Experimental Design: Patient survival data were determined by Kaplan–Meier analysis. Relationship between TGFβ and ATF4, their effects on aggressiveness (tumor proliferation, metastasis, and stemness), and the underlying pathways were analyzed in three TNBC cell lines and in vivo using patient-derived xenografts (PDX). Results: ATF4 overexpression correlated with TNBC patient survival decrease and a SMAD-dependent crosstalk between ATF4 and TGFβ was identified. ATF4 expression inhibition reduced migration, invasiveness, mammosphere-forming efficiency, proliferation, epithelial–mesenchymal transition, and antiapoptotic and stemness marker levels. In PDX models, ATF4 silencing decreased metastases, tumor growth, and relapse after chemotherapy. ATF4 was shown to be active downstream of SMAD2/3/4 and mTORC2, regulating TGFβ/SMAD and mTOR/RAC1–RHOA pathways independently of stress. We defined an eight-gene signature with prognostic potential, altered in 45% of 2,509 patients with breast cancer. Conclusions: ATF4 may represent a valuable prognostic biomarker and therapeutic target in patients with TNBC, and we identified a cell signaling pathway–based gene signature that may contribute to the development of combinatorial targeted therapies for breast cancer. Clin Cancer Res; 24(22); 5697–709. ©2018 AACR.

Triple negative breast cancer (TNBC) still remains a challenge to treat in the clinic due to a lack of good targets for treatment. Although TNBC lacks expression of ERα, the expression of ERβ and its variants are detected quite frequently in this cancer type and can represent an avenue for treatment. We show that the variants of ERβ, namely ERβ1, ERβ2, ERβ4, and ERβ5, regulate aggressiveness of TNBC by regulating hypoxic signaling. RNA-seq of patient derived xenografts (PDX) from TNBC show expression of ERβ4 and ERβ5 variants in more than half of the samples. Furthermore, expression of ERβ4 in the immortalized, normal mammary epithelial cell line MCF-10A that is resistant to mammosphere formation caused transformation and development of mammospheres. By contrast, ERβ1, ERβ2 or ERβ5 were unable to support mammosphere formation. We have previously shown that all variants except ERβ1 stabilizes HIF-1α but only ERβ4 appear to have the ability to transform normal mammary epithelial cells, pointing towards a unique property of ERβ4. We propose that ERβ variants may be good diagnostic tools and also serve as novel targets for treatment of breast cancer. Citation Format: Faria M, Karami S, Granados-Principal S, Dey P, Verma A, Choi DS, Elemento O, Bawa-Khalfe1 T, Chang JC, Gustafsson J-A, Strom AM. The ERβ4 variant induce transformation of the normal breast mammary epithelial cell line MCF-10A; the ERβ variants ERβ2, ERβ4 and ERβ5 increase aggressiveness of TNBC by regulation of hypoxic signaling [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-10.

Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation.

Purpose: Pan-class I/II histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. ACY-1215 (ricolinostat) is a first-in-class selective HDAC6 inhibitor. To better understand the discrete function of HDAC6 and its role in lymphoma, we developed a lymphoma cell line resistant to ACY-1215. Experimental Design: The diffuse large B-cell lymphoma cell line OCI-Ly10 was exposed to increasing concentrations of ACY-1215 over an extended period of time, leading to the development of a resistant cell line. Gene expression profiling (GEP) was performed to investigate differentially expressed genes. Combination studies of ACY-1215 and ibrutinib were performed in cell lines, primary human lymphoma tissue, and a xenograft mouse model. Results: Systematic incremental increases in drug exposure led to the development of distinct resistant cell lines with IC50 values 10- to 20-fold greater than that for parental lines. GEP revealed upregulation of MAPK10, HELIOS, HDAC9, and FYN, as well as downregulation of SH3BP5 and LCK. Gene-set enrichment analysis (GSEA) revealed modulation of the BTK pathway. Ibrutinib was found to be synergistic with ACY-1215 in cell lines as well as in 3 primary patient samples of lymphoma. In vivo confirmation of antitumor synergy was demonstrated with a xenograft of DLBCL. Conclusions: The development of this ACY-1215–resistant cell line has provided valuable insights into the mechanistic role of HDAC6 in lymphoma and offered a novel method to identify rational synergistic drug combinations. Translation of these findings to the clinic is underway. Clin Cancer Res; 23(12); 3084–96. ©2016 AACR.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.

Metastatic melanoma is one of the most deadly and evasive types of cancer. On average, cancer patients with metastatic melanoma survive only 6–9 months after diagnosis. Epidemiological and animal studies suggest that obesity increases the metastatic ability of malignant melanoma, though the mechanism is not known. In the present studies, we assessed the ability of 3T3L1 adipocytes to modulate B16BL6 melanoma cell invasion and the Epithelial-to-Mesenchymal Transition (EMT). For this purpose, we induced the differentiation of 3T3L1 fibroblasts to adipocytes. Then, we collected the cell culture media from both fibroblasts and adipocytes and determined their effect on the invasive ability and EMT gene expression of B16BL6 melanoma cells. Results show that adipocyte media increased that ability of B16BL6 cells to invade. The higher invasive ability of B16BL6 melanoma cells was associated with increased expression of EMT genes such as Snai1, MMP9, Twist, and Vimentin. Additionally, the expression of the cell-to-cell adhesion protein E-cadherin and the metastasis suppressor gene Kiss1 were down-regulated in these B16BL6 cells. Also, adipocytes had high levels of the pro-inflammatory cytokine Interleukin 6 (IL-6). Treatment of B16BL6 cells with IL-6 elicited similar effects as the adipocyte media; IL-6 promoted the invasive ability of B16BL6 melanoma cells, increased the expression of Snai1, and decreased Kiss1 expression. IL-6 neutralization, however, did not have a visible effect on adipocyte media-induced invasion and snai1 staining. In summary, adipocytes may increase the invasive ability of B16BL6 melanoma cells by promoting EMT and decreasing the expression of genes such as E-cadherin and Kiss1.

7546Background: EZH2 is the catalytic subunit of PRC2, and induces methylation of H3K27. Activating mutations and overexpression of EZH2 are found in NHL. Inactivating mutations in histone acetyltr...

Prostate cancer is the most commonly diagnosed male cancer in the United States. Taxanes are the only established chemotherapy drugs proven to be effective in improving survival of men with advanced prostate cancer through disruption of the AR-signaling axis downstream of microtubule stabilization. However, there is significant heterogeneity in how patients respond to taxanes and most patients ultimately become refractory due to the development of drug resistance. Currently, the molecular basis of clinical taxane resistance in PC is poorly understood. Prostate cancer circulating tumor cells (P-CTCs) are often found in the peripheral blood of patients suffering from metastatic prostate cancer and have been clinically used as prognostic biomarker for metastatic progression and treatment outcome. The objective of this study is to identify clinically relevant mechanisms of taxane resistance through conducting RNA-Seq analysis in P-CTCs isolated from patients before, during and after they become refractory to taxane chemotherapy. To show feasibility of RNA-Seq experiments with limiting samples such as CTCs and given the presence of contaminating leucocytes, a pilot experiment was performed in which limiting numbers of prostate cancer cells (LNCaP) either pure or enriched following spiking into healthy donor blood, were analyzed by RNA-Seq. Matching healthy donor blood processed with the same enrichment protocol was used as germline control as well as control for the presence of contaminating leucocytes following CTC enrichment. Trimmed reads were aligned to human reference genome (hg38) using STAR. Determination of Fragments Per Kilobase of exon per Million mapped fragments (FPKM) was performed using Cufflinks and heat map was built based on the value of log10(FPKM+1). Gene expression analysis showed that markers of prostate (such as AR, PSMA, KLK3, KLK2, and AMACR) or epithelial lineage (such as EpCAM, CDH1, KRT8 and KRT18) were detected in both pure LNCaP cells-regardless of amount- as well as limited number of captured LNCaP cells in the presence of contaminating leucocytes. In contrast, healthy donor blood was negative for the prostate and epithelial lineage markers and positive for the leucocyte specific markers (such as CD45 and CD16). Gene set enrichment analysis (GSEA) indicated significant enrichment for Andorgen response, MYC, MTOR and RB related pathways in pure and captured LNCap cells compared with healthy donor blood. These data clearly show that by using RNA-Seq we can detect the prostate and epithelial specific gene signatures of limited number of spiked prostate cancer cells using the microfluidic device. Ongoing work includes RNA-Seq analysis of P-CTCs isolated from patients before and after taxane treatment, in order to detect differentially expressed genes, pathways, and potentially driver somatic mutations associated with clinical taxane resistance. Citation Format: Jiaren Zhang, Ada Gjyrezi, Prashant Thakkar, Giuseppe Galletti, Akanksha Verma, Olivier Elemento, Paraskevi Giannakakou. Elucidation of taxane resistance in prostate cancer through RNA-Seq analysis of circulating tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3489.

Systemic Anaplastic Large Cell Lymphomas (ALCLs) is among the common subtypes of T-cell lymphomas. The prognosis of ALCLs patients has been proven to be associated with translocations of the anaplastic lymphoma kinase (ALK) gene, having ALK negative patients (ALK-ALCL) a poorer 5-years survival rate (30-40%). We now recognize that ALK- ALCL includes multiple subsets some of which are bear unique genetic defects and unique clinical outcomes. Long non coding RNAs (lncRNAs) are regulators of gene expression controlling critical processes (epigenetic regulation, imprinting, cell cycle, apoptosis) frequently deregulated in cancer. In T-cells, more than 500 lncRNAs are associated with lymphocyte signatures, control cell differentiation and cell identity. While lncRNAs are found to be differentially expressed in solid cancer and directly linked to the different stages of carcinogenesis, their role in lymphoid neoplasms is largely unknown. To identify novel lncRNAs maintaining the neoplastic phenotype of ALCL, we prepared cDNA libraries representative of coding and non-coding RNA. High coverage and directional RNA-sequencing was performed in 30 purified human T lymphocytes from multiple healthy donors, corresponding to different stages of differentiation, 22ALK+ ALCL and 20 ALK-ALCL patients' samples. Seven bona fide ALCL cell lines were included. By de novo transcriptome reconstruction and using a new bioinformatic pipeline, we identified 83lncRNAs exclusively expressed in ALCL patient's samples. Among them, 82 lncRNAs were coshared between ALK+ and ALK- ALCL and only one was exclusively expressed in ALK-ALCL samples. We named this new lncRNA BlackMamba. We selected the 10 top-scoring ALCL-associated lncRNAs and BlackMamba and determined their expression by RT-PCR, in a validation set of ALCL (ALK+: n=3, ALK-: n=3), T-ALL (n=8), PTCL (n=9), and AITL (n=9) samples. Resting and activated PBMCs from two additional donors were included. Our data confirmed that the 10 top-scoring ALCL-associated lncRNAs were expressed exclusively in ALCLs without differences in the expression among ALCL subtypes. Moreover, BlackMamba was detected only in ALCL samples with a preferential expression in ALK-ALCL samples. All lncRNAs were not expressed in normal T-lymphocytes. We next determine the BlackMamba expression in ALCL (n=6), T-ALL (n=3), cutaneous T-lymphomas (n=3) cell lines by RT-PCR. FedP, MAC1 and MAC2a (ALK-ALCL) cell lines showed the highest expression. Because the cellular localization of lncRNAs affects their function, we assessed the sub-cellular localization of BlackMamba by nucleo/cytoplasm cell fractionation and RT-PCR in FedP and MAC2A cell lines. BlackMamba displayed a preferential nuclear/chromatin-associated localization. Having proven that JAK/STAT signaling pathway plays a key role in both ALK+ and ALK-ALCL, we test whether the expression of BlackMamba could be modulated after treatment with JAK1/2 (ruxolitinib) TKi. Our data showed that, in FedP and MAC2A cell lines, BlackMamba expression was 50% down-regulated relative to control. Next, to identify whether BlackMamba may have a unique associated to specific chromatin remodel proteins, we verified its association with several candidates and found the physical interaction with the histone-lysine N-methyltransferase enzyme EZH2 by RIP in the FedP cells. Notably, after BlackMamba knock down, the proliferation of FedP cells was reproducibly reduced (25% at 84hrs using multiple RNAi oligonucleotides). Collective, our data indicate that ALCL aberrantly express novel and uncharacterized lncRNAs and that BlackMamba is a novel lncRNA associated with ALK-ALCL. Moreover, BlackMamba may contributes to the maintenance of ALK-ALCL neoplastic phenotype. Disclosures Merli: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva Pharmaceuticals Industries: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.