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Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.

Waldenstrom macroglobulinemia (WM) and its precursor IgM gammopathy are distinct disorders characterized by clonal mature IgM-expressing B-cell outgrowth in the bone marrow. Here, we show by high-dimensional single-cell immunogenomic profiling of patient samples that these disorders originate in the setting of global B-cell compartment alterations, characterized by expansion of genomically aberrant extrafollicular B cells of the nonmalignant clonotype. Alterations in the immune microenvironment preceding malignant clonal expansion include myeloid inflammation and naïve B- and T-cell depletion. Host response to these early lesions involves clone-specific T-cell immunity that may include MYD88 mutation–specific responses. Hematopoietic progenitors carry the oncogenic MYD88 mutations characteristic of the malignant WM clone. These data support a model for WM pathogenesis wherein oncogenic alterations and signaling in progenitors, myeloid inflammation, and global alterations in extrafollicular B cells create the milieu promoting extranodal pattern of growth in differentiated malignant cells.Significance:These data provide evidence that growth of the malignant clone in WM is preceded by expansion of extrafollicular B cells, myeloid inflammation, and immune dysfunction in the preneoplastic phase. These changes may be related in part to MYD88 oncogenic signaling in pre–B progenitor cells and suggest a novel model for WM pathogenesis.This article is highlighted in the In This Issue feature, p. 549

Purpose: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. Experimental Design: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for 1–6 months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA sequencing (RNA-seq) and assay for transpose accessible chromatin sequencing (ATAC-seq). Chromatin accessible regions were used to identify regulatory RNA expression in more than 700 samples from newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass trial (NCT01454297). Results: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. More than 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with immunoglobulin heavy chain translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. Conclusions: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.

Because most patients with multiple myeloma (MM) develop resistance to current regimens, novel approaches are needed. Genetically modified, replication-competent oncolytic viruses exhibit high tropism for tumor cells regardless of cancer stage and prior treatment. Receptors of oncolytic herpes simplex virus 1 (oHSV-1), NECTIN-1, and HVEM are expressed on MM cells, prompting us to investigate the use of oHSV-1 against MM. Using oHSV-1-expressing GFP, we found a dose-dependent increase in the GFP+ signal in MM cell lines and primary MM cells. Whereas NECTIN-1 expression is variable among MM cells, we discovered that HVEM is ubiquitously and highly expressed on all samples tested. Expression of HVEM was consistently higher on CD138+/CD38+ plasma cells than in non-plasma cells. HVEM blocking demonstrated the requirement of this receptor for infection. However, we observed that, although oHSV-1 could efficiently infect and kill all MM cell lines tested, no viral replication occurred. Instead, we identified that oHSV-1 induced MM cell apoptosis via caspase-3 cleavage. We further noted that oHSV-1 yielded a significant decrease in tumor volume in two mouse xenograft models. Therefore, oHSV-1 warrants exploration as a novel potentially effective treatment option in MM, and HVEM should be investigated as a possible therapeutic target., Graphical Abstract, Receptors of oncolytic herpes simplex virus (oHSV-1), NECTIN-1, and HVEM are expressed on multiple myeloma (MM) cells. Whereas NECTIN-1 expression is variable among MM cells, HVEM is ubiquitously and highly expressed on all samples tested. However, no viral replication occurred. Instead, oHSV-1 induced MM cell apoptosis via caspase-3 cleavage.

Venetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain–binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).

Background/Objectives: Despite intensive multimodal therapy, greater than 50% of children with high-risk neuroblastoma (HR NB) relapse with incurable disease. Next generation sequencing of primary HR NB tumors identified an increase in activating mutations in the RAS/RAF/MAPK pathway. Moreover, gene set enrichment analyses showed a significant decrease in expression of genes suppressed by the Yes-Associated Protein (YAP) at relapse, suggesting increased YAP transcriptional repression. YAP binds with TEAD family transcription factors to regulate gene expression. We have shown that YAP promotes chemotherapy and MEK inhibitor resistance in RAS-mutated NB tumors in vivo by suppressing the expression of Harakiri (HRK), a BH3-only pro-apoptotic protein activated in response to tumor environmental stress such as serum starvation. Our overall objective is to elucidate how YAP represses HRK and tumor suppressor genes globally, and to enhance MEK inhibitor potency by combining MEK inhibition with agents that inhibit YAP or induce HRK to restore the tumor environmental stress response and apoptosis in RAS-mutated NB. Design/Methods: We used publicly available databases to identify TEAD binding sites on the HRK gene locus in NB. To assess the global state of methylation, we treated NB cells, SK-N-AS (NRASQ61K mutation, MYCN non-amplified) and NLF (NF1 deletion, MYCN amplified), with demethylating agent azacitidine and evaluated HRK expression. To identify the specific TEAD (1-4) binding partner to YAP, we performed siRNA and co-immunoprecipitation studies. We further tested novel YAP-TEAD small molecule inhibitors with varying TEAD1-4 inhibition specificity in SK-N-AS and NLF cells in vitro. Results: We observed that TEAD binds near cis-regulatory regions on the HRK gene locus in NB. We found that HRK expression is restored when SK-N-AS and NLF cells are treated with azacitidine despite YAP expression increasing. We also identified TEAD2 as the specific binding partner to YAP in NB and found that TEAD2 is necessary for HRK regulation. Novel YAP-TEAD small molecule inhibitors affect NB cell viability under serum-deprived conditions in vitro, especially the inhibitor with highest specificity against TEAD2, and affect YAP-TEAD downstream targets. Conclusions: YAP-TEAD2 binding is essential for HRK regulation in RAS-mutated NB and thus is a logical therapeutic target to restore therapy response. Further studies are ongoing to test YAP-TEAD small molecule inhibitors in combination with MEK inhibitors and in vivo. Citation Format: Jenny Shim, Andrew Ho, Hunter C. Jonus, Adeiye A. Pilgrim, Benjamin G. Barwick, Tracy T. Tang, Lawrence H. Boise, Kelly C. Goldsmith. YAP-TEAD2 binding mediates therapy resistance in RAS-driven neuroblastoma. [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 3552.

The molecular mechanisms underlying chemoresistance in some newly diagnosed multiple myeloma (MM) patients receiving standard therapies (lenalidomide, bortezomib, and dexamethasone) are poorly understood. Identifying clinically relevant gene networks associated with death due to MM may uncover novel mechanisms, drug targets, and prognostic biomarkers to improve the treatment of the disease. This study used data from the MMRF CoMMpass RNA-seq dataset (N = 270) for weighted gene co-expression network analysis (WGCNA), which identified 21 modules of co-expressed genes. Genes differentially expressed in patients with poor outcomes were assessed using two independent sample t-tests (dead and alive MM patients). The clinical performance of biomarker candidates was evaluated using overall survival via a log-rank Kaplan–Meier and ROC test. Four distinct modules (M10, M13, M15, and M20) were significantly correlated with MM vital status and differentially expressed between the dead (poor outcomes) and the alive MM patients within two years. The biological functions of modules positively correlated with death (M10, M13, and M20) were G-protein coupled receptor protein, cell–cell adhesion, cell cycle regulation genes, and cellular membrane fusion genes. In contrast, a negatively correlated module to MM mortality (M15) was the regulation of B-cell activation and lymphocyte differentiation. MM biomarkers CTAG2, MAGEA6, CCND2, NEK2, and E2F2 were co-expressed in positively correlated modules to MM vital status, which was associated with MM’s lower overall survival.

Multiple myeloma is a malignancy of antibody-secreting plasma cells. Most patients benefit from current therapies, however, 20% of patients relapse or die within two years and are deemed high risk. Here we analyze structural variants from 795 newly-diagnosed patients as part of the CoMMpass study. We report translocations involving the immunoglobulin lambda (IgL) locus are present in 10% of patients, and indicative of poor prognosis. This is particularly true for IgL-MYC translocations, which coincide with focal amplifications of enhancers at both loci. Importantly, 78% of IgL-MYC translocations co-occur with hyperdiploid disease, a marker of standard risk, suggesting that IgL-MYC-translocated myeloma is being misclassified. Patients with IgL-translocations fail to benefit from IMiDs, which target IKZF1, a transcription factor that binds the IgL enhancer at some of the highest levels in the myeloma epigenome. These data implicate IgL translocation as a driver of poor prognosis which may be due to IMiD resistance., Multiple myeloma is frequently characterised by translocation of genes next to the immunoglobulin heavy chain locus. In this study, the authors sequence a large cohort of high risk myeloma samples and find translocations of cMyc to the immunoglobulin heavy chain locus and this is associated with poor prognosis.

BackgroundMedulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes.ResultsWe investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout ofTet1in the smoothened (SmoA1)mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacologicalTet1inhibition reduces cell viability andplatelet-derived growth factorsignaling pathway-associated genes.ConclusionsThese results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

Advanced prostate cancer (PCa) often develops bone metastasis, for which therapies are very limited and the underlying mechanisms are poorly understood. We report that bone-borne TGF-β induces the acetylation of transcription factor KLF5 in PCa bone metastases, and acetylated KLF5 (Ac-KLF5) causes osteoclastogenesis and bone metastatic lesions by activating CXCR4, which leads to IL-11 secretion, and stimulating SHH/IL-6 paracrine signaling. While essential for maintaining the mesenchymal phenotype and tumorigenicity, Ac-KLF5 also causes resistance to docetaxel in tumors and bone metastases, which is overcome by targeting CXCR4 with FDA-approved plerixafor. Establishing a mechanism for bone metastasis and chemoresistance in PCa, these findings provide a rationale for treating chemoresistant bone metastasis of PCa with inhibitors of Ac-KLF5/CXCR4 signaling., The therapies for bone metastatic prostate cancer are limited and the underlying mechanisms are unclear. Here, the authors show that bone derived TGF-β induces acetylation of KLF5 (Ac-KLF5), and Ac-KLF5 promotes prostate cancer bone metastasis and resistance to docetaxel by upregulating CXCR4.

Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup– and IL-6–induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup–induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.

Introduction The clinical and biological significance of the newly described SCLC subtypes, SCLC-A, SCLC-N, SCLC-Y, and SCLC-P, defined by the dominant expression of transcription factors ASCL1, NeuroD1, YAP1, and POU2F3, respectively, remain to be established. Methods We generated new RNA sequencing expression data from a discovery set of 59 archival tumor samples of neuroendocrine tumors and new protein expression data by immunohistochemistry in 99 SCLC cases. We validated the findings from this discovery set in two independent validation sets consisting of RNA sequencing data generated from 51 SCLC cell lines and 81 primary human SCLC samples. Results We successfully classified 71.8% of SCLC and 18.5% of carcinoid cases in our discovery set into one of the four SCLC subtypes. Gene set enrichment analysis for differentially expressed genes between the SCLC survival outliers (top and bottom deciles) matched for clinically relevant prognostic factors revealed substantial up-regulation of interferon-γ response genes in long-term survivors. The SCLC-Y subtype was associated with high expression of interferon-γ response genes, highest weighted score on a validated 18-gene T-cell–inflamed gene expression profile score, and high expression of HLA and T-cell receptor genes. YAP1 protein expression was more prevalent and more intensely expressed in limited-stage versus extensive-stage SCLC (30.6% versus 8.5%; p = 0.0058) indicating good prognosis for the SCLC-Y subtype. We replicated the inflamed phenotype of SCLC-Y in the two independent validation data sets from the SCLC cell lines and tumor samples. Conclusions SCLC subtyping using transcriptional signaling holds clinical relevance with the inflamed phenotype associated with the SCLC-Y subset.

Human deep space and planetary travel is limited by uncertainties regarding the health risks associated with exposure to galactic cosmic radiation (GCR), and in particular the high linear energy transfer (LET), heavy ion component. Here we assessed the impact of two high-LET ions 56Fe and 28Si, and low-LET X rays on genome-wide methylation patterns in human bronchial epithelial cells. We found that all three radiation types induced rapid and stable changes in DNA methylation but at distinct subsets of CpG sites affecting different chromatin compartments. The 56Fe ions induced mostly hypermethylation, and primarily affected sites in open chromatin regions including enhancers, promoters and the edges (“shores”) of CpG islands. The 28Si ion-exposure had mixed effects, inducing both hyper and hypomethylation and affecting sites in more repressed heterochromatic environments, whereas X rays induced mostly hypomethylation, primarily at sites in gene bodies and intergenic regions. Significantly, the methylation status of 56Fe ion sensitive sites, but not those affected by X ray or 28Si ions, discriminated tumor from normal tissue for human lung adenocarcinomas and squamous cell carcinomas. Thus, high-LET radiation exposure leaves a lasting imprint on the epigenome, and affects sites relevant to human lung cancer. These methylation signatures may prove useful in monitoring the cumulative biological impact and associated cancer risks encountered by astronauts in deep space.

PTEN (phosphatase and tensin homolog) is a known tumor suppressor gene (TSG) in solid tumors and hematologic malignancies, including multiple myeloma (MM), yet several aspects of its biology and clinical implications remain incompletely understood in MM. For instance, PTEN mutations/deletions are infrequent in newly diagnosed MM in the CoMMpass study (6 and 9 cases with heterozygous mutations or loss of one PTEN gene copy, respectively in 957 patients, IA16; no evidence of bi-allelic loss), and there is no enrichment for PTEN transcript suppression or biallelic PTEN loss at relapse. Moreover, while MM cell lines are considered to reflect advanced relapsed/refractory MM, nearly all are PTEN-proficient (with notable exceptions e.g. biallelic loss in OPM2). These considerations raise a question whether PTEN loss is indeed functionally associated with a competitive advantage for MM cells compared to their PTEN-proficient counterparts. In genome-scale CRISPR-Cas9 gene editing (knockout, KO) screens we observed that 16/19 MM cell lines exhibited CERES or CHRONOS scores >0.4, consistent with the proposed role of PTEN as a negative regulator of survival and proliferation: the 3 outlier cell lines included OPM2 (biallelic PTEN deletion) and 2 other lines (SKMM2 and AMO1) with no obvious genetic events suppressing PTEN function or causing constitutive activation of PI3K/Akt/mTOR signaling. We performed subgenome-scale CRISPR KO screens with focused libraries of single guide RNAs (sgRNAs) for several known (including PTEN) or candidate TSGs and control sgRNAs from MM and other tumors in vitro and confirmed that in vitro enrichment of PTEN KO in several PTEN-proficient (but not -deficient lines), consistent with the genome-scale CRISPR study results. These observations were further confirmed with in vitro competition assays where isogenic Cas9+ cells transduced with sgRNAs against PTEN or olfactory receptor (OR) genes (as DNA-cutting negative controls) were mixed, cultured for 4-12 weeks and DNA sequenced for the distribution of their sgRNAs, and determined a significant relative enrichment of PTEN KOs compared to their OR KO counterparts. Importantly, when cells harboring focused sub-genome scale sgRNA library were implanted in vivo in a bicalcium phosphate scaffold-based bone marrow (BM)-like model with a "humanized" mesenchymal bone stromal cell compartment, we observed that CRISPR KO of PTEN represented the top enriched gene perturbation in the PTEN-proficient MM cell lines MM.1S and KMS11 (not the AMO1 line) in vivo, consistent with the in vitro results. Collectively, these observations indicate that while PTEN deficiency is infrequent in newly diagnosed MM patient samples and in preclinical cell line models that reflect advanced disease, PTEN KO is associated with major "fitness" advantage for MM cells in preclinical in vitro and in vivo xenograft models, i.e. systems in which tumor cell "fitness" does not account for potential immunogenicity of human tumor cells. We hypothesized that PTEN KO remain highly immunogenic which curtails their "fitness" advantage: indeed, we observed in a series of CRISPR KO screens in PTEN-proficient cell lines from MM, other hematologic malignancies or solid tumors, PTEN loss was not associated with increased resistance to allogeneic donor-derived NK cells (in-house studies) or T-cell treatment (e.g. 41T, RENCA cells; GSE149933). In fact, for some MM cell lines (e.g. MM1S, KMS11), PTEN KO led to more pronounced sensitivity to NK cells (compared to control OR KOs) in genome-scale or focused CRISPR screens, and in validation experiments with individual sgRNAs for PTEN (vs. OR gene sgRNAs). While our functional genomic studies document that PTEN loss confers a generalizable "fitness advantage" for MM lines in vitro and in immunocompromised in vivo models, the limited, if any, association of advanced MM with PTEN loss raises intriguing questions about the potentially complex role of PTEN as a TSG. While alternative hypotheses are plausible, our results point to the immune responses of PTEN-deficient tumor cells as an additional factor that can reconcile our integrated observations in molecular annotated clinical samples and preclinical functional genomic studies. Disclosures Tsherniak: Cedilla Therapeutics: Consultancy; Foghorn Therapeutics: Consultancy; The Center for Protein Degradation: Consultancy; SAB: Membership on an entity's Board of Directors or advisory committees. Licht: Epizyme: Research Funding. Boise: Abbvie: Consultancy; AstraZeneca: Consultancy, Research Funding. Mitsiades: Arch Oncology: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; EMD Serono: Research Funding; Abbvie: Research Funding; Nurix: Research Funding; Janssen/Johnson & Johnson: Research Funding; BMS: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria; Fate Therapeutics: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; TEVA: Research Funding; Novartis: Research Funding; H3 Biomedicine: Research Funding.

Multiple myeloma (MM) is an incurable form of plasma cell cancer in which primary and secondary chromosomal translocations routinely juxtapose oncogenes to plasma cell-specific super-enhancers. Coincidentally, drugs which target super-enhancers have had success clinically. For example, immunomodulatory imide drugs (IMiDs) degrade super-enhancer-binding pioneer factors IKAROS and AIOLOS, while glucocorticoids (Dexamethasone) and proteasome inhibitors (Bortezomib) have the ability to transrepress or block the processing of super-enhancer-forming NF-κB proteins, respectively. Currently, alternative enhancer-targeting drugs are also in clinical development, like p300 inhibitors which target the acetyl-binding bromodomains and/or histone acetyl transferase activity of the chromatin-regulating coactivator homologs CBP and EP300. Despite showing therapeutic promise, our understanding of how these drugs function, alone or together, remains incomplete. Case in point, we find that IMiD-induced degradation of its target proteins IKAROS and AIOLOS does not guarantee a therapeutic response in vitro, and patients successfully treated with IMiDs eventually relapse; meanwhile, coactivator-targeting therapies like p300 inhibitors are often too toxic in vivo, and lack a therapeutic window. To improve the outcomes of MM patients we need to understand the heterogeneous genetics and transcription-factor milieus of the myeloma enhancer landscape, as well as how to increase the precision of enhancer-disrupting drugs. To accomplish this, our lab utilizes more than 60 human myeloma cell lines that have been extensively characterized at the genetic, proteomic, and drug-therapeutic-response levels. Additionally, we have generated a highly-predictive immunocompetent mouse model (Vk*MYC hCRBN+) that develops human-like MM and is sensitive to both IMiDs and a new class of therapeutics termed "degronimids" (normal mice do not respond to IMiDs or degronimids). Our central hypothesis is that combining a broad coactivator-targeting drug (e.g., the p300 inhibitor GNE-781), with a MM-specific transcription factor-targeting drug (e.g., IMiDs) restricts toxicities to myeloma cells and thus improves the therapeutic window. Currently, we are testing a variety of coactivator-targeting compounds alongside traditional IMiD therapies and other preclinical transcription factor-targeting drugs both in vivo and in vitro. We show that Vk*MYC hCRBN+ mice are exquisitely sensitive to GNE-781, requiring one fourth of the dose needed to treat other cancers and therefore avoiding the neutropenia and thrombocytopenia seen at higher doses. Second, we show that although IMiDs and GNE-781 induce an effective but transient response in vivo as single agents, the combination of the two drugs proved curative, with a progressive deepening of the anti-tumor response occurring even after therapy is discontinued. Ongoing experiments aim to determine how this drug combination, and other coactivator + transcription factor-targeting combinations, permanently disrupt myeloma-specific super-enhancers. Disclosures Neri: BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis: Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Genentech: Consultancy. Boise: AstraZeneca: Honoraria, Research Funding; AbbVie/Genentech: Membership on an entity's Board of Directors or advisory committees. Chesi: Abcuro: Patents & Royalties: Genetically engineered mouse model of myeloma; Pi Therapeutics: Patents & Royalties: Genetically engineered mouse model of myeloma; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties: human CRBN transgenic mouse; Palleon Pharmaceuticals: Patents & Royalties: Genetically engineered mouse model of myeloma.

Background: Moderate mitochondrial stress induced by multiple mediators but most notably ROS can lead to activation of persistent mito-protective mechanisms termed "Mitohormesis". As a result of massive protein synthesis, malignant plasma cells (PCs) from MM patients (pts) undergo substantial ER stress but in addition high rates of Ig synthesis contributes to overproduction of ROS. We hypothesized that MM cells exploit mitohormesis to maintain ROS in the hometic zone, thereby increasing mitochondrial fitness to avoid apoptosis. We therefore set out to determine if the processes of mitohormesis are activated in MM and whether unmitigated mitochondrial stress can be exploited as a therapeutic strategy in MM. Results: Protective stress mechanisms of mitohormesis include the activation of the mitochondrial UPR (UPR MT),a mitochondrial-to-nuclear signaling pathway mediated by CHOP and ATF5 that upregulates mitochondrial import proteins, chaperones and proteases to maintain mitochondrial proteastasis. We first demonstrated that UPR MT activation occurs with progression from precursor to overt MM. Using a UPR MTgene signature derived from published gene-sets we observed upregulation of UPR MT genes in single-cell RNA sequencing (scRNA-seq) data generated from PCs derived from Vκ*MYC mice (a transgenic mouse model of MM) spanning the spectrum of the disease. UPR MT gene signature scores in PCs from mice increased with disease progression with the highest levels found in late-MM> int-MM> early MM>wild type mice. Similarly, analysis of publicly available gene expression datasets (GSE6477) that includes normal donors, MGUS and newly diagnosed MM (NDMM) revealed higher expression of UPR MT genes in the majority of NDMM, weak expression in MGUS and absence in normal PCs. To assess the impact of UPR MT expression on pt outcomes we calculated a UPR MT index score derived from the median expression of 12 mtUPR classifier genes across the MMRF CoMMpass dataset of NDMM pts. Stratifying pts by UPR MT expression score we found that pts in the top quartile had a significantly shorter PFS and OS compared to pts with the lowest quartile weighted score. Next, we postulated that perturbation of the mitochondrial import protein, Translocase of the Inner Membrane 23 (TIM23) would exaggerate mitochondrial stress as mitochondrial import efficiency is a key regulator of the UPR MT. First, we demonstrated that TIM23 complex genes are enriched in pts from the CoMMpass dataset with poor risk (1q gain and PR gene signature) and that shorter PFS and OS is associated with a higher weighted score of TIM23 complex genes. We then demonstrated that genetic (shRNA) knockdown or pharmacologic inhibition of TIM23 with MB-10, a small molecule inhibitor of TIM23 induced apoptosis of MM cell lines and primary pt PCs. Further non-transformed cell lines, CD138 - non-MM cells and normal donor hematopoietic progenitor cells were less susceptible to the effects of MB-10. Consistent with activation of the UPR MT, treatment of MM cells resulted in increased cytosolic ATF4, CHOP and a shift of ATF5 to the nuclear fraction. Activation of the CHOP-dependent branch of the UPR MT resulted in in upregulation of mitochondrial-targeted proteins, cpn10 and ClpP. Interestingly, MB-10 also induced XBP1 splicing demonstrating that inhibition of TIM23 complex can simultaneously activate the IRE1/XBP1 branch of integrated stress response (ISR), This led us to hypothesize that targeting TIM23 as an alternative means of activating the ISR could overcome acquired resistance to proteosome inhibitors (PIs). Indeed, PI-resistant and parental isogenic cell lines were equally susceptible to MB-10 as measured by IC50 values of cell growth. Finally, we demonstrated that doxycycline inducible knockdown of TIM23 in a mouse xenograft model induced tumor regression with significantly small tumor volumes at the end of 17 days of doxycycline treatment compared to tumors expressing an inducible control vector. Conclusions: These data demonstrate that mitohormesis and UPR MT activation is associated with MM progression and worse clinical outcomes. Further we show that disrupting mitochondrial protein import results in unmitigated mitochondrial stress that switches the UPR MT from an adaptive cytoprotective to cytotoxic proapoptotic response. Thus, targeting mitochondrial import proteins such as TIM23 may represent novel therapeutic targets for MM. Disclosures Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Trudel: Janssen: Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Sanofi: Honoraria; Pfizer: Honoraria, Research Funding; Genentech: Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding.

Background We have previously demonstrated that the cytoplasmic region of CD86 confers a survival phenotype in myeloma cells and regulates IRF4 and Integrin β7 expression. Here, we show that the cytoplasmic tail is important for surface expression of CD86. Methods We transfected HEK293T with either full length CD86 (CD86FL) or a mutant lacking the cytoplasmic tail (CD86TL). CD86FL can properly traffic to the cell surface while CD86TL cannot effectively export from the Golgi apparatus. We developed additional truncation mutants of the CD86 cytoplasmic tail and identified that several regions of the tail are required for proper trafficking out of the Golgi. While the truncation mutants traffic CD86 more effectively than CD86TL, they never fully phenocopy CD86FL surface expression. One specific region for proper transport is a three amino acid-long PDZ binding motif at the C-terminus of the tail. Using BioID analysis, we identified two PDZ-domain containing proteins, SCRIB and DLG1 as proximal to the CD86 cytoplasmic tail. Results Using the CoMMpass dataset, we identified that high SCRIB expression is a poor prognostic indicator for progression free (p=0.00022) and overall (p=0.000132) survival. We observed co-localization of SCRIB and DLG1 with CD86 in myeloma using confocal microscopy. Using a doxycycline-inducible CRISPR-Cas9 system, we deleted SCRIB and DLG1 in two myeloma cell lines, KMS18 and RPMI8226. Ablation of SCRIB or DLG1 decreased CD86 surface expression 3 days following activation of the Cas9 enzyme via doxycycline addition. Additionally, we found that knockout of SCRIB or DLG1 results in significantly decreased cell proliferation and viability in these cell lines. SCRIB and DLG1 have been classically studied in the context of apical-basal polarity indicating a possible role for regulating CD86 expression in time and space. We found decreased localization of SCRIB/DLG1 with CD86 at areas of cell-cell contact, presumably where CD86 binds to CD28 to signal for myeloma cell survival. These regions of contact have increased surface expression of CD86 relative to areas where there is no cell contact. This is only prevalent in myeloma cells, and we observed uniform distribution of expression of CD86 throughout the membrane of CD86FL-transfected HEK293T. This suggests that SCRIB and DLG1 transport CD86 to the cell surface, and binding with CD28 may help to stabilize CD86 surface expression. Ablation of SCRIB and DLG1 also decreases IRF4 and Integrin β7 expression, suggesting that SCRIB and DLG1 may contribute to CD86-mediated growth and survival signaling. Conclusion Our data supports a role for PDZ proteins as regulators of myeloma cell signaling and viability. Study of PDZ-proteins and their binding motifs may be warranted as numerous surface proteins in myeloma cells such as ICAM-1 and CD138 contain PDZ-binding motifs. The PDZ-binding motif may represent a specific region that can effectively be targeted and may be an attractive strategy for novel therapeutics.

14-3-3 proteins are a family of master regulators of intracellular signaling, yet their impact on proteasome function is unknown. We demonstrate that 14-3-3ζ binds the 11S proteasome activator, limiting proteasome assembly and cellular capacity for protein degradation. To define the functional impact of 14-3-3ζ proteasomal binding in myeloma cells, silencing and overexpression experiments are performed. We find that downregulation of 14-3-3ζ impairs myeloma cell growth and confers resistance to clinically used proteasome inhibitors. In a large cohort of newly diagnosed myeloma patients, elevated expression of 14-3-3ζ is associated with high risk myeloma genetic subtypes and worse prognosis overall. Our work demonstrates the important role of 14-3-3ζ in regulating proteasome function, myeloma cell growth and sensitivity to therapeutics, and suggests regulation of 14-3-3ζ as a new approach in myeloma therapy.

High dose melphalan and autologous stem cell transplantation is standard of care for the upfront treatment of multiple myeloma (MM). Several studies have shown upregulation of single DNA repair genes and whole DNA repair pathways as associated with melphalan resistance and poor outcomes after ASCT. Here we set out to identify the most important DNA repair enzymes and pathways to predict outcomes after ASCT using the MMRF CoMMpass dataset but instead found DNA repair gene expression to be a poor prognostic feature regardless of treatment. Of the 561 MM patients who received ASCT as part of frontline therapy, 378 (67%) patients had whole transcriptome sequencing data available for analysis in this study. The majority of patients in this cohort received a 3-drug novel agent-based regimen for first line treatment. We selected 81 genes of known function related to DNA damage repair across multiple pathways. Using the median mRNA expression as cutoff for each gene, we first compared PFS for high vs. low expressers and found 38 genes in non-homologous end joining (NHEJ), homologous recombination (HR), nucleotide excision repair (NER), base excision repair (BER) and Fanconi anemia (FA) pathway which predicted for inferior survival with higher expression (p≤0.05). We subsequently analyzed each gene individually in a multivariate Cox proportional hazards models adjusted for known prognostic and treatment-related factors (age, gender, race, ISS stage, LDH, normal/abnormal cytogenetics, high risk FISH/cytogenetics, cycles to first response, frontline treatment, and time to transplant) and found that 9 genes retained significance including 3 genes in NHEJ (POLL, PRKDC, NHEJ1), 3 in FA pathway (BRIP1, RMI1, FANCE) and 3 in MMR (MLH3, MSH2 and PMS1). In addition, increased pathway level gene overexpression for NHEJ (p=0.02) and MMR (0.009) pathways conferred worse PFS. Genes involved in NER did not retain significance despite this pathway being involved with repair of melphalan-induced bulky adducts formation. Additionally, high MMR pathway expression was significant despite this pathway not having a known role in repair of melphalan-induced DNA damage. To validate the specific importance of these genes in predicting response to ASCT, we tested whether high expression predicted outcomes in patients who did not undergo ASCT. In 387 non-ASCT patients with whole transcriptome sequencing available, NHEJ and MMR gene expression were significant predictors of PFS confirming that this finding was not specific to patients undergoing ASCT. We next correlated the expression of NHEJ and MMR genes and found significant co-expression of these genes (r=0.55-0.8), suggesting a common mediator leading to global upregulation of DNA repair. As increased MYC activity is a common finding in MM and is a master regulator of transcription, we hypothesized the DNA damage repair genes were upregulated by increased MYC activity. Gene Set Enrichment Analysis of hallmark gene sets confirmed that patients with increased DNA repair were also enriched for MYC targets (FDR=0.0063). Based on this, we hypothesized that oncogene-mediated constitutive DNA damage and replication stress (RS) are a hallmark of high risk and aggressive myeloma. Here we present novel findings to show that global DNA repair upregulation occurs in high risk disease as evidenced by the inferior PFS. We hypothesize that this could be attributed to MYC-related increased gene transcription resulting in DNA damage and RS which in turn recruits several DNA repair pathways. These specific DNA repair pathways and signal activation pathways involved with replication stress represent novel therapeutic targets in myeloma. Disclosures Calip: Flatiron Health: Current Employment. Hofmeister:Sanofi: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Nektar: Honoraria, Research Funding; Imbrium: Honoraria; Janssen: Honoraria, Research Funding; Oncopeptides: Honoraria; Karyopharm: Honoraria, Research Funding; Oncolytics Biotech: Research Funding. Patel:Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

B cell activation and differentiation yields plasma cells with high affinity antibodies to a given antigen in a time-frame that allows for host protection. Although the end product is most commonly humoral immunity, the rapid proliferation and somatic mutation of the B cell receptor also results in oncogenic mutations that cause B cell malignancies including plasma cell neoplasms such as multiple myeloma. Myeloma is the second most common hematological malignancy and results in over 100,000 deaths per year worldwide. The genetic alterations that occur in the germinal center, however, are not sufficient to cause myeloma, but rather impart cell proliferation potential on plasma cells, which are normally non-dividing. This pre-malignant state, referred to as monoclonal gammopathy of undetermined significance or MGUS, provides the opportunity for further genetic and epigenetic alterations eventually resulting in a progressive disease that becomes symptomatic. In this review, we will provide a brief history of clonal gammopathies and detail how some of the key discoveries were interwoven with the study of plasma cells. We will also review the genetic and epigenetic alterations discovered over the past 25 years, how these are instrumental to myeloma pathogenesis, and what these events teach us about myeloma and plasma cell biology. These data will be placed in the context of normal B cell development and differentiation and we will discuss how understanding the biology of plasma cells can lead to more effective therapies targeting multiple myeloma.

Aberrant DNA methylation is a critical feature of cancer. Epigenetic therapy seeks to reverse these changes to restore normal gene expression. DNA demethylating agents, including 5-aza-2′-deoxycytidine (DAC), are currently used to treat certain leukemias, and can sensitize solid tumors to chemotherapy and immunotherapy. However, it has been difficult to pin the clinical efficacy of these agents to specific demethylation events, and the factors that contribute to the durability of response remain largely unknown. Here we examined the genome-wide kinetics of DAC-induced DNA demethylation and subsequent remethylation after drug withdrawal in breast cancer cells. We find that CpGs differ in both their susceptibility to demethylation and propensity for remethylation after drug removal. DAC-induced demethylation was most apparent at CpGs with higher initial methylation levels and further from CpG islands. Once demethylated, such sites exhibited varied remethylation potentials. The most rapidly remethylating CpGs regained >75% of their starting methylation within a month of drug withdrawal. These sites had higher pretreatment methylation levels, were enriched in gene bodies, marked by H3K36me3, and tended to be methylated in normal breast cells. In contrast, a more resistant class of CpG sites failed to regain even 20% of their initial methylation after 3 months. These sites had lower pretreatment methylation levels, were within or near CpG islands, marked by H3K79me2 or H3K4me2/3, and were overrepresented in sites that become aberrantly hypermethylated in breast cancers. Thus, whereas DAC-induced demethylation affects both endogenous and aberrantly methylated sites, tumor-specific hypermethylation is more slowly regained, even as normal methylation promptly recovers. Taken together, these data suggest that the durability of DAC response is linked to its selective ability to stably reset at least a portion of the cancer methylome.

Advanced prostate cancer most commonly spreads to bones. Although denosumab or zoledronic acid is recommended for prostate cancer patients with bone metastases to prevent or delay skeletal-related events, no significant benefits in terms of cancer-specific and overall survival have been observed. Some chemotherapies are available for the treatment of bone metastasis, but eventually all such tumors develop resistance. Additionally, the underlying mechanisms of prostate cancer bone metastasis and its resistance to chemotherapy are still poorly understood. In this study, we report that TGF-β-induced acetylation of the basic transcription factor Krüppel-like factor 5 (KLF5) frequently occurred in prostate cancer cells located in both the mouse and human bone microenvironments. Using multiple in vitro and in vivo systems, we demonstrated that tumor cells expressing acetylated KLF5 (Ac-KLF5) enhanced prostate cancer-induced bone lesions by promoting osteoclast differentiation. As revealed by RNA-Seq and ChIP-Seq analyses, Ac-KLF5 transcriptionally activated CXCR4 expression, the silence of which attenuated Ac-KLF5-enhanced osteoclast differentiation. A further screening on the paracrine signaling indicates that IL-11 mediated the promoting effect of the Ac-KLF5/CXCR4 axis on osteoclast differentiation. These findings indicate that the TGF-β/Ac-KLF5/CXCR4/IL-11 signaling axis from bone to tumor and then to bone again is a crucial pathway responsible for TGF-β mediated vicious cycle in bone metastasis. More importantly, targeting CXCR4 using FDA approved drugs AMD3100 selectively sensitizes Ac-KLF5-expressing tumors and bone metastases to docetaxel treatment. These findings provide a rationale for targeting Ac-KLF5/CXCR4 signaling as a novel therapeutic strategy in advanced prostate cancer patients with bone metastases. Citation Format: Baotong Zhang, Yixiang Li, Qiao Wu, Lin Xie, Benjamin Barwick, Jin-Tang Dong. KLF5 acetylation promotes bone metastatic growth of prostate cancer by activating CXCR4/IL-11 and subsequent osteoclast differentiation [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 3840.

Multiple myeloma is a malignancy of antibody-secreting plasma cells. Most patients benefit from current therapies, however, 20% of patients relapse or die within two years and are deemed ‘high-risk’. To better understand and identify high-risk myeloma, we analyzed the translocation landscape of 826 newly-diagnosed patients by whole genome sequencing as part of the CoMMpass study. Translocations at the IgL locus were present in 10% of myeloma patients, and corresponded with poor prognosis. Importantly, 70% of IgL translocations co-occurred with hyperdiploid disease, a marker of standard risk, which is routinely diagnosed clinically whereas IgL-translocations are not. Thus, it is likely that the majority of IgL-translocated myeloma is being misclassified. The IgL enhancer is among the strongest in myeloma cells, indicating it can robustly drive oncogene expression when translocated. Consistent with this, IgL-translocated patients failed to benefit from immunomodulatory imide drugs (IMiDs), which target the lymphocyte-specific transcription factor Ikaros. These data implicate the IgL enhancer as resistant to IMiD-inhibition, and when translocated, as a driver of poor prognosis.

Multiple myeloma (MM) is a commonly occurring hematologic malignancy in the United States with poor prognosis. Among all treatments, proteasome inhibitor (PI) based regimens have been a major breakthrough for patients' outcomes. Available PIs all target 20S proteasome core complex, and the duration of response is limited by toxicity and resistance development. Until now, the underlying mechanism of drug resistance remains unclear. The proteasome is the major proteolytic machinery in protein homeostasis which is pivotal for myeloma cell survival. A functional proteasome consists of 20S proteasome core particle with regulatory particle on one or both ends. There are 3 types of proteasome regulators that could activate a 20S proteasome, PA700 (19S), 11S REG (PA28) and PA200. The 11S REG (PA28) protein family consists of three members, α, β, and γ. PA28 α/β are IFN-γ inducible and with higher expression in antigen presenting cells. Currently, the function of 11S subunit remains largely unknown. Our analysis of plasma cells from MM patients and healthy donors has demonstrated that expression of 11S proteasome is higher in myeloma cells than normal plasma cells and progressively upregulated with disease progression. To further identify the function of 11S proteasome especially PA28α in MM, we generate PA28α knockdown stable MM cell lines. We have found that knockdown of PA28α inhibits MM cell growth and proliferation, also induces myeloma cell resistance to PIs. The mechanism of PI resistance is different from knocking down of 19S or 20S proteasome subunits. Silencing of PA28α inhibits proteasome activity and decreases proteasome work load concurrently, resulting in a favorable proteasome load vs capacity ratio. Altogether, in this report, we describe the function of PA28α in MM cells, also provide novel insights into regulating PIs sensitivity through modulation of the 11S proteasome subunit PA28α. Disclosures Hofmeister: Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Kaufman:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Amgen: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Celgene: Consultancy; Winship Cancer Institute of Emory University: Employment; AbbVie: Consultancy; Takeda: Consultancy; TG Therapeutics: Consultancy. Nooka:Amgen: Honoraria, Other: advisory board participation; GSK: Honoraria, Other: advisory board participation; Celgene: Honoraria, Other: advisory board participation; Takeda: Honoraria, Other: advisory board participation; Spectrum pharmaceuticals: Honoraria, Other: advisory board participation; BMS: Honoraria, Other: advisory board participation; Janssen: Honoraria, Other: advisory board participation; Adaptive technologies: Honoraria, Other: advisory board participation. Boise:Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. Lonial:Takeda: Consultancy, Research Funding; Amgen: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; GSK: Consultancy; Karyopharm: Consultancy; Genentech: Consultancy; Celgene Corporation: Consultancy, Research Funding.

Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal plasma cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation. However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these contribute to disease etiology. To this end, we have analyzed the DNA methylome of 119 myeloma specimens from the CoMMpass study (NCT01454297) by whole genome bisulfite sequencing (WGBS) and more than 90% of these same specimens were also characterized for structural variants (long-insert whole genome sequencing) and gene expression (RNA-seq). Unsupervised hierarchical clustering grouped together specimens with t(4;14) translocations that upregulate the H3K36 dimethyltransferase NSD2 (also known as MMSET and WHSC1), which likely impacts the DNA methylation state through epigenetic cross-talk. These data also revealed a dramatic genome-wide hypomethylation where myeloma samples had a median global CpG methylation level of 41% as compared to 71% and 89% in normal plasma cells and B cells, respectively. Demethylation of the myeloma methylome occurred in large megabase domains encompassing genes that were devoid of gene expression. Conversely, DNA methylation remained mostly unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, many regions of variable methylation exist and these differences corresponded with proximal gene expression differences. These variably methylated regions were compared to PFS and OS and this identified 6,314 CpG loci where the level of DNA methylation was prognostic of outcome (P≤0.00001). These loci were clustered into discrete regions and in the majority of cases (79%), reduced DNA methylation at these loci corresponded with poor outcome. For example, several loci in the gene bodies of PRKCE, MGMT, FHIT, WWOX were prognostic of poor survival. Interestingly, myeloma t(14;16) translocations disrupt the tumor suppressor WWOX and induce the oncogene MAF. These data suggest that WWOX expression may also be lost by epigenetic mechanisms. Finally, we analyzed the DNA methylome of primary and relapsed samples for 22 patients, including multiple relapsed samples for 2 patients. These data identified genome-wide DNA methylation remodeling with a median of 1.9 million differential methylated CpGs between the newly diagnosed and relapsed specimens. These relapse differentially methylated loci coincided at the same regions in several patients and significantly overlapped loci where the DNA methylation level was prognostic of outcome. Integrative genetic, epigenetic, and transcriptional analyses for 120 myeloma samples will be presented. Citation Format: Benjamin G. Barwick, Doris R. Powell, Daniel Penaherrera, Sheri Skerget, Jonathan J. Keats, Daniel Auclair, Sagar Lonial, Lawrence H. Boise, Paula M. Vertino. Whole genome DNA methylation analysis of multiple myeloma identifies pervasive hypomethylation and biomarkers of survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 839.

The differentiation of CD8 T cells in response to acute infection results in the acquisition of hallmark phenotypic effector functions; however, the epigenetic mechanisms that program this differentiation process on a genome-wide scale are largely unknown. In this article, we report the DNA methylomes of Ag-specific naive and day-8 effector CD8 T cells following acute lymphocytic choriomeningitis virus infection. During effector CD8 T cell differentiation, DNA methylation was remodeled such that changes in DNA methylation at gene promoter regions correlated negatively with gene expression. Importantly, differentially methylated regions were enriched at cis-elements, including enhancers active in naive T cells. Differentially methylated regions were associated with cell type–specific transcription factor binding sites, and these transcription factors clustered into modules that define networks targeted by epigenetic regulation and control of effector CD8 T cell function. Changes in the DNA methylation profile following CD8 T cell activation revealed numerous cellular processes, cis-elements, and transcription factor networks targeted by DNA methylation. Together, the results demonstrated that DNA methylation remodeling accompanies the acquisition of the CD8 T cell effector phenotype and repression of the naive cell state. Therefore, these data provide the framework for an epigenetic mechanism that is required for effector CD8 T cell differentiation and adaptive immune responses.

Monoallelic point mutations of the NADP+-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1R132H allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1R132H/WT mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1R132H/WT-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1R132H/WT knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1R132H/WT mutants in driving epigenetic instability in human cancer cells.

Background: Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT–PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel. Methods: The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data. Results: Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearson's R2=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R2=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes. Conclusion: We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE tissues on the DASL platform. Importantly, we have identified a 30-biomarker gene set that can classify breast cancer into subtypes and have shown that a subset of these markers is prognostic of OS and RFS.

Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal B cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation (Barwick et al., 2016, 2018). However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these may contribute to pathogenesis. To this end, we are analyzing the DNA methylome of myeloma specimens from the MMRF CoMMpass trial (NCT01454297), which has already characterized the mutational, structural, and transcriptional landscape of nearly 1,000 myelomas from newly diagnosed patients. CoMMpass specimens were obtained from a centralized biobank with approval from the CoMMpass Tissue Use Committee and Emory IRB. DNA isolated from CD138+ myeloma specimens was subjected to reduced representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS). In total, DNA methylation was derived for over 24 million CpGs with an average of 18x coverage. WGBS data from normal B cells and plasma cells was obtained with permission from the BluePrint project (Agirre et al., 2015) via the European Genome Archive. DNA methylation levels were associated with PFS and OS using a cox proportional regression. We have determined the DNA methylome for 36 primary myeloma specimens and an additional 84 specimens are currently being sequenced. Relative to normal B cells that had an average DNA methylation level of 89.1%, plasma cells and myelomas exhibited a progressive demethylation with mean levels of 71.3% and 43.7%, respectively. While this is consistent with previous observations (Agirre et al., 2015; Salhia et al., 2010), WGBS revealed that myeloma in particular was characterized by large hypomethylated domains. These large hypomethylated domains encompassed genes that were devoid of gene expression whereas DNA methylation remained unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, there existed many regions where methylation levels varied between myelomas and these differences commonly corresponded with local gene expression differences. To understand if these specific patterns of DNA methylation were indicative of disease pathogenesis, DNA methylation levels were compared to PFS and OS. This identified 2,594 regions where the level of DNA methylation was prognostic of outcome (P≤0.001). Reduced DNA methylation corresponded with poor outcome at 88.5% (N=2,298) of these regions, which included loci proximal to cell cycle genes such as MYC, E2F3, CCND1, and CCNE1. Only 11.5% (N=296) of regions associated with outcome had higher levels of DNA methylation associated with poor prognosis. These regions tended to be proximal to genes involved in B cell receptor signaling, such as PLCG2 and VAV2. Although the expression of several of these genes was also prognostic of survival, the majority were not, indicating that the epigenetic state contains a unique prognostic value. These data indicate that myeloma undergoes profound epigenetic remodeling that is co-ordinate with changes in gene expression. Perhaps the most striking feature were megabase domains of hypomethylation. That DNA methylation was preferentially retained in the bodies of expressed genes suggests that a molecular mechanism and/or cellular selection occurs to maintain methylation at genes whose expression is required for myeloma cell survival. Despite the small number (N=36) of myeloma specimens analyzed thus far, the large number of regions associated with survival indicates the potential prognostic value of DNA methylation in myeloma. Furthermore, DNA methylation indicative of outcome only partially overlapped with the prognostic value of gene expression, indicating DNA methylation has independent value as a biomarker of outcome in myeloma. This may be due, in part, to the fact that DNA methylation is a very stable modification that not only reflects the current gene expression program, but is also indicative of the cell history and potential. Integrative genetic, epigenetic, and transcriptional analysis from WGBS of 120 CoMMpass myeloma specimens will be presented, including matched baseline and relapsed specimens from 25 patients. Disclosures Lonial: Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.

Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Multiple myeloma is the second most common hematological malignancy with an estimated 30,000 new diagnoses in 2017. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, they have gained the capacity to proliferate, causing harmful bone lesions in patients, and in advanced stages become extramedullary. Therefore, we are investigating molecular mechanisms of myeloma cells that allow them to survive independently of the bone marrow microenvironment. We have identified syntenin as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin has been reported to bind to and regulate levels of the plasma cell marker, CD138. We used the CoMMpass dataset, a longitudinal study of myeloma patients following their transcriptomic expression throughout treatment, and discovered that patients expressing in the top quartile for syntenin trend toward lower overall survival, progression-free survival, and shorter response duration than those in the bottom quartile. To determine whether it is involved in myeloma cell survival, we used short hairpin RNA to knock down syntenin (shsyn) in RPMI 8226 and MM1.s myeloma cell lines and determined cell death using Annexin-V staining four days following lentiviral infection. We observed increased death in syntenin-silenced cells compared to our empty vector control in both RPMI 8226 (control=57.83% viable, shsyn=29.47% viable, p=0.04) and MM1.s cell lines (control=91.43% viable, shsyn=70.1% viable, p=0.04) suggesting that syntenin is important for myeloma cell survival. Syntenin is a PDZ-domain containing protein that has been shown to regulate expression of several transmembrane proteins. We, therefore, wanted to look at correlation of syntenin expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin had increased expression of CD86 while syntenin-low patients had decreased CD86 expression. This correlation does not exist with CD138 as syntenin-low patients express higher levels of CD138. The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin in myeloma cells. Our lab has shown that expression of CD86 is necessary for myeloma cell survival, and signals to confer drug resistance. When we silenced syntenin, we observed decreased CD86 expression. Moreover, knockdown of CD86 showed increased expression of syntenin. Taken together, these data suggest that syntenin may regulate CD86 expression on the cell surface. We now show a novel role for syntenin in myeloma cell viability and as a potential regulator of CD86 expression. Syntenin has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an effective target for therapeutic treatment of the disease. Citation Format: Tyler Moser-Katz, Catherine M. Gavile, Benjamin G. Barwick, Sagar Lonial, Lawrence H. Boise. Syntenin regulates multiple myeloma cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 319.

Background: Recent publications have described an important role for cross talk between PI-3 kinase and sonic hedgehog signaling pathways in the pathogenesis of medulloblastoma. Methodology/Principal Findings: We crossed mice with constitutive activation of Smoothened, SmoA1, with Pten deficient mice. Both constitutive and conditional Pten deficiency doubled the incidence of mice with symptoms of medulloblastoma and resulted in decreased survival. Analysis revealed a clear separation of gene signatures, with up-regulation of genes in the PI-3 kinase signaling pathway, including downstream activation of angiogenesis in SmoA1+/2; Pten +/2 medulloblastomas. Western blotting and immunohistochemistry confirmed reduced or absent Pten, Akt activation, and increased angiogenesis in Pten deficient tumors. Down-regulated genes included genes in the sonic hedgehog pathway and tumor suppressor genes. SmoA1+/2; Pten +/+ medulloblastomas appeared classic in histology with increased proliferation and diffuse staining for apoptosis. In contrast, Pten deficient tumors exhibited extensive nodularity with neuronal differentiation separated by focal areas of intense staining for proliferation and virtually absent apoptosis. Examination of human medulloblastomas revealed low to absent PTEN expression in over half of the tumors. Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent PTEN expression. Conclusions/Significance: This suggests that PTEN expression is a marker of favorable prognosis and mouse models with activation of PI-3 kinase pathways may be important tools for preclinical evaluation of promising agents for the treatment of medulloblastoma.

Background: Recent studies have indicated that prostate cancer patients with the TMPRSS2–ERG gene fusion have a higher risk of recurrence. To identify markers associated with TMPRSS2–ERG fusion and prognostic of biochemical recurrence, we analysed a cohort of 139 men with prostate cancer for 502 molecular markers. Methods: RNA from radical prostatectomy tumour specimens was analysed using cDNA-mediated, annealing, selection, extension and ligation (DASL) to determine mRNAs associated with TMPRSS2–ERG T1/E4 fusion and prognostic of biochemical recurrence. Differentially expressed mRNAs in T1/E4-positive tumours were determined using significance analysis of microarrays (false discovery rate (FDR)

Mutations of genes in tumor cells of Triple Negative subset of Breast Cancer (TNBC) deregulate pathways of signal transduction. The loss of tumor suppressor gene PTEN is the most common first event associated with basal-like subtype (Martins, De, Almendro, Gonen, and Park, 2012). Here we report for the first time that the functional upregulation of secreted-MMP7, a transcriptional target of Wnt-β-catenin signature pathway in TNBC is associated to the loss of PTEN. We identified differential expression of mRNAs in several key-components genes, and transcriptional target genes of the Wnt-β-catenin pathway (WP), including beta-catenin, FZD7, DVL1, MMP7, c-MYC, BIRC5, CD44, PPARD, c-MET, and NOTCH1 in FFPE tumors samples from TNBC patients of two independent cohorts. A similar differential upregulation of mRNA/protein for beta-catenin, the functional readout of WP, and for MMP7, a transcriptional target gene of beta-catenin was observed in TNBC cell line models. Genetic or pharmacological attenuation of beta-catenin by SiRNA or WP modulators (XAV939 and sulindac sulfide) and pharmacological mimicking of PTEN following LY294002 treatment downregulated MMP7 levels as well as enzymatic function of the secreted MMP7 in MMP7 positive PTEN-null TNBC cells. Patient data revealed that MMP7 mRNA was high in only a subpopulation of TNBC, and this subpopulation was characterized by a concurrent low expression of PTEN mRNA. In cell lines, a high expression of casein-zymograph-positive MMP7 was distinguished by an absence of functional PTEN. A similar inverse relationship between MMP7 and PTEN mRNA levels was observed in the PAM50 data set (a correlation coefficient of -0.54). The PAM50 subtype and outcome data revealed that the high MMP7 group had low pCR (25%) and High Rd (74%) in clinical stage T3 pathologic response in contrast to the high pCR (40%) and low residual disease (RD) (60%) of the low MMP7 group.

A plethora of genetic changes occurring in the triple negative (TN) breast tumors (BT) contribute to the different phenotypes exhibited by the tumor cells via deregulation of the specific signaling-pathways. Our group identified a differential upregulation of the Wnt-beta-catenin pathway in TN subset of BT (Barwick etal, 2008, BCS; Dey etal. 2009, AACR) and reported that the poor prognosis and higher incidence of metastasis is associated with this pathway in TNBT (Barwick etal., 2009; PNAS, revised). Of late, we demonstrated a role of the pathway in the control of integrin-mediated migration in TNBT cells (Dey etal. 2009, Adv. BCR). Since integrin-directed migration is instrumental for the invasion (directional movement across extracellular-matrix following matrix degradation) of the tumor cells and TN tumor cells are highly invasive in nature, we hypothesized that the upregulation of Wnt-beta-catenin pathway in TNBT is functionally connected to the control of invasion. Our hypothesis is strengthened by our recent identification of MMP7 (key target-gene of this pathway), as a selective-biomarker of TN subtype (Abramovitz etal, 2009, AACR). In an attempt to define the significance of the pathway in the regulation of invasion, we examined (a) the expression of MMP7 and CTNNB1 in tumor samples and 15 TNBT cell lines, (b) the activity of MMP7 in high MMP7-expressing TNBT cell lines, and (c) effects of pharmacological intervention of the pathway on the invasion of MMP7-expressing TNBT cells. We demonstrated that MMP7 is selectively upregulated in TNBT tumors (our data sets; Montreal-91, Georgia-137) and published data set (MSK-96). We validated the DASL-data by RT-PCR in tumors and cell lines. The expression of MMP7 and beta-catenin (mRNA) in tumor samples from TN patients was comparable to the pattern of expression of MMP7 and beta-catenin (protein) in TNBT cell lines. Zymography of MMP7 demonstrated that the secreted protein is enzymatically active. MDA-MB231, MDA-MB468 and SUM149 TNBT cell lines invaded more in vitro than non-TNBT cells. Administration of sulindac to attenuate the Wnt-beta-catenin pathway showed, 1) a decrease in the levels of beta-catenin (total and active) and MMP7, 2) an inhibition of invasion of cells which corroborated with the integrin-directed cell movement (real-time-video-microscopy), 3) an altered distribution-pattern of filamentous-actin, 4) over 50% attenuation of the activation of RAC1-GTPase and 5) a blockade of migration of TNBT cells. The status of activation of cofilin is directly related to cell migration, invasion, and metastasis in many tumor cells including mammary tumors. We are currently studying the state of phosphorylation of cofilin in the context of fibronectin-dependent activation of the RAC1 (upstream of cofilin) in TNBT cells following the intervention of Wnt-beta-catenin pathway, the results of which will be presented in the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5287.

Clinical Significance: Among 1 million cases of newly diagnosed breast cancer worldwide each year, over 170,000 cases will have a distinct type of triple-negative breast cancer (TNBC), which lacks expression of estrogen (ER), progesterone (PR) and human epidermal growth factor receptor 2 (Her-2/neu). The patients with TNBC are usually detected at the late stage, have an aggressive tumor type, an increased likelihood of local and distant recurrence, and a poorer prognosis compared to women with other types of breast cancer. TNBC patients usually receive neoadjuvant therapy to reduce local and distant recurrence. However, more than 50% of TNBC patients are resistant to conventional chemotherapy. Therefore, novel methods for effective therapy are an urgent need for improving survival of the TNBC patients. Methods: We have developed a targeted therapy for TNBC by systemic delivery of biodegradable theranostic magnetic iron oxide nanoparticles (IONPs) that combine targeted drug delivery and non-invasive tumor imaging capabilities. These theranostic IONPs are targeted to urokinase plasminogen activator receptor (uPAR) by conjugating to the amino-terminal fragment (ATF) of the high affinity receptor binding domain of uPA. Results: 1). Our results from analysis of the gene expressing profile of a cohort of 143 breast cancer tissues revealed that uPAR, a potential cell surface target for the development of targeted therapeutics in TNBC, is highly expressed in the TNBC tissues compared to the luminal type of breast cancer (p < 0.0001). The major advantage of targeting uPAR is that the receptor is highly expressed in tumor cells and angiogenic endothelial cells, which facilitates transporting nanoparticles across the endothelium lining tumor vessels. Internalization of ATF-nanoparticle complexes by tumor cells further increases intracellular drug concentration; 2). We demonstrated that systemic delivery of uPAR-targeted theranostic IONPs carrying a chemotherapy drug, doxorubicin, significantly inhibited the growth of primary tumors through anti-tumor and anti-angiogenesis effects, and delayed local and distant recurrence for 8 weeks as compared mice-treated with free doxorubicin in a TNBC-like 4T1 mouse mammary tumor model. The improved therapeutic effect using these targeted theranostic nanoparticles has also been demonstrated in a human breast cancer xenograft model; 3). Targeting theranostic nanoparticles into tumors allow for monitoring intratumoral drug delivery and therapeutic response in the tumors by MRI; and 4). Targeted delivery of doxorubicin using theranostic IONPs reduces systemic toxicity of the drug in normal organs, such as heart and liver. Conclusion: uPAR-targeted theranostic nanoparticles have potential for targeted therapy, timely assessment of therapeutic response of a given therapy, and ultimately, increased survival of TNBC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5482.

Malignant plasma cells alter the bone microenvironment resulting in osteolytic lesions, a typical feature of multiple myeloma (MM). Using a cytokine array in MM patients with bone disease, we identified activin A, a member of the TGFsuperfamily as a critical pathway with bone catabolic effects. By ELISA assay, we studied activin A expression in bone marrow (BM) plasma of MM patients and ex vivo–derived BM cell supernatant. In vitro and in vivo effects of activin A inhibition were investigated using a soluble receptor, RAP-011 (Acceleron Pharma Inc., Cambridge). The in vivo studies were performed using the SCID-hu mouse model, consisting of INA6 cells engrafted in a human fetal bone chip. MM patients with osteolytic lesions had a 4-fold increase in activin A expression compared with MM patients with < 1 lesion and non-MM patients (average 123.6 136 vs. 26.4 21.4 vs. 30.6 25.1 pg/mL, respectively, P < .05). The main source of activin were BM stromal cells (BMSCs), followed by osteoclasts, and osteoblasts (average levels in 72h supernatant were 1884, 1300, 299 pg/mL, respectively). In contrast, MM cells did not secrete activin but stimulated its secretion in co-culture by BMSC (1.3to 2-fold increase). Activin A inhibited OB differentiation (30% reduction of ALP activity; P < .05) and function (80% inhibition of mineralization; P < .01). RAP-011 enhanced OB differentiation and activity (5-fold increase in calcium deposition at day 21; P < .05) and reversed the inhibitory effects of MM cell lines (INA6 and MOLP5) as well as patient-derived MM cells on OB. Enhanced OB differentiation by RAP-011 resulted in inhibition of MM cell proliferation compared to BMSCs and mature OB. In vivo, treatment with RAP-011 decreased the number of osteolytic lesions and improved bone density assessed by CT imaging. An inhibition of tumor growth measured by soluble human IL-6 receptor levels (P < .02) was also noted. These data suggest that activin A is involved in development of MM bone disease and can be effectively inhibited by a soluble receptor, RAP-011. Our results support the use of the humanized clinical-grade form ACE-011 as a novel therapeutic approach for the treatment of MM.

Abstract #2027 Background: DASL (cDNA-mediated Annealing, Selection, extension Ligation) is a recently described, RT-PCR-based, multiplex gene expression assay developed by Illumina specifically for use with fragmented RNA prepared from formalin-fixed paraffin-embedded (FFPE) samples. For use with this assay, we have designed a custom 512-gene breast cancer panel, validated this panel with FFPE breast tumor samples, and identified novel genes associated with the triple negative (TN) subtype. Material and Methods: The DASL assay was used to measure mRNA levels from RNA isolated from 5 um FFPE breast tumor sections using the Roche High Pure Kit. Samples that passed our quality control tests were run in the assay. Arrays were imaged using a BeadArray Reader and data was analyzed using BeadStudio software. Differential mRNA regulation was identified by Significance Analysis of Microarrays software using a false discover rate (q-value < 0.01) or Bonferroni correction of the two-tailed Student's t-test. IHC concordance was measured by mRNA expression fold-change for positive versus negative IHC categories. Results: To validate our custom breast cancer panel, we performed the DASL assay on 187 samples from 87 patients with differing receptor status as previously determined by IHC (ER, PR, HER2) and/or FISH (HER2). Correlation between 152 common genes on Illumina's 502-gene standard cancer panel and our custom panel was very high; R2 value of 0.88. Concordance between IHC/FISH and DASL data for ESR1, PGR and ERBB2 was > 90%. We identified 73 genes differentially expressed between TN and the other tumor samples. Of these, 20 were in common between the standard and custom panels, 53 were uniquely expressed on the custom panel. These included genes associated with the TN/Basal subtype identified in previous microarray studies; CXCL1, CDH3, ANXA8, KRT5, TRIM29, KRT17, MFGE8, CX3CL1, FZD7, CHI3L2, B3GNT5 (PNAS 100:8418, 2003) and CDH3, CRABP1, CX3CL1, FOXC1, MMP7 (BMC Genomics 7:96, 2006) further validating both DASL assay and custom panel. In addition, we identified genes not previously linked to the TN subtype, highlighting the potential utility of the custom panel for profiling breast tumor FFPE specimens in the DASL assay. Discussion: We have designed and validated a custom 512-gene breast cancer panel for expression profiling studies using RNA prepared from FFPE tumor specimens on the DASL platform. Thus far, we have identified a 73-gene set with the custom panel (significantly more genes than with the standard panel) that can discriminate TN from other receptor expressing subtypes. We are now applying this platform to samples from clinical trials in order to identify both prognostic and predictive breast tumor gene sets. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2027.

Abstract #2089 Background: A disparity in prognosis of triple negative (TN) breast cancer (BC) has been observed between African American (AA) and Caucasian (CAU) race/ethnicities afflicted with this aggressive and invasive BC subtype. Etiological understanding of these differences involves accounting for several factors associated with phenotype and genotype. Here, we address the latter using the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) assay to quantify mRNA expression of 512 breast cancer related genes in a cohort of 24 CAU and 56 AA TN BC tissues sourced from formalin-fixed, paraffin-embedded (FFPE) blocks. Material and Methods: The DASL assay was used to measure mRNA expression levels from FFPE sourced tissues in both cohorts of self-identified patients. CAU BC patients were obtained from St. Mary's Hospital, Montreal, Quebec and AA BC patients were obtained from Grady Hospital, Atlanta, Georgia. RNA extraction used the RNA High Pure Kit (Roche) and was taken from archival FFPE tissues either 5µm tissue sections or cores. Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR) less than 1% and a two fold-change criteria to determine differential regulation. Results: In all, 33 genes were found differentially expressed between AA and CAU TN BC tumor samples, 32 of which were upregulated in the AA cohort, only 1 of which was upregulated in the CAU group. The upregulated gene in CAU TN BC was TFF1. Upregulated genes in the AA cohort (order of statistical significance according to SAM software) were KIF20A, EP300, AURKB, FGF4, C14orf155, USP22, EPOR, ZNF668, SCNN1G, MAPT, FLNB, EP400, LTA, ACOT11, RBP3, CSF3, E2F2, TGFB1, CCNE1, L1CAM, NDP, VWF, RHOB, FEN1, BIN1, KRT17, CDC42EP4, SERPINF1, CHI3L2, NES, BCL2, and RERG. Discussion: TFF1 upregulated in the CAU population, has been indicated as biomarker of favorable prognosis in endocrine therapy in clinical studies which is consistent with race/ethnicity disparities. The remaining genes upregulated in the AA cohort include transcription factors E2F2 and RBP3/E2F1 both with cyclin binding domains which may interact with CCNE1, extracellular and adhesion related genes KRT17, L1CAM and FGF4, genes associated with cell cycle AURKB, EP400, and EP300 (activator of HIF-1A). Several RAS related genes were also found differentially expressed in the AA cohort including RHOB, RERG, BIN1, and EPOR. Moreover, it is worth mentioning that BCL2 which is expressed in the aggressive mammary cancer cell line MCF-7 was also found upregulated in the AA cohort. These initial findings suggest that several differentially regulated genes between AA and CAU race/ethnicities may account for the disparity in outcomes resultant in these populations. These initial data warrant further investigation which is currently ongoing. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2089.

Abstract #43 Background: Patients afflicted with triple negative (TN) breast cancer (BC) are typically younger and have the worst prognosis of any BC subtype as a result of its aggressive and invasive phenotype. TN BC has been well studied with respect to the associative and prognostic values of its mRNA expression profiles. We have used the Illumina DASL (cDNA mediated, Annealing, Selection, Extension, and Ligation) platform to characterize 502 genes from 96 BC patients sourced from formalin-fixed, paraffin-embedded (FFPE) tissues. RNA extracted from FFPE tissues is highly fragmented and typically unsuited for expression platforms, but recent advances have allowed for accurate expression profiling of RNA from FFPE tissues. This 96 patient cohort included 21 TN BC patients identified by immunohistochemistry (IHC); differential analysis of TN expression was integrated with known pathway mechanisms and identified activation of the Wnt pathway in TN tumors. Material and Methods: The Illumina DASL assay was used to measure mRNA expression levels from BC patients obtained from St. Mary's Hospital, Montreal, Quebec. RNA was extracted from three 5µm tissue sections from FFPE blocks using the RNA High Pure Kit (Roche). Differential mRNA regulation was identified by Significance Analysis of Microarrays (SAM) software using a false discover rate (FDR, q-value < 0.01) or a Bonferroni corrected two-tailed student's t-test (p-value < 0.05). IHC concordance was measured by mRNA expression fold-change for positive versus negative IHC categories. Results: 165 samples from 96 patients were characterized for mRNA expression on 1,488 probes across 502 genes in the DASL assay. Quality control limited the analysis to 150 samples from 91 patients over 500 genes. Comparison of the TN subtype relative to HER2 positive and hormone positive breast carcinomas identified up-regulation [fold-change] of MMP7 [2.9], MMP9 [2.0], MMP1 [1.8], NOTCH1 [1.5], FZD7 [1.5], PPARD [1.3], CCND2 [1.2], MET [1.3], MYC [1.5], and MYCN [2.0] in the TN subtype. Most of these genes are either a component or downstream target of the Wnt signaling pathway. Down-regulated relevant genes included ESR1 [-8.4], PGR [-3.9], ERBB2 [-3.4] as expected, as well as ERRB3, ERBB4, GLI3, TFF1, LAF4/AFF3, and AR. Discussion: Down-regulation of TFF1 suggests inhibition of the ERK/MAPK pathways which have complex effects on cell-cell adhesion. In combination with over-expression of MMPs 1, 7, and 9, these changes may contribute to the invasive nature of TN BCs. GLI3 down-regulation may attenuate Hedgehog signaling potentially contributing to Wnt activation, further leading to cell migration, invasion, and decrease in cell adhesion to cognate integrin-matrix interaction Up-regulation of both the FZD7 receptor as well as the Wnt targets MMP7, MYC, MYCN, PPARD, and CCND2 strongly suggest Wnt pathway activation in the TN subtype. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 43.

5056 Background: Nam et al (BJC 97:1690, 2007) have recently shown that of 165 patients, the subgroup of patients who expressed the TMPRSS2:ERG fusion gene product, had a significantly higher risk ...

Background Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies. Methods We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease. Results The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/β-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/β-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or β-catenin (functional read out of Wnt/β-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from β-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/β-catenin classifier had a greater risk of lung and brain, but not bone metastases. Conclusion These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.