Proliferative Gene Signatures Research Articles

151 Exceptional Responders to Base Excision Repair (BER) Inhibition for Recurrent Glioblastoma Display Enrichment for DNA Damage Response Pathways: RNA Sequencing Analysis From a Multicenter Trial

INTRODUCTION: Therapies for glioblastoma recurrence after surgery and chemoradiation are warranted. TRC102 (methoxyamine), a small molecule DNA base-excision repair (BER) inhibitor, reverses temozolomide (TMZ) resistance in preclinical glioma models. We had investigated efficacy of TRC102+TMZ for recurrent glioblastoma (rGBM) through multicenter trial. METHODS: A two-arm, two-stage, non-randomized, Phase II trial of TRC102+TMZ for adults with rGBM was conducted. Arm 1 included bevacizumab-naive patients at first recurrence, with primary aim of estimating response rates using RANO criteria. If sufficient activity was identified, arm 2 was planned in bevacizumab-refractory patients. Secondary aims were to determine overall survival (OS), progression-free survival (PFS), PFS at six months, and toxicity using CTCAE v4.0. Exploratory aims were correlating clinical outcomes with MPG expression and MGMT methylation. Differential gene expression profiling was analyzed using DESeq2 GSVA (RRID:SCR_015687) and transformed into enrichment scores for 'hallmark of DNA repair' pathways from Molecular Signatures Database (MSigDB, RRID:SCR_016863) to assign mechanistic signatures of therapeutic vulnerability. RESULTS: Arm 1 enrolled 19 patients with median of two treatment cycles, but objective responses were not observed. Hence, arm 2 did not open. Median OS was 11.1 months (95%CI 8.2-17.9). Median PFS was 1.9 months (95%CI 1.8-3.7). MGMT promoter was unmethylated in 14/19 patients, and MPG expression was present in 8/14 cases with available tumor tissue. MGMT methylation and MPG non-expression were associated with better OS and PFS. However, two ‘exceptional responders’ had PFS = 11 months, with MGMT methylated and MPG expressed. RNA sequencing of their tumor tissue demonstrated significant enrichment for DNA damage response (DDR) pathways (MSigDB), chromosomal instability gene signatures (CIN70,CIN25), and proliferative gene signatures (PCNA25). CONCLUSIONS: rGBM patients with elevated levels of MPG and DDR molecular signature may have impaired BER and respond better to TRC102+TMZ.

High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.

ATP1A1 is a promising new target for melanoma treatment and can be inhibited by its physiological ligand bufalin to restore targeted therapy efficacy

Despite advancements in treating metastatic melanoma, many patients exhibit resistance to targeted therapies. Our study focuses on ATP1A1, a sodium pump subunit associated with cancer development. We aimed to assess ATP1A1 prognostic value in melanoma patients and examine the impact of its ligand, bufalin, on melanoma cell lines in vitro and in vivo. High ATP1A1 expression (IHC) correlated with reduced overall survival in melanoma patients. Resistance to BRAF inhibitor was linked to elevated ATP1A1 levels in patient biopsies (IHC, qPCR) and cell lines (Western blot, qPCR). Additionally, high ATP1A1 mRNA expression positively correlated with differentiation/pigmentation markers based on data from The Cancer Genome Atlas (TCGA) databases and Verfaillie proliferative gene signature analysis. Bufalin specifically targeted ATP1A1 in caveolae, (proximity ligation assay) and influenced Src phosphorylation (Western blot), thereby disrupting multiple signaling pathways (phosphokinase array). In vitro, bufalin induced apoptosis in melanoma cell lines by acting on ATP1A1 (siRNA experiments) and, in vivo, significantly impeded melanoma growth using a nude mouse xenograft model with continuous bufalin delivery via an osmotic pump. In conclusion, our study demonstrates that ATP1A1 could serve as a prognostic marker for patient survival and a predictive marker for response to BRAF inhibitor therapy. By targeting ATP1A1, bufalin inhibited cell proliferation, induced apoptosis in vitro, and effectively suppressed tumor development in mice. Thus, our findings strongly support ATP1A1 as a promising therapeutic target, with bufalin as a potential agent to disrupt its tumor-promoting activity.

A High-Risk Subgroup Multiple Myeloma Classification Based on the Detection of PR Minor Subclones

In the last two decades several classifications of Multiple Myeloma (MM) have been proposed based on gene expression signatures. These include the supervised translocation and cyclin D (TC) molecular subgroup approach or unsupervised methods to elucidate the molecular heterogeneity of MM and identify high risk subgroups. In particular, two RNA based gene expression studies have identified 8 to 10 unique subtypes, including the MS, MAF, CD1, CD2, and PR subtypes. The PR subgroup represents a highly proliferative gene signature that is associated with very poor survival outcomes. However, assigning this PR subgroup based on bulk transcriptome signatures can be challenging giving the mixture of genetic backgrounds that can be observed within this subtype. Furthermore, the presence of a minor PR subclone can often be missed with bulk genomic studies leading to the misclassification of a patient as standard risk. We here postulated that minor PR subclones expand under selective therapeutic pressures to become the predominant clones at subsequent relapses and the presence of PR subclones can impact any of the molecular subgroups pertaining poor survival outcomes. Bulk RNA or single cell RNA (scRNAseq) analysis were performed in bone marrow sorted primary MM cells obtained from 718 patients from the CoMMpass trialand 32 relapse/refractory MM patients, respectively. For scRNAseq, serial samples with paired baseline and relapse samples were available in 18 cases. Bone marrow plasma cells samples were separated from bone marrow aspirates using positive sorting selection of CD138 + cells. For bulk RNA-seq we used the mRNA HyperPrep kit (Kapa Biosystems) with RNA from 50,000 cells.For scRNA studiesunbiased mRNA profiling was conducted using the GemCode system (10x Genomics). Cell Ranger and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3' gene and data analysis. Sequencing was performed on Illumina. By performing GSEA at the single cell level using the Zhan gene data set we have classified patients in the appropriate TC groups. FISH data was used to insure accurate calls. For the survival analysis OS was analyzed by the Kaplan-Meier method. In the CoMMpass dataset, with bulk RNA sequencing, the PR subtype contained 7.1% of patients with an admixture of classic genetic subtypes and very poor clinical outcome, with a median OS of 21.3 months. High proliferation index scores were concentrated in this subtype. There was also a strong tendency for patients to transition to the PR subtype at progression, with 25.5% of patients in a non-PR subtype at diagnosis transitioning to PR. Regardless of original subtype, patients that transitioned to the PR subtype rapidly succumbed to their disease with a median OS after the detected progression of 88 days. Using subtype prediction weight we could identify non-PR patients with PR subtype weights above 5% and these patients have a significantly shorter OS of 50 months compared to patients with <5% PR. At the single cell level, investigating the relapsed patients cohort, we have analyzed 92,139 cells and identified 59 clusters. A PR signature (defined as present in ≥15% of cells) was identified in 38% of cases despite baseline favorable prognostic group by FISH cytogenetics (7% HP, 28% t(11;14), and 57% normal FISH). A good concordance with FISH data was observed. In serially relapsed paired samples, all the PR patients maintain their signature overtime and 44% of patients transitioned to PR at subsequent relapse (Fig1A). Survival analyses were performed to evaluate how the presence of minor PR clones at the single cell level relate to clinical outcomes. Notably, the overall survival of patients classified as PR was dramatically reduced with a median OS of 6 months compared to 34 months for non-PR patients (Fig1B). In conclusion, we here show that minor PR clones defined by transcriptional signature through bulk or scRNA may be detectable in patients with variable MM molecular subgroups, including those classified as standard risk by the currently adopted classifications. We also show that the presence of these PR minor clones pertains adverse outcomes with very poor survivals. At relapse, most acquired PR cases originated from standard-risk presentations consistent with a Darwinian clonal evolution. Future classifications should account for the presence of these PR subclones for a better disease prognostication and developments of novel therapeutics.

DNAR-05. EXCEPTIONAL RESPONDERS TO BASE EXCISION REPAIR (BER) INHIBITION FOR RECURRENT GLIOBLASTOMA DISPLAY ENRICHMENT FOR DNA DAMAGE RESPONSE PATHWAYS: RNA SEQUENCING ANALYSIS FROM A MULTICENTER TRIAL

Abstract INTRODUCTION Therapies for glioblastoma recurrence after surgery and chemoradiation are warranted. TRC102 (methoxyamine), a small molecule DNA base-excision repair (BER) inhibitor, reverses temozolomide (TMZ) resistance in preclinical glioma models. We had investigated efficacy of TRC102+TMZ for recurrent glioblastoma (rGBM) through a multicenter trial. This work sought to report translational findings from RNA sequencing performed on patients with available tumor tissue. METHOD: A two-arm, two-stage, non-randomized, Phase II trial of TRC102+TMZ for adults with rGBM named BERT was conducted. Arm 1 included bevacizumab-naive patients at first recurrence, with primary aim of estimating response rates using RANO criteria. If sufficient activity was identified, arm 2 was planned in bevacizumab-refractory patients. Secondary aims were to determine overall survival (OS), progression-free survival (PFS), 6-month PFS, and toxicity. Exploratory aims were correlating clinical outcomes with MPG expression and MGMT methylation. Differential gene expression profiling was analyzed using DESeq2 GSVA (RRID:SCR_015687) and transformed into enrichment scores for 'hallmark of DNA repair' pathways from Molecular Signatures Database (MSigDB, RRID:SCR_016863) to assign mechanistic signatures of therapeutic vulnerability. RESULTS Arm 1 enrolled 19 patients with median of two treatment cycles, but objective responses were not observed. Hence, arm 2 did not open. Median OS was 11.1 months (95%CI 8.2-17.9). Median PFS was 1.9 months (95%CI 1.8-3.7). MGMT promoter was unmethylated in 14/19 patients, and MPG expression was present in 8/14 cases with available tumor tissue. MGMT methylation and MPG non-expression were associated with better OS and PFS. However, two ‘exceptional responders’ had PFS ≥11 months, with MGMT methylated and MPG expressed. RNA sequencing of their tumor tissue demonstrated significant enrichment for DNA damage response (DDR) pathways (MSigDB), chromosomal instability gene signatures (CIN70, CIN25), and proliferative gene signatures (PCNA25). CONCLUSION rGBM patients with elevated levels of MPG and DDR molecular signature may have impaired BER and respond better to TRC102+TMZ.

Prostate cancer radiogenomics reveals proliferative gene expression programs associated with distinct MRI-based hypoxia levels

Background and purposeThe biology behind individual hypoxia levels in patient tumors is poorly understood. Here, we used radiogenomics to identify associations between magnetic resonance imaging (MRI)-based hypoxia levels and biological processes derived from gene expression data in prostate cancer. Materials and methodsFor 85 prostate cancer patients, MRI-based hypoxia images were constructed by combining diffusion-weighted images reflecting oxygen consumption and supply. The ability to differentiate hypoxia levels in these images was verified by comparison with matched biopsy sections stained for the hypoxia marker pimonidazole. For MRI-defined hypoxia levels, corresponding hypoxic fractions were calculated and correlated with biopsy gene expression profiles. Biological processes were predicted by gene set enrichment analysis (GSEA) and validated by immunohistochemistry (Ki67 proliferation marker, reactive stroma grade) and RT-PCR (MYC). ResultsGenes with correlation between expression level and hypoxic fraction were identified for 56 MRI-based hypoxia levels. At all levels, GSEA identified proliferation as the predominant biological process enriched among the correlating genes. Two independent proliferative gene signatures were developed. The Peak1 signature, upregulated at moderate/severe hypoxia, reflected MYC upregulation and high Ki67-proliferation index of cancer cells in pimonidazole-positive regions. The Peak2 signature, upregulated at mild to non-hypoxic levels, was associated with fibroblast gene signature and reactive stroma grade. High scores of both Peak1 and Peak2 indicated elevated risk of biochemical recurrence in multiple cohorts. ConclusionRadiogenomics identified two gene expression programs activated at different hypoxia levels, reflecting proliferation of cancer cells and stroma cells. Genes involved in these programs could be candidate targets for intervention.

Systems medicine dissection of chr1q-amp reveals a novel PBX1-FOXM1 axis for targeted therapy in multiple myeloma

AbstractUnderstanding the biological and clinical impact of copy number aberrations (CNAs) on the development of precision therapies in cancer remains an unmet challenge. Genetic amplification of chromosome 1q (chr1q-amp) is a major CNA conferring an adverse prognosis in several types of cancer, including in the blood cancer multiple myeloma (MM). Although several genes across chromosome 1 (chr1q) portend high-risk MM disease, the underpinning molecular etiology remains elusive. Here, with reference to the 3-dimensional (3D) chromatin structure, we integrate multi-omics data sets from patients with MM with genetic variables to obtain an associated clinical risk map across chr1q and to identify 103 adverse prognosis genes in chr1q-amp MM. Prominent among these genes, the transcription factor PBX1 is ectopically expressed by genetic amplification and epigenetic activation of its own preserved 3D regulatory domain. By binding to reprogrammed superenhancers, PBX1 directly regulates critical oncogenic pathways and a FOXM1-dependent transcriptional program. Together, PBX1 and FOXM1 activate a proliferative gene signature that predicts adverse prognosis across multiple types of cancer. Notably, pharmacological disruption of the PBX1-FOXM1 axis with existing agents (thiostrepton) and a novel PBX1 small molecule inhibitor (T417) is selectively toxic against chr1q-amp myeloma and solid tumor cells. Overall, our systems medicine approach successfully identifies CNA-driven oncogenic circuitries, links them to clinical phenotypes, and proposes novel CNA-targeted therapy strategies in MM and other types of cancer.

CD116+ fetal precursors migrate to the perinatal lung and give rise to human alveolar macrophages.

Despite their importance in lung health and disease, it remains unknown how human alveolar macrophages develop early in life. Here we define the ontogeny of human alveolar macrophages from embryonic progenitors in vivo, using a humanized mouse model expressing human cytokines (MISTRG mice). We identified alveolar macrophage progenitors in human fetal liver that expressed the GM-CSF receptor CD116 and the transcription factor MYB. Transplantation experiments in MISTRG mice established a precursor-product relationship between CD34-CD116+ fetal liver cells and human alveolar macrophages in vivo. Moreover, we discovered circulating CD116+CD64-CD115+ macrophage precursors that migrated from the liver to the lung. Similar precursors were present in human fetal lung and expressed the chemokine receptor CX3CR1. Fetal CD116+CD64- macrophage precursors had a proliferative gene signature, outcompeted adult precursors in occupying the perinatal alveolar niche, and developed into functional alveolar macrophages. The discovery of the fetal alveolar macrophage progenitor advances our understanding of human macrophage origin and ontogeny.

Working together: Heterotypic clusters and collective cell migration in melanoma metastasis.

In this issue of Developmental Cell, Campbell etal. (2021) show that melanoma cells with distinct invasive or proliferative gene signatures can form heterotypic clusters that extravasate collectively and readily seed the growth of metastatic lesions. These findings highlight interactions between heterogenous tumor cells as being critical for metastasis.

Combination of gene set signatures correlates with response to nivolumab in platinum-resistant ovarian cancer

Based on our previous phase II clinical trial of anti-programmed death-1 (PD-1) antibody nivolumab for platinum-resistant ovarian cancer (n = 19, UMIN000005714), we aimed to identify the biomarkers predictive of response. Tumor gene expression was evaluated by proliferative, mesenchymal, differentiated, and immunoreactive gene signatures derived from high-grade serous carcinomas and a signature established prior for ovarian clear cell carcinoma. Resulting signature scores were statistically assessed with both univariate and multivariate approaches for correlation to clinical response. Analyses were performed to identify pathways differentially expressed by either the complete response (CR) or progressive disease (PD) patient groups. The clear cell gene signature was scored significantly higher in the CR group, and the proliferative gene signature had significantly higher scores in the PD group where nivolumab was not effective (respective p values 0.005 and 0.026). Combinations of gene signatures improved correlation with response, where a visual projection of immunoreactive, proliferative, and clear cell signatures differentiated clinical response. An applicable clinical response prediction formula was derived. Ovarian cancer-specific gene signatures and related pathway scores provide a robust preliminary indicator for ovarian cancer patients prior to anti-PD-1 therapy decisions.

Mesenchymal Niche-Derived Neuregulin-1 Drives Intestinal Stem Cell Proliferation and Regeneration of Damaged Epithelium

Epidermal growth factor (EGF) maintains intestinal stem cell (ISC) proliferation and is a key component of organoid growth media yet is dispensable for intestinal homeostasis, suggesting roles for multiple EGF family ligands in ISC function. Here, we identified neuregulin 1 (NRG1) as a key EGF family ligand that drives tissue repair following injury. NRG1, but not EGF, is upregulated upon damage and is expressed in mesenchymal stromal cells, macrophages, and Paneth cells. NRG1 deletion reduces proliferation in intestinal crypts and compromises regeneration capacity. NRG1 robustly stimulates proliferation in crypts and induces budding in organoids, in part through elevated and sustained activation of mitogen-activated protein kinase (MAPK) and AKT. Consistently, NRG1 treatment induces a proliferative gene signature and promotes organoid formation from progenitor cells and enhances regeneration following injury. These data suggest mesenchymal-derived NRG1 is a potent mediator of tissue regeneration and may inform the development of therapies for enhancing intestinal repair after injury.

ACTR-43. GENOMIC ANALYSIS OF RESPONDERS OF PHASE II TRIAL OF TEMOZOLOMIDE AND TRC-102 (BASE EXCISION REPAIR INHIBITOR) IN BEVACIZUMAB-NAÏVE GLIOBLASTOMA AT FIRST RECURRENCE

Abstract BACKGROUND Temozolomide forms O6-methylguanine (O6mG), 7-methylguanine (N7mG), and 3- methyladenine (N3mA) DNA adducts. The O6mG DNA adduct is repaired by MGMT. N7mG and N3mA DNA adducts are removed by the base excision repair (BER) pathway, initiated by N- methylpurine DNA glycosolase (MPG). TRC-102 is a BER inhibitor that binds to the apurinic site created through the action of MPG. METHODS A phase II study of adult glioblastoma in first recurrence was performed in the Adult Brain Tumor Consortium with temozolomide, 150 mg/ m2 and TRC-102, 150 mg (1–5/ 28 days). Primary objective included radiographic response rate. Secondary objectives included safety and PFS-6. Exploratory objectives included tumor expression of N-methylpurine DNA glycosylase (MPG). The study tested hypothesis that combination therapy will achieve 30% RR. To understand the context of vulnerability to TRC102 we performed RNA sequencing on treatment naïve tissue from 7 patients. RESULTS Nineteen patients were enrolled in first stage. Median age was 60 years (range: 48–76), 53% females, median KPS was 80 (range: 70–90). Median cycles of treatment was 2 (range: 1–12). No responses were observed. Median OS was 11.0 months (95% CI: 8–18 months), median PFS was 2.0 months (95% CI: 1.8–3.6 months). PFS-6 rate was 10.5 % (2/19). The combination was safe. MPG staining was negative in six, 1+ in five and 2+ in three patients. PFS of 11 + months in two patients (exceptional responders) was associated with MPG expression. Preliminary analysis on RNA sequencing revealed significant enrichment for DNA Damage Response pathways (MsigDB), chromosomal instability gene signature (CIN70 and CIN25), and proliferative gene signature (PCNA25) in these 2 patients. CONCLUSIONS TRC 102 with temozolomide has acceptable safety but did not meet the primary endpoint of response. Gene signature of MsigDB, CIN70, CIN25 and PCNA25 was seen in exceptional responders and biomarker driven study is planned.

Maternal embryonic leucine zipper kinase is a novel target for proliferation-associated high-risk myeloma.

Treatment of high-risk patients is a major challenge in multiple myeloma. This is especially true for patients assigned to the gene expression profiling-defined proliferation subgroup. Although recent efforts have identified some key players of proliferative myeloma, genetic interactions and players that can be targeted with clinically effective drugs have to be identified in order to overcome the poor prognosis of these patients. We therefore examined maternal embryonic leucine zipper kinase (MELK) for its implications in hyper-proliferative myeloma and analyzed the activity of the MELK inhibitor OTSSP167 both in vitro and in vivo. MELK was found to be significantly overexpressed in the proliferative subgroup of myeloma. This finding translated into poor overall survival in patients with high vs. low MELK expression. Enrichment analysis of upregulated genes in myeloma cells of MELKhigh patients confirmed the strong implications in myeloma cell proliferation. Targeting MELK with OTSSP167 impaired the growth and survival of myeloma cells, thereby affecting central survival factors such as MCL-1 and IRF4. This activity was also observed in the 5TGM.1 murine model of myeloma. OTSSP167 reduced bone marrow infiltration and serum paraprotein levels in a dose-dependent manner. In addition, we revealed a strong link between MELK and other proliferation-associated high-risk genes (PLK-1, EZH2, FOXM1, DEPDC1) and MELK inhibition also impaired the expression of those genes. We therefore conclude that MELK is an essential component of a proliferative gene signature and that pharmacological inhibition of MELK represents an attractive novel approach to overcome the poor prognosis of high-risk patients with a proliferative expression pattern.

Abstract LB-098: Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC

Abstract Combination epigenetic treatment as a therapeutic approach holds much promise. However, to unlock the true potential of these combinatorial paradigms, further optimization is required. We have elucidated in our current study, a treatment schedule combining Azacitidine with Histone Deacetylase Inhibition (HDACi) which considers both schedule and isoform specificity. We have deployed a sequential schedule with HDACi administered post Azacitidine in a chronic low dose manner. This schedule takes advantage of two key parameters altered by Azacitidine: dsRNA induced immune signature and MYC depletion. In our system, HDACis amplify an existing immune signature and the MYC depleted environment acts as a potent sensitizer. HDACi induced augmentation of immune gene transcription seems driven predominantly through the inhibition of HDAC1/2 and HDAC6 isoforms, with broad spectrum HDACi inhibitors demonstrating the greatest potency. While the combination treatment induced perturbation of proliferation is driven by inhibition of HDAC1/2/3 isoforms. Applying this concept to a genetically engineered mouse model of NSCLC we observe a potent anti-tumor response as evidenced by a reduction in tumor burden, progression and proliferation. These phenotypic parameters are correlated with modification of both immune and proliferative gene signatures as well as alteration of key immune populations in the tumor microenvironment. We propose that this combinatorial alteration of both immune and proliferative parameters holds the promise to be a more robust treatment for NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Michael Topper, Michelle Vaz, Katherine Chiappinelli, Christina DeStefano Shields, Alyssa Wenzel, Cynthia Zahnow, Pam Strissel, Reiner Strick, Stephen Baylin. Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-098. doi:10.1158/1538-7445.AM2017-LB-098

Abstract ES9-2: ES9-2 Metastatic TNBC: New targets/new strategies

Abstract Triple negative breast cancer (TNBC) is a diagnosis of exclusion as these tumors lack ER, PR and HER2. Through advances in molecular profiling, the heterogeneity of TNBC has been revealed as subsets with molecular features targetable with either investigational or existing FDA approved drugs. Basal-like TNBCs (BL1 and BL2) are associated with proliferative gene signatures. BL1 tumors demonstrate at higher rate of pCR to standard chemotherapy compared to other subtypes. The BL2 subset demonstrates gene ontologies involving growth factor signaling such as EGFR and IGF1R. Preclinical models demonstrate that basal-like TNBCs have high sensitivity to cisplatin and relative resistance to PI3K inhibitors in cell culture and xenografts. Germ line aberrations in BRCA1 and BRCA2 lead to a defect in DNA repair in cancers harboring such mutationsand these tumors appear to be more sensitive to therapy inducing DNA breaks, such as those induced by platinum based therapies or PARP inhibition. Approximately 30% of TNBCs display mesenchymal features and carry a high rate of molecular aberrations that activate the PI3K/Akt/mTOR axis with clinical data suggesting that this subgroup may be responsive to therapeutic regimens targeting this pathway. Approximately 10-15% of TNBCs are associated with expression of androgen receptor (AR+ or LAR) and are characterized by the expression of luminal genes and a high rate of PI3K activation/mutation. Response to AR and/or PI3K directed therapy has been demonstrated in preclinical models and AR inhibition has demonstrated response in early phase clinical trials. Lastly, though breast cancer is generally considered a non-immunogenic malignancy, some TNBCs have tumor infiltrating lymphocytes. Novel agents to enrich immune response have been associated with durable responses in a subset of patients with metastatic TNBC. In summary, molecular characterization has identified subsets of TNBC that show promising response to existing or emerging targeted agents. Citation Format: Moulder SL. ES9-2 Metastatic TNBC: New targets/new strategies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES9-2.

PPARδ induces estrogen receptor-positive mammary neoplasia through an inflammatory and metabolic phenotype linked to mTOR activation.

The peroxisome proliferator-activated receptor-δ (PPARδ) regulates a multitude of physiological processes associated with glucose and lipid metabolism, inflammation, and proliferation. One or more of these processes are potential risk factors for the ability of PPARδ agonists to promote tumorigenesis in the mammary gland. In this study, we describe a new transgenic mouse model in which activation of PPARδ in the mammary epithelium by endogenous or synthetic ligands resulted in progressive histopathologic changes that culminated in the appearance of estrogen receptor- and progesterone receptor-positive and ErbB2-negative infiltrating ductal carcinomas. Multiparous mice presented with mammary carcinomas after a latency of 12 months, and administration of the PPARδ ligand GW501516 reduced tumor latency to 5 months. Histopathologic changes occurred concurrently with an increase in an inflammatory, invasive, metabolic, and proliferative gene signature, including expression of the trophoblast gene, Plac1, beginning 1 week after GW501516 treatment, and remained elevated throughout tumorigenesis. The appearance of malignant changes correlated with a pronounced increase in phosphatidylcholine and lysophosphatidic acid metabolites, which coincided with activation of Akt and mTOR signaling that were attenuated by treatment with the mTOR inhibitor everolimus. Our findings are the first to show a direct role of PPARδ in the pathogenesis of mammary tumorigenesis, and suggest a rationale for therapeutic approaches to prevent and treat this disease.

Abstract 3569: Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.

Abstract Progesterone receptors (PR) are emerging as important breast cancer drivers. Elevated mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2) signaling is common in breast cancer and these kinases drive direct PR-B, but not PR-A, Ser294 phosphorylation. Post-translational modifications, including phosphorylation and SUMOylation, dramatically impact PR transcriptional activity. Phospho-Ser294 PRs are less SUMOylated on Lys388 (i.e. a repressive modification). Thus, active mitogenic kinase signaling provides a mechanism for PR derepression (i.e. transcriptional activation) during breast cancer progression. A broad range of PR protein expression is observed clinically; PR-negative tumors are generally more aggressive. However, clinical (IHC) assays do not measure phospho-PRs, which turnover rapidly and may appear undetectable, confounding the true level of activated PR within tumors. A PR gene signature would provide a valuable marker of PR contribution to breast cancer progression. Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild type or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Target genes sensitive to changes in PR SUMOylation included those required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors were preferentially recruited to enhancer regions of derepressed genes. SUMO-deficient (i.e. phospho-Ser294) PR gene signatures were significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Furthermore, treatment with antiprogestin or MEK inhibitors abrogated expression of SUMO-sensitive PR target-genes and inhibited progestin-driven proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells. Similarly, treatment of T47D cells expressing deSUMOylated PR with ERBB2 inhibitors blocked progestin-dependent growth. Potential mechanisms of differential promoter selection were investigated. Notably, genes preferentially up-regulated by SUMO-deficient PR contain RUNX binding motifs located near their transcriptional start sites. Herein, we demonstrated that RUNX2 directly interacts with PR. Further, RUNX2 knockdown in breast cancer cells reduced SUMO-deficient PR dependent gene expression. Our studies suggest that deSUMOylated PR cooperates with RUNX2 to mediate promoter selective gene expression. We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters PR target gene selection in breast cancer cells, in part via regulation of PR interactions with gene-specific co-factors, such as RUNX2. Patients whose ER+/PR+ tumors are driven by hyperactive phospho-PRs may benefit from endocrine (antiestrogen) therapies that also contain an antiprogestin. Citation Format: Todd P. Knutson, Andrea R. Daniel, Danhua Fan, Kevin A. Silverstein, Kyle R. Covington, Suzanne A. Fuqua, Carol A. Lange. Phosphorylated and SUMO-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3569. doi:10.1158/1538-7445.AM2013-3569

Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression.

IntroductionProgesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression.MethodsGlobal gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature.Results'SUMO-sensitive' PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (that is, MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at 'closed' enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells.ConclusionsWe conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.

Biologic markers determine both the risk and the timing of recurrence in breast cancer

Breast cancer has a long natural history. Established and emerging biologic markers address overall risk but not necessarily timing of recurrence. 346 adjuvant naïve breast cancer cases from Guy's Hospital with 23years minimum follow-up and archival blocks were recut and reassessed for hormone-receptors (HR), HER2-receptor and grade. Disease-specific survival (DSS) was analyzed by recursive partitioning. To validate insights from this analysis, gene-signatures (proliferative and HR-negative) were evaluated for their ability to predict early versus late metastatic risk in 683 node-negative, adjuvant naïve breast cancers annotated with expression microarray data. Risk partitioning showed that adjuvant naïve node-negative outcome risk was primarily partitioned by tumor receptor status and grade but not tumor size. HR-positive and HER2-negative (HRpos) risk was partitioned by tumor grade; low grade cases have very low early risk but a 20% fall-off in DSS 10 or more years after diagnosis. Higher grade HRpos cases have risk over >20years. Triple-negative (Tneg) and HER2-positive (HER2pos) cases DSS events occurred primarily within the first 5years. Among node-positive cases, only low grade conferred late risk, suggesting that proliferative gene signatures that identify proliferation would be important for predicting early but not late recurrence. Using pooled data from four publicly available data sets for node-negative tumors annotated with gene expression and outcome data, we evaluated four prognostic gene signatures: two proliferation-based and two immune function-based. Tumor proliferative capacity predicted early but not late metastatic risk for HRpos cases. The immune function or HRneg specific signatures predicted only early metastatic risk in Tneg and HER2pos cases. Breast cancer prognostic signatures need to inform both risk and timing of metastatic events and may best be applied within subsets. Current signatures predict for outcome risk within 5years of diagnosis. Predictors of late risk for HR positive disease are needed.

MAGE-A Inhibits Apoptosis In Proliferating Multiple Myeloma Cells

AbstractAbstract 785The type I Melanoma Antigen GEne (MAGE) MAGE-A3 is commonly present in primary multiple myeloma cells and its expression is correlated with advanced disease and proliferation. MAGE-A3 belongs to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins, which are present in many cancers, but their normal expression is limited to developing germ cells and placental trophoblast. This unique expression pattern fuels speculation on a role for CTAg in oncogenesis; however, very little is known about their function. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome, suggesting they contribute to the pathogenesis or progression of this disease through effects on survival and/or proliferation of myeloma cells. To investigate this, we examined the impact of MAGE-A on disease progression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). MAGE-A3 protein expression was examined by immunohistochemistry in a new, independent set of myeloma bone marrow specimens from two critical clinical milestones, newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003). The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating a correlation between MAGE-A3, progression of disease and proliferation. The mechanisms for MAGE-A3 activity were investigated by silencing this gene in primary myeloma cells and HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA and protein in MM.1r (p53+/+) and ARP-1 (p53−/−) HMCL and in primary myeloma cells by 48 hours, and this effect was maintained up to 96 hours. Silencing of MAGE-A did not affect cell cycling, as this intervention did not affect the phosphorylation of the Retinoblastoma gene product (Rb) that is required for progression through the G1 cell cycle checkpoints and entry into S phase. In contrast, MAGE-A was required for survival of proliferating myeloma cells. Silencing of MAGE-A led to a precipitous loss of viable cells within 48–72 hrs compared to controls. This was due to activation of intrinsic apoptosis, as demonstrated by increased annexin V staining, loss of mitochondrial membrane polarization, and cleavage/activation of caspase-9. These effects of MAGE-A knock-down were completely reversed by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh. Apoptosis after MAGE-A silencing appeared to be mediated by at least two distinct mechanisms; p53-dependent activation of pro-apoptotic Bax and Bak expression and reduced expression of the Inhibitor of Apoptosis Protein survivin through both p53-dependent and independent mechanisms. These results demonstrate that MAGE-A plays a role in the survival of proliferating multiple myeloma cells through the regulation of two critical apoptotic mechanisms. Disclosures:No relevant conflicts of interest to declare.