Gene Expression Changes In Tumors Research Articles

The Effect of Exposure to Neighborhood Violence on Glucocorticoid Receptor Signaling in Lung Tumors.

Exposure to neighborhood violent crime is correlated with glucocorticoid signaling and lung tumor gene expression changes associated with increased tumor aggressiveness, suggesting social conditions have downstream biophysical consequences that contribute to lung cancer disparities.

Abstract 1293: AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer

Abstract Leukemic inhibitory factor (LIF) is an IL-6 family member involved in embryonic stem cell maintenance and protection from maternal immune-attack. Emerging evidence indicates that LIF biology is hijacked by some tumors to promote immunosuppressive tumor associated macrophages, cancer initiating cells and epithelial-mesenchymal transition (EMT). In patients treated with anti-PD-1/anti-PD-L1 antibodies (IO), baseline high LIF expression correlated with poor outcome (Loriot et al. 2021). In preclinical models, LIF blockade has separately been reported to sensitize tumors to IO and to chemotherapy (chemo), but the impact of LIF blockade on chemo/IO combination activity is unknown. We therefore evaluated the hypothesis that LIF blockade sensitizes tumors to chemo/IO combinations. AZD0171 is a monoclonal antibody that binds to LIF and prevents signaling through LIF receptor. A murine surrogate of AZD0171 (mAZD0171) was generated and tested in vivo using three syngeneic murine cancer models, and AZD0171 was tested in patient derived xenograft models. Monotherapy mAZD0171 induced tumor gene expression changes consistent with its proposed mechanism of action in the syngeneic CT26 colon tumor model; including elevated CD8 attracting chemokines (CXCL9/10), cDC1 transcription factor (Batf3) and reduced stem cell (CD44), EMT (CY61) and hypoxic/angiogenic signatures, including CXCL12. Flow cytometry analysis highlighted changes consistent with suppressive macrophage re-education (reduced CD206/elevated MHCII). mAZD0171 sensitized mouse tumors to both IO and chemo. mAZD0171 + anti-PD-L1 treatment reversed CD8 T cell exclusion in the D2A1-m2 model; a LIF expressing cancer associated fibroblast rich, anti-PD-L1 resistant mouse mammary tumor syngeneic model (Jungwirth et al. 2018). mAZD0171 + anti-PD-L1 treatment further inhibited D2A1-m2 growth versus (vs) anti-PD-L1 treatment alone. In a non-small cell lung carcinoma patient derived xenograft model, AZD0171 improved the tumor growth inhibitory effect of Carboplatin + Paclitaxel. The triplet combination of mAZD0171 plus chemo and IO had the most marked impact on both tumor microenvironment and tumor growth. mAZD0171 addition to oxaliplatin (OHP) or Docetaxel (DTX) chemotherapy plus anti-PD-L1 significantly elevated CD8 abundance and granzyme B expression in MC38 colon tumors. mAZD0171 + anti-PD-L1 + OHP or DTX improved inhibition of MC38 tumor growth (63% for OHP triplet vs 34% for anti-PD-L1 + OHP; 49% for DTX triplet vs 36% for anti-PD-L1 + DTX) and extended median survival times (P<0.05 vs anti-PD-L1+OHP/DTX). Together these data support the hypothesis that AZD0171 has the potential to sensitize solid tumors to chemotherapy/IO combinations. The safety and activity of AZD0171 is currently being assessed in a Ph2 clinical trial in combination with durvalumab (anti-PD-L1) and chemotherapy in metastatic pancreatic cancer (NCT04999969). Citation Format: Juliana Candido, Gozde Kar, Bairu Zhang, Grace Opoku-Ansah, Amit Grover, Elizabeth A. Kuczynski, Tim Slidel, Doug Palmer, Robert W. Wilkinsom, Nadia Luheshi, Jim Eyles. AZD0171 (anti-LIF) combines productively with chemotherapy and anti-PD-L1 in mouse models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1293.

Intranasal Iloprost Prevents Tumors in a Murine Lung Carcinogenesis Model.

Lung cancer chemoprevention with the prostacyclin analogue iloprost is the most promising approach to date for intercepting progression of premalignant lung lesions in former smokers. Previous preclinical studies of iloprost used oral delivery, but a study modeling delivery directly to the target organ was needed. In vivo and in vitro studies have identified gene expression changes following iloprost treatment, including increased e-cadherin and Ppargγ and decreased COX2 and vimentin. We used tumor counts and gene expression to demonstrate the effectiveness of intranasal delivery of iloprost in a murine model of premalignant adenomas. Intranasal delivery of iloprost reduced adenoma multiplicity 14 weeks after urethane exposure in FVB/N mice compared with untreated urethane controls. Intranasal iloprost reversed urethane-induced gene expression changes in tumors and whole lung. These results correspond to previous studies of oral iloprost and in vitro treatment of human bronchial epithelial cells. This study demonstrates that intranasal delivery of iloprost in a mouse model of lung premalignant lesions is effective chemoprevention. This will be an essential tool for exploring mechanisms and outcomes of iloprost chemoprevention, along with supporting ongoing clinical trials of inhaled iloprost chemoprevention. PREVENTION RELEVANCE: Iloprost is a promising chemoprevention agent for lung cancer and this work describes a new delivery approach in vivo.

232 INCB090244, a potent small molecule that inhibits the PD-L1/PD-1 axis and functions similarly to PD-L1 antibodies

BackgroundBlocking the PD-L1 immune checkpoint axis with therapeutic antibodies against either the ligand or PD-1 has proven to be an effective treatment modality for multiple cancer histologies. Small molecules targeting the PD-L1/PD-1 axis represent an alternate modality of blocking this pathway. INCB090244 is a small molecule that blocks the PD-L1/PD-1 interaction and restores T cell function similar to the clinical stage PD-L1 inhibitor INCB086550.MethodsMDA-MB-231 or CHO cells overexpressing PD-L1 were used to investigate effects of INCB090244 on PD-L1 dimerization, and intracellular trafficking. In vivo, CD34+ humanized mice harboring MDA-MB-231 tumors or C57Bl/6 mice bearing GL261 subcutaneous or orthotopic tumors were used to investigate the efficacy, biodistribution, and pharmacodynamic effects of INCB090244. Human specific gene expression changes in tumors from MDA-MB-231 bearing humanized mice were analyzed by RNA sequencing.ResultsIn vitro, INCB090244 potently disrupted the PD-L1:PD-1 interaction, induced PD-L1 dimerization, and inhibited PD-1-mediated negative signaling, resulting in enhanced IFN gamma and IL-2 production in primary human immune cells. Following dimerization, INCB090244 induced internalization of PD-L1 resulting in co-localization with the Golgi apparatus and partial localization in the nucleus. After cell treatment and washing, full restoration of PD-L1 at the cell surface was observed after 5 days of culture in vitro. In vivo, INCB090244 reduced tumor growth in CD34+ humanized mice bearing MDA-MB-231 tumors, to similar levels as atezolizumab. Antitumor activity was completely abrogated in immunodeficient mice, confirming the pharmacologic dependency on a competent immune system. RNA sequencing analysis on tumors from these mice demonstrated similar T cell activation gene signatures as clinical checkpoint blockade antibodies. Biodistribution studies in mice bearing both subcutaneous and orthotopically implanted GL261 glioma tumors demonstrated higher accumulation of INCB090244 in tumor tissue compared to PD-L1 antibodies.ConclusionsINCB090244 effectively disrupted the PD-L1/PD-1 interaction, induced dimerization and internalization of PD-L1, restored immunity in in vitro and in vivo tumor models, and is a suitable surrogate for the clinical candidate INCB086550. RNA sequencing demonstrated T cell activation signatures similar to those observed in patients receiving checkpoint blockade antibodies. Biodistribution studies demonstrated higher subcutaneous and brain tumor penetration by INCB090244 compared to PD-L1 antibodies, suggesting a potential advantage of small molecule PD-L1 inhibitors in accessing intratumoral regions. These data further support the clinical evaluation of small molecule PD-L1 inhibitors as an alternative approach to immune therapy.

Evaluating the Effect of 3'-UTR Variants in DICER1 and DROSHA on Their Tissue-Specific Expression by miRNA Target Prediction.

Untranslated gene regions (UTRs) play an important role in controlling gene expression. 3′-UTRs are primarily targeted by microRNA (miRNA) molecules that form complex gene regulatory networks. Cancer genomes are replete with non-coding mutations, many of which are connected to changes in tumor gene expression that accompany the development of cancer and are associated with resistance to therapy. Therefore, variants that occurred in 3′-UTR under cancer progression should be analysed to predict their phenotypic effect on gene expression, e.g., by evaluating their impact on miRNA target sites. Here, we analyze 3′-UTR variants in DICER1 and DROSHA genes in the context of myelodysplastic syndrome (MDS) development. The key features of this analysis include an assessment of both “canonical” and “non-canonical” types of mRNA-miRNA binding and tissue-specific profiling of miRNA interactions with wild-type and mutated genes. As a result, we obtained a list of DICER1 and DROSHA variants likely altering the miRNA sites and, therefore, potentially leading to the observed tissue-specific gene downregulation. All identified variants have low population frequency consistent with their potential association with pathology progression.

Phase I/Ib study of pembrolizumab and vorinostat in patients with metastatic NSCLC (mNSCLC): Updated results.

9073 Background: Histone deacetylase inhibitors (HDACi) enhance tumor immunogenicity through several mechanisms and may augment response to immune checkpoint inhibitors (ICI). We report updated results from a phase I/Ib trial testing the combination of oral HDACi vorinostat (V) with PD-1 inhibitor pembrolizumab (P) in mNSCLC. Methods: In phase I, pts with ICI-naïve or ICI-pretreated mNSCLC were treated with P (200mg IV q3 wk) + V (200 or 400 mg PO daily). In phase Ib expansion, pts were required to have progressed on prior ICI treatment. Primary endpoints were safety/tolerability; secondary endpoints included RR, PFS, DOR, and OS. Tissue and blood specimens from pre- and on-treatment were collected for correlative analyses to determine tumor gene expression changes, T cell density and levels of myeloid-derived suppressor cells. Results: Between 3/2016 - 12/2018, Phase I: 13 pts were treated (4 at 200mg, and 9 at 400mg V dose); and Phase Ib: 20 pts were treated. Median age: 68 (range 38-82); Females: 11 (33%); ECOG 1: 31 (94%); and never/former/current smokers: 3/22/8 (9%/67%/24%). PD-L1 expression was < 1% in 8/33 (18%), ≥1-49% in 7/33 (21%), ≥ 50% in 9/33 (27%) and unknown in 11/30 (33%). No DLTs or treatment related deaths were observed. The RP2D was P 200mg and V 400mg. Most common any grade AEs was fatigue (11%) and nausea/vomiting (8%). 2 (6%) patients had treatment discontinued due to toxicity. 30 pts are evaluable for response, 6 ICI-naïve and 24 ICI-pretreated. 4 (13%) had PR (2 confirmed), 16 (53%) had SD, and 10 (33%) had PD for a disease control rate of 67%. In the ICI-pretreated Ib cohort, 3 pts (1 confirmed; 2 unconfirmed) had a PR and 10 had SD (8 confirmed). For ICI-pretreated pts, mPFS was 3.2 and mOS was 7.3 months, and 1-year PFS was 17% (4 pts). For ICI-naïve, mPFS was 7.6 months and mOS was 16 months. CD8 T cell presence in tumor stromal regions was associated with benefit to P + V treatment. Conclusions: P + V were well tolerated. The combination demonstrates preliminary anti-tumor activity despite progression on prior ICI treatment and stromal CD8 T cells may be associated with benefit from P + V treatment. A randomized phase II portion of this study, examining P combined with V vs. placebo in immunotherapy naïve pts, is ongoing. Clinical trial information: NCT02638090.

Abstract 712: Immune checkpoint inhibitor induced tumor gene expression changes in murine syngeneic colon cancer models

Abstract The success of immune checkpoint therapy with anti-PD-1 and anti-CTLA-4 antibodies has generated broad interest in immuno-based interventions for oncology. Preclinical efficacy assessment of these novel immune-oncology therapies requires a functional immune system and therefore is frequently performed in murine syngeneic tumor models. In addition to tumor growth inhibition, syngeneic tumor models provide a platform to investigate the mechanism of action for a given novel therapeutic target. Characterization and mechanism of action for immune-based cancer therapies in murine syngeneic tumor models is often assessed by flow cytometry; measuring changes in tumor infiltrating leukocyte (TIL) populations following treatment induction. We assessed whether transcriptomic gene expression analysis could identify additional relevant biomarkers for immunotherapy response. Mice bearing subcutaneous syngeneic colon cancer tumors were dosed intraperitoneally with either vehicle alone, anti-CTLA-4, anti-PD-1, or a combination of the two immune checkpoint inhibitors on days 1, 4, and 8. Tumors were harvested on day 9 and half from each group were assessed for TIL population composition by flow cytometry and half by Clariom D mouse transcriptome array analysis. We identified a number of immune cell-related genes with differential expression that correlated to the therapeutic response and were consistent with changes identified in TIL populations by flow cytometry. These results provide an additional metric for assessing immunotherapy response in vivo. In addition, these data suggest that a smaller, defined panel of gene targets may be developed as biomarkers for specific immune system responses to immune checkpoint therapies in murine syngeneic models. Citation Format: Patrick Fadden, Douglas Weitzel, Kelli Davis, Thi Bui, David Hurtado, Hannah Haines, Rhiannon Roark, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Katherine Krontz, Cristina Sokolowski, Aidan Synnott, Edgar Wood. Immune checkpoint inhibitor induced tumor gene expression changes in murine syngeneic colon cancer models [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 712.

Early loss of mitochondrial complex I and rewiring of glutathione metabolism in renal oncocytoma

Renal oncocytomas are benign tumors characterized by a marked accumulation of mitochondria. We report a combined exome, transcriptome, and metabolome analysis of these tumors. Joint analysis of the nuclear and mitochondrial (mtDNA) genomes reveals loss-of-function mtDNA mutations occurring at high variant allele fractions, consistent with positive selection, in genes encoding complex I as the most frequent genetic events. A subset of these tumors also exhibits chromosome 1 loss and/or cyclin D1 overexpression, suggesting they follow complex I loss. Transcriptome data revealed that many pathways previously reported to be altered in renal oncocytoma were simply differentially expressed in the tumor's cell of origin, the distal nephron, compared with other nephron segments. Using a heuristic approach to account for cell-of-origin bias we uncovered strong expression alterations in the gamma-glutamyl cycle, including glutathione synthesis (increased GCLC) and glutathione degradation. Moreover, the most striking changes in metabolite profiling were elevations in oxidized and reduced glutathione as well as γ-glutamyl-cysteine and cysteinyl-glycine, dipeptide intermediates in glutathione biosynthesis, and recycling, respectively. Biosynthesis of glutathione appears adaptive as blockade of GCLC impairs viability in cells cultured with a complex I inhibitor. Our data suggest that loss-of-function mutations in complex I are a candidate driver event in renal oncocytoma that is followed by frequent loss of chromosome 1, cyclin D1 overexpression, and adaptive up-regulation of glutathione biosynthesis.

A global transcriptional network connecting noncoding mutations to changes in tumor gene expression.

Although cancer genomes are replete with noncoding mutations, the effects of these mutations remain poorly characterized. Here we perform an integrative analysis of 930 tumor whole genomes and matched transcriptomes, identifying a network of 193 noncoding loci in which mutations disrupt target gene expression. These “somatic eQTLs” (expression Quantitative Trait Loci) are frequently mutated in specific cancer tissues, and the majority can be validated in an independent cohort of 3,382 tumors. Among these, we find that the effects of noncoding mutations on DAAM1, MTG2 and HYI transcription are recapitulated in multiple cancer cell lines, and that increasing DAAM1 expression leads to invasive cell migration. Collectively the noncoding loci converge on a set of core pathways, permitting a classification of tumors into pathway-based subtypes. The somatic eQTL network is disrupted in 88% of tumors, suggesting widespread impact of noncoding mutations in cancer.

Bioinformatics analysis of gene expression alterations conferring drug resistance in tumor samples from melanoma patients with EGFR-activating BRAF mutations.

Melanoma is a highly malignant tumor of the skin melanocytes. Patients with this cancer have a high frequency (~50%) of oncogenic BRAF mutations, particularly BRAF V600E. Treatments for melanoma often target BRAF mutations or involve mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitors. A major challenge in melanoma treatment is resistance to BRAF inhibitor treatment, which may be enhanced by the BRAF mutation itself and/or epidermal growth factor receptor (EGFR) activation, leading to poor prognosis. However, no effective clinical treatment exists for patients with EGFR-activating feedback. The aim of the present study was to analyze gene expression changes in tumors from patients with EGFR-activating BRAF mutations during development of drug resistance. RNA-seq data was downloaded from the Gene Expression Omnibus (GEO) database for pre- and post-treatment tumor samples from three melanoma patients with EGFR-activating BRAF V600E mutations, and from The Cancer Genome Atlas (TCGA) melanoma database for tumor and non-tumor samples from patients with the BRAF V600E mutation and unknown EGFR activation status. Using functional enrichment and KEGG pathway analyses, the present study analyzed differentially expressed genes (DEGs) between pre- vs. post-treatment data from the GEO database and tumor or non-tumor sample data from the TCGA database. The results of the present study indicated that functional and structural changes to the plasma membrane may be associated with drug resistance. The present study identified 9 DEGs that were significantly different between tumor and non-tumor samples and also between prior to and following treatment. Thus, it was confirmed that patients with EGFR-activating BRAF V600E mutations undergo gene expression changes during disease development, and during therapy. These findings may provide potential directions for melanoma-specific therapy.

Targeting Hypoxic Prostate Tumors Using the Novel Hypoxia-Activated Prodrug OCT1002 Inhibits Expression of Genes Associated with Malignant Progression

Purpose: To understand the role of hypoxia in prostate tumor progression and to evaluate the ability of the novel unidirectional hypoxia-activated prodrug OCT1002 to enhance the antitumor effect of bicalutamide.Experimental Design: The effect of OCT1002 on prostate cancer cells (LNCaP, 22Rv1, and PC3) was measured in normoxia and hypoxia in vitroIn vivo, tumor growth and lung metastases were measured in mice treated with bicalutamide, OCT1002, or a combination. Dorsal skin fold chambers were used to image tumor vasculature in vivo Longitudinal gene expression changes in tumors were analyzed using PCR.Results: Reduction of OCT1002 to its active form (OCT1001) decreased prostate cancer cell viability. In LNCaP-luc spheroids, OCT1002 caused increased apoptosis and decreased clonogenicity. In vivo, treatment with OCT1002 alone, or with bicalutamide, showed significantly greater tumor growth control and reduced lung metastases compared with controls. Reestablishment of the tumor microvasculature following bicalutamide-induced vascular collapse is inhibited by OCT1002. Significantly, the upregulation of RUNX2 and its targets caused by bicalutamide alone was blocked by OCT1002.Conclusions: OCT1002 selectively targets hypoxic tumor cells and enhances the antitumor efficacy of bicalutamide. Furthermore, bicalutamide caused changes in gene expression, which indicated progression to a more malignant genotype; OCT1002 blocked these effects, emphasizing that more attention should be attached to understanding genetic changes that may occur during treatment. Early targeting of hypoxic cells with OCT1002 can provide a means of inhibiting prostate tumor growth and malignant progression. This is of importance for the design and refinement of existing androgen-deprivation regimens in the clinic. Clin Cancer Res; 23(7); 1797-808. ©2016 AACR.

70. Safety, Toxicity and Efficacy of Systemic Recombinant VSV Therapy in Pet Dogs With Spontaneous Cancer

Clinical translation of oncolytic therapy to benefit cancer patients is dependent upon extensive evaluation of viral safety, toxicity and therapeutic efficacy in disease bearing animals. Comparative oncology, the study of naturally occurring cancer in animals, allows evaluation of novel cancer therapies in pet dogs that are similar in size, genetic make-up, physiology and exposure to environmental risk factors to humans. We have previously shown single shot systemic therapy with recombinant Vesicular stomatitis virus (VSV) engineered to express Interferon-beta (IFN) and the sodium-iodide symporter (NIS) in tumor bearing mice results in tumor-specific virus replication that can be monitored noninvasively by SPECT/CT imaging using NIS specific radio-tracer, induces tumor remission and confers significant survival benefit. A comparative oncology approach was utilized to investigate safety, clinical toxicities and therapeutic efficacy of systemic VSV therapy in dogs with spontaneous cancer. A pre-clinical rapid dose-escalation study was performed in healthy lab beagles defined a safe systemic dose range. We initiated evaluation of systemic VSV administration in dogs with spontaneous cancer. 8 dogs with various cancers were treated with VSV expressing either human or canine IFN-beta and NIS. We demonstrate that systemic VSV therapy at a dose of 10(10) TCID is well tolerated in dogs with spontaneous disease. We detected transient hepatotoxicity that resolved spontaneously in 2/8 dogs following systemic VSV-IFN-NIS treatment. No infectious virus was detected in urine or buccal swab samples. Monitoring of disease response indicated that 2/2 dogs with T-cell lymphoma had dramatic disease remission following systemic VSV-hIFN-NIS treatment resulting in partial response, though remission was transient and disease eventually relapsed. Virus pharmacokinetics (PK) studies indicate that dogs with robust responses also had highest viral PK in blood, suggesting therapeutic response is PK dependent. A dose optimization protocol was adopted where gradual dose escalation and multi-dosing protocols are being evaluated. Initial findings indicate two-dose systemic VSV-IFN-NIS therapy in a dog with metastatic Osteosarcoma (OSA) is well tolerated, delays viral decay in blood, and has resulted in improved health of the treated dog with no major disease progression to date. RNAseq analysis of tumor biopsies from treated animals reveals changes in tumor gene expression following systemic VSV therapy. Overall the data demonstrate that recombinant VSV can be safely administered systemically in cancer bearing dogs. Our data suggest that therapeutic response is PK dependent, prompting dose optimization studies that will directly inform and support planned clinical studies to evaluate systemic VSV therapy in patients with relapsed or metastatic cancer.

Amplification and deletion of the RAPH1 gene in breast cancer patients

Lamellipodin protein (Lpd), encoded by the RAPH1 gene, modulates the assembly of actin cytoskeleton through its binding to the Ena/VASPs proteins, and acts in cellular motility and lamelipodial protrusion. The region where RAPH1 gene is located (2q33) is deleted in various types of cancer and the gene expression changes in tumors when compared to normal tissues. Amplifications and deletions of the RAPH1 gene were investigated in breast carcinoma samples, in order to determine the possible relationship of the gene with breast cancer tumorigenesis and lymph node metastasis. RAPH1 gene alterations were determined by relative quantification, standard curve method using Real-time PCR technique in samples of tumor and peripheral blood from 52 patients. Regression and correlation analyses were conducted using gene alterations and clinicopathological data. All samples analyzed were altered, with 63.5% deletion cases and 36.5% amplification cases. The logistic regression and correlation analysis with clinicopathological data did not show significant results. The results suggest that although the RAPH1 gene was deleted or amplified in all samples, the Lpd does not seem to play a major role in tumorigenesis of mammary carcinomas and probably other proteins, also involved in the process of cellular motility and metastasis, are acting more effectively for or against the migration of breast tumor cells.

Immunotherapy Exposes Cancer Stem Cell Resistance and a New Synthetic Lethality

Development of resistance to targeted drugs and immunotherapy limits improvement in response rates and disease-free survival of cancer patients. In advanced melanoma, for example, multiple mechanisms of resistance emerge during BRAF inhibitor drug therapy that affect both tumor cell properties and microenvironment.1 In addition, just as immunoediting during the de novo immune response against cancer selects for immune-resistant tumor variants,2 active and passive immunotherapies can create selective pressures that lead to outgrowth of resistant tumor cells or new metastases (Figure 1). Understanding the mechanisms of resistance is critical to developing therapeutic strategies to counteract them as well as preventive measures to avoid their onset. In this issue of Molecular Therapy, Boisgerault and colleagues3 nicely elucidate these points using a tumor vaccination model to induce a T-cell response that places a selective pressure on the tumor facilitating the emergence or selective survival of resistant cancer stem cells or cells undergoing epithelial–mesenchymal transition (EMT). The study further shows how acquired or intrinsic mechanisms of resistance can reveal molecular vulnerabilities that may allow responses even to chemotherapy drugs that would have been considered ineffectual.

Genomic Landscape of Copy Number Aberrations Enables the Identification of Oncogenic Drivers in Hepatocellular Carcinoma

Cancer is a genetic disease with frequent somatic DNA alterations. Studying recurrent copy number aberrations (CNAs) in human cancers would enable the elucidation of disease mechanisms and the prioritization of candidate oncogenic drivers with causal roles in oncogenesis. We have comprehensively and systematically characterized CNAs and the accompanying gene expression changes in tumors and matched nontumor liver tissues from 286 hepatocellular carcinoma (HCC) patients. Our analysis identified 29 recurrently amplified and 22 recurrently deleted regions with a high level of copy number changes. These regions harbor established oncogenes and tumor suppressors, including CCND1 (cyclin D1), MET (hepatocyte growth factor receptor), CDKN2A (cyclin-dependent kinase inhibitor 2A) and CDKN2B (cyclin-dependent kinase inhibitor 2B), as well as many other genes not previously reported to be involved in liver carcinogenesis. Pathway analysis of cis-acting genes in the amplification and deletion peaks implicates alterations of core cancer pathways, including cell-cycle, p53 signaling, phosphoinositide 3-kinase signaling, mitogen-activated protein kinase signaling, Wnt signaling, and transforming growth factor beta signaling, in a large proportion of HCC patients. We further credentialed two candidate driver genes (BCL9 and MTDH) from the recurrent focal amplification peaks and showed that they play a significant role in HCC growth and survival. We have demonstrated that characterizing the CNA landscape in HCC will facilitate the understanding of disease mechanisms and the identification of oncogenic drivers that may serve as potential therapeutic targets for the treatment of this devastating disease.

Abstract 2718: REGN1400, a fully-human ERBB3 antibody, potently inhibits tumor growth in preclinical models, both as a monotherapy and in combination with EGFR or HER2 blockers

Abstract ERBB3 is a member of the ERBB family of receptor tyrosine kinases, that includes EGFR and HER2, well-established drivers of tumorigenesis. Recent preclinical studies indicate that both EGFR and HER2 cooperate with ERBB3 to activate the phosphatidylinositol-3 kinase/Akt survival pathway and to promote tumor cell survival. Furthermore, ERBB3 activation has been demonstrated to mediate resistance to EGFR inhibitors in non-small cell lung cancer cell lines and to HER2 inhibitors in breast cancer cell lines. Consistent with these findings, anti-ERBB3 monoclonal antibodies have been shown to inhibit the growth of human tumor xenografts, demonstrating the importance of ERBB3 for tumor growth in vivo. REGN1400 is a fully-human anti-ERBB3 monoclonal antibody that binds human ERBB3 with high affinity (KD ∼50pM) and potently inhibits binding of the ERBB3 ligand neuregulin 1 (IC50 ∼30pM). REGN1400 inhibited phosphorylation of ERBB3 and Akt in multiple human tumor cell lines in vitro, including A431 (epidermoid carcinoma), MDA-MB-175-VII (breast cancer) and FaDu (head and neck cancer). In addition, REGN1400 strongly inhibited the growth of these cell lines in vitro. Consistent with its potent effects on tumor cell growth in vitro, REGN1400 strongly inhibited the growth of A431 and FaDu tumor xenografts in a dose-dependent manner, providing 70% and 97% tumor growth inhibition, respectively. Inhibition of FaDu tumor growth by REGN1400 was accompanied by a significant decrease in tumor ERBB3 phosphorylation, as assessed by both western blot and immunohistochemistry, confirming target inhibition in vivo. While treatment of FaDu tumors with single agent REGN1400 provided almost complete tumor growth inhibition, but not regression, the combination of REGN1400 plus anti-EGFR antibody caused significant tumor regression. Furthermore, combination treatment with REGN1400 plus the anti-HER2 antibody trastuzumab inhibited the growth of BT474 breast tumor xenografts more potently than either single agent. These findings indicate that ERBB3-directed therapies might be more effective when combined with agents that inhibit other ERBB family members. Finally, gene profiling studies in REGN1400-treated tumor cells revealed upregulation of several genes that could promote resistance to REGN1400, e.g., ERBB3 itself and ERBB4. Further analysis of tumor gene expression changes induced by REGN1400 could provide insight into potentially useful combination regimens. In summary, REGN1400, a novel anti-ERBB3 antibody, exhibits significant antitumor activity in preclinical models, suggesting the possibility that it could provide benefit to patients with multiple types of cancer, either as a single agent or when combined with therapies targeting other ERBB family members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2718. doi:1538-7445.AM2012-2718

Inhibitory Effects of Calcitriol on the Growth of MCF-7 Breast Cancer Xenografts in Nude Mice: Selective Modulation of Aromatase Expression in vivo

Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active metabolite of vitamin D, exerts many anticancer effects in breast cancer (BCa) cells. We have previously shown using cell culture models that calcitriol acts as a selective aromatase modulator (SAM) and inhibits estrogen synthesis and signaling in BCa cells. We have now examined calcitriol effects in vivo on aromatase expression, estrogen signaling, and tumor growth when used alone and in combination with aromatase inhibitors (AIs). In immunocompromised mice bearing MCF-7 xenografts, increasing doses of calcitriol exhibited significant tumor inhibitory effects (~50% to 70% decrease in tumor volume). At the suboptimal doses tested, anastrozole and letrozole also caused significant tumor shrinkage when used individually. Although the combinations of calcitriol and the AIs caused a statistically significant increase in tumor inhibition in comparison to the single agents, the cooperative interaction between these agents appeared to be minimal at the doses tested. Calcitriol decreased aromatase expression in the xenograft tumors. Importantly, calcitriol also acted as a SAM in the mouse, decreasing aromatase expression in the mammary adipose tissue, while increasing it in bone marrow cells and not altering it in the ovaries and uteri. As a result, calcitriol significantly reduced estrogen levels in the xenograft tumors and surrounding breast adipose tissue. In addition, calcitriol inhibited estrogen signaling by decreasing tumor ERα levels. Changes in tumor gene expression revealed the suppressive effects of calcitriol on inflammatory and growth signaling pathways and demonstrated cooperative interactions between calcitriol and AIs to modulate gene expression. We hypothesize that cumulatively these calcitriol actions would contribute to a beneficial effect when calcitriol is combined with an AI in the treatment of BCa.

Dietary intervention strategies to modulate prostate cancer risk and prognosis

There is increasing interest in complementary and holistic approaches for cancer prevention and management. We sought to review the latest literature regarding dietary interventions for prostate cancer with a special emphasis on dietary fat and carbohydrate intake for modulating prognosis among men with prostate cancer. Several recent prospective trials have investigated various dietary and lifestyle investigations on malignant prostate tissue biology. These interventions included a very low-fat (12% fat kcals) vegan diet with various supplements and lifestyle changes, a more traditional low-fat diet (25% fat kcals) with flaxseed supplementation, and a low-glycemic index diet. Low-glycemic index and very low-fat vegan diets (with supplements and lifestyle changes) alter tumor biology as assessed by tumor gene expression changes, with a common mechanism perhaps being weight loss whereas no effects were seen with a traditional low-fat diet. In mice, either very low-fat or low-carbohydrate diets significantly slow tumor growth independent of weight loss. Epidemiologic and preclinical data also suggest cholesterol intake and serum cholesterol levels may be linked with the development and progression of prostate cancer. Small clinical trials suggest that tumor biology can be altered by either a vegan low-fat diet or eliminating simple carbohydrates accompanied by weight loss. Larger and longer term studies are needed to determine the clinical relevance of these findings.

Chemotherapy-induced tumor gene expression changes in human breast cancers

Studying chemotherapy-induced gene expression changes in vivo, which could provide insights into mechanisms of chemotherapy resistance. We analyzed and compared tumor gene expression changes of about 38 500 genes before and 3 weeks after doxorubicin or docetaxel treatment in 47 breast cancer patients. By using the median expression level of each probe set as the parameter, less than 5% of genes were upregulated or downregulated by more than 50% after treatment with either drug. Doxorubicin and docetaxel concordantly induced 251 genes predominantly involved in protein and macromolecule metabolism (upregulated), and cell cycle and DNA/RNA metabolism (downregulated). Doxorubicin treatment resulted in coregulation of a cluster of 345 probe sets involved in focal adhesion, Jak-Stat signaling pathway, cell adhesion molecules, and natural killer cell mediated cytotoxicity, whereas docetaxel treatment resulted in coregulation of a cluster of 448 probe sets involved in focal adhesion, neurodegenerative disorders, sphingolipid metabolism, and cell cycle. Tumors that were intrinsically sensitive or resistant to doxorubicin or docetaxel evoked distinct gene expression changes in response to the drug; doxorubicin-resistant tumors upregulated genes that were enriched for ErbB signaling, ubiquitin-mediated proteolysis, TGF-beta signaling, and MAP-kinase signaling pathways, whereas docetaxel-resistant tumors upregulated genes that were enriched for focal adhesion and regulation of actin cytoskeleton. The drug-specific tumor gene expression changes were validated in independent in-vitro and in-vivo datasets. Gene expression alterations of breast cancer were specific to doxorubicin and docetaxel treatment, and yielded mechanistic insights into resistance to either drug. Gene expression analysis provides more global perspectives on resistance pathways that could be exploited for therapeutic selection.

LBH589, a Novel Deacetylase Inhibitor (DACi), Treatment of Patients with Cutaneous T-Cell Lymphoma (CTCL). Skin Gene Expression Profiles in the First 24 Hours Related to Clinical Response Following Therapy.

Background: LBH589 is a novel DACi in Phase I trials. Pre-clinical studies have demonstrated that DACi alter gene expression and other DACi have induced disease regression in CTCL. Indeed, CTCL is an ideal disease to assess variation in tumor gene expression over time following drug administration. In this study we evaluated the safety and activity of LBH589 in CTCL and examined changes in tumor gene expression in the first 24 hours following oral LBH589.Methods: Pts with advanced-stage CTCL, who had progressed following prior systemic therapy were entered into the oral DLT dose level 30 mg M,W,F cohort (n=1), the subsequent MTD dose level 20 mg M,W, F weekly (n=9). LBH589 was continued until disease progression or unacceptable toxicity. Intensive cardiac monitoring was performed. Six pts had 3 mm punch biopsies from CTCL-involved skin lesions at 0, 4, 8 and 24 h after administration, which were subjected to gene expression profiling using Affymetrix U133 plus 2.0 GeneChips with 47,000 probesets. Alteration in gene expression patterns was confirmed by QRT-PCR of selected genes. Individual gene expression analysis is underway, utilizing set enrichment analysis to elucidate the functional categories which correlate with degree of patient response.Results: 10 pts are currently evaluable for response. 2 of the pts attained a complete response (CR), 4 attained a partial response (PR), 1 achieved stable disease (SD) with ongoing improvement, and 2 progressed on treatment (PD). (RR = 6/10; 60%). Microarray data on 5 pts demonstrated distinct gene expression response profiles between pts. Individual gene expression within patient tumors varied over the timepoints in the first 24 hours following treatment. To demonstrate effects of LBH589 as an epigenetic modulator, global changes in gene expression patterns in responding versus progressing patients have been delineated. In addition, functional categories of genes which correlate with degree of patient response have been identified.Conclusions: LBH589 induces CR's in CTCL pts. Preliminary microarray analysis of tumor samples have identified distinct gene expression profiles.