Supplementary Diagrams 1-2 from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Supplementary Figures 3-5 from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Supplementary Tables 1-4, Figures 1-2 from Identification of Biomarkers Modulated by the Rexinoid LGD1069 (Bexarotene) in Human Breast Cells Using Oligonucleotide Arrays

Supplementary Materials 3 from Identification of Modulated Genes by Three Classes of Chemopreventive Agents at Preneoplastic Stages in a p53-Null Mouse Mammary Tumor Model

e14055 Background: In preclinical studies eribulin has shown significant effects on epithelial-mesenchymal transition (EMT) in human breast cancer cells in vitro and in vivo, inducing a switch from mesenchymal to epithelial states, decreasing migration and invasiveness of human breast cancer cells in vitro, as well as experimental metastases in vivo. HBI-8000 is a novel, orally bioavailable Class I selective HDAC inhibitor (HDACi). In addition to its demonstrated positive effects on antitumor immunity, it also exerts significant effects on EMT pathway components, enhancing CDH1 (e-cadherin) expression and inhibiting TGF-β induced metastasis. Methods: The syngeneic, orthotopic 4T1 model is thought to be a close representation of the clinical situation; similar to human mammary carcinoma, the major cause of morbidity and mortality is development of spontaneous metastases. 4T1-implanted mice treated with combinations of eribulin and HBI-8000 were monitored for primary tumor growth inhibition (TGI), survival, and metastatic lung foci. Tumor samples were analyzed by Q-PCR for expression of genes involved in EMT or in the metastatic process. Results: Eribulin alone had no effect on primary TGI. Eribulin combined with HBI-8000 resulted in significant (P < 0.05) reductions in median tumor volume, tumor volume distribution (TMD) and survival (P < 0.005). Single agent HBI-8000 had a similar effect on TGI, TMD (P < 0.05) and survival (P < 0.005). Whereas single agent HBI-8000 or eribulin failed to significantly reduce metastases, the eribulin-HBI-8000 combination produced significant (P < 0.01) and synergistic inhibition of metastases, decreasing the number of lung foci approximately 10-fold. Gene expression analyzed by Q-PCR revealed correlations between metastasis blockade and increases in E-Cadherin, Occludin and Claudin1. There was a strong correlation with inhibition of HMGA2 (High Mobility Group AT-Hook 2) gene expression. Conclusions: Eribulin combined with HBI-8000 inhibited primary tumor growth and inhibited metastasis development and increased survival. This effect was correlated and consistent with changes in genes relevant to EMT, tumor plasticity and stemness.

Abstract Immunomodulatory antibodies targeting the immune checkpoint receptors CTLA-4, PD-1 and its ligand PD-L1, have resulted in significantly improved clinical benefits over the standard of care in many cancer types. While these immune checkpoint inhibitors have successfully exploited the adaptive immune system to produce remarkable clinical responses in various cancers, a significant number of patient's tumors are either innately resistant or develop resistance, and the disease eventually progresses. For this reason, rational drug combinations with checkpoint inhibitors and other immunomodulating agents are imperative to improve the rate of durable responses and patient survival. Histone deacetylase inhibitors (HDACi) have been successful as monotherapy for hematologic malignancies, but their clinical effectiveness against solid tumors has been limited to date. The long-held belief that HDACi are immunosuppressive has been challenged recently by data demonstrating the ability of HDACi to enhance anti-tumor immunity through effects on tumor immunogenicity, regulatory T cells, myeloid-derived suppressor cells (MDSCs), as well as effector cell functions. HBI-8000 (chidamide) is a novel, orally bioavailable Class I (HDAC 1,2,3) and Class II (HDAC 10) selective HDACi with a demonstrated positive effects on antitumor immunity, enhancing the activity of CTL and NK cells, as well as having direct activity against tumor cells through reversal of apoptosis resistance. We report here that HBI-8000 enhances the activity of both PD-1 and PD-L1 blocking antibody (Ab) in the MC38 syngeneic mouse model of colon cancer. Combinations of HBI-8000 with PD-1 or PD-L1 Ab resulted in statistically significant decreases in tumor progression and increases in survival compared to single agent therapy. Analysis of tumor infiltrating lymphocytes (TIL) revealed a significant increase in the percentage of intratumoral CD8+ T cells in tumors treated with both HBI-8000/PD-1 Ab and HBI-8000/PD-L1 Ab combinations. The TIL analysis also revealed a trend towards lower percentages of Tregs and granulocytic MDSCs. To evaluate the potential role of this combination as a 2nd line treatment option, i.e., in subjects failing 1st line checkpoint antibody therapy, we developed a model of immune checkpoint antibody failure, where tumor-bearing mice were initially treated (1st line) with either PD-1 or PD-L1 blocking Ab. Mice which had either slow disease progression or stable disease followed by progression, were randomized into one of six 2nd line treatment groups, including combinations of anti PD-1 or PD-L1 blocking Ab with HBI-8000. Fifty percent of mice failing 1st line PD-1 Ab treatment experienced a complete response, and were rescued by treatment with HBI-8000 combined with PD-L1 Ab. Altogether our data suggest that the addition of the HDACi HBI-8000 can improve the anti-tumor response to either PD-1 or PD-L1 blocking antibodies, increase the percentage of intratumoral immune effector cells and potentially counter innate resistance to checkpoint inhibitors. Our data also suggests that combination therapy with HBI-8000 and a checkpoint inhibitor can rescue tumor-bearing mice that have failed 1st line therapy with different checkpoint inhibitor. These results provide a strong rationale for the evaluation of therapeutic regimens utilizing PD-L1 or PD-1 blocking antibodies in combination with HBI-8000. Citation Format: Reid P. Bissonnette, Alain Rolland, Bob Goodeneow, Mireille Gillings. The HDAC inhibitor HBI-8000 enhances immunotherapy with either PD-1 or PD-L1 blockade in the MC38 model of colon cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B108.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell lymphotropic virus 1. Treatment options for acute ATL patients include chemotherapy, stem cell transplantation, and recently the anti-chemokine (C-C motif) receptor 4 antibody, although most patients still have a poor prognosis and there is a clear need for additional options. HBI-8000 is a novel oral histone deacetylase inhibitor with proven efficacy for treatment of T-cell lymphomas that recently received approval in China. In the present study, we evaluated the effects of HBI-8000 on ATL-derived cell lines and primary cells obtained from Japanese ATL patients. In most cases HBI-8000 induced apoptosis in both primary ATL cells and cell lines. In addition, findings obtained with DNA microarray suggested Bim activation and, interestingly, the contribution of the NLR family, pyrin domain containing 3 (NLRP3) inflammasome pathway in HBI-8000-induced ATL cell death. Further investigations using siRNAs confirmed that Bim contributes to HBI-8000-induced apoptosis. Our results provide a rationale for a clinical investigation of the efficacy of HBI-8000 in patients with ATL. Although the role of NLRP3 inflammasome activation in ATL cell death remains to be verified, HBI-8000 may be part of a novel therapeutic strategy for cancer based on the NLRP3 pathway.

In pursuit of effective therapeutic agents for the estrogen receptor (ER)-negative breast cancer, we previously showed that bexarotene reduced mammary tumor development by 75% in ErbB2 mice. To further improve the effectiveness of breast cancer prevention, we have now investigated the effects of a combinatorial therapy consisting of two cancer preventive drugs. On the basis of the hypothesis, rexinoid LG100268 plus tamoxifen would more effectively prevent the development of both ER-positive and ER-negative breast cancer. We treated p53-null mammary gland mice with tamoxifen and LG100268, individually and in combination. By 60 weeks of age, vehicle-treated mice developed tumors in 52% of transplanted mammary glands, whereas mice treated with tamoxifen and LG100268 developed tumors in only 13% of transplanted mammary glands. To further define the mechanistic effects of this combinatorial treatment, we investigated the effects of tamoxifen and LG100268 on mammary tissue biomarkers. In mammary tissue harvested before tumor development, the proliferation markers Ki67 and cyclin D1 were significantly reduced in mice treated with the combination therapy. In addition, the rexinoid target genes ABCA1 and ABCG1 were induced in both the rexinoid and combination treatment groups, whereas expression remained constant in tamoxifen group. These results show that tamoxifen-LG100268 combinatorial treatment is more effective in preventing mammary tumors than either agent alone. In addition, these studies have identified relevant tissue biomarkers that can be used to show the effect of these agents on mammary tissue. These results support the development of clinical trials of antiestrogen and rexinoid combinatorial therapy for the prevention of patients with high-risk breast cancer. Cancer Prev Res; 5(10); 1195–202. ©2012 AACR.

Angiogenesis is essential for tumor growth and metastasis, and controlling tumor-associated angiogenesis is a proven therapeutic intervention to limit tumor progression. Several pathways have been involved in tumor-associated angiogenesis and the predominant target of current interventions is the vascular endothelial growth factor (VEGF) pathway. Recently, Activin-receptor like kinase type 1 (ALK-1) has been proven to be a novel and potent target of tumor angiogenesis. Our results indicate that inhibiting VEGF and ALK1 have different effects on tumor vasculature, suggesting that targeting both pathways could have a more potent effect than targeting either pathway alone. We have created a bi-functional antibody conjugate targeting both ALK-1 and VEGF by conjugating VEGF-trap peptides to an anti-ALK1 monoclonal antibody. We demonstrate that our bi-functional molecule binds both ALK-1 and VEGF with high affinity in vitro, blocks both ALK-1 and VEGF pathways in HUVEC primary human endothelial cells and has good pharmacokinetics properties in mice. Finally, in a MDA-MB-231 xenograft model in SCID mice, the ALK-1/VEGF bi-functional antibody conjugate slowed tumor growth and prolonged survival with greater efficacy than anti-VEGF therapy alone. 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 2327. doi:1538-7445.AM2012-2327

Background: Recent research in breast biology supports the cancer stem cell hypothesis which asserts that malignancies arise in tissue stem cells through dysregulation of self-renewal. The cancer stem cell hypothesis has important implications for early detection, prevention and treatment of breast cancer. If breast cancers arise from the transformation of normal stem cells, it may be possible to target stem cells for the prevention of cancer. We have previously shown that rexinoids prevent the development of ER-negative breast cancer in transgenic mouse models. Previous studies have demonstrated a role of retinoid signaling in the regulation of normal breast stem cell self-renewal and differentiation. Bexarotene and LG100268 are rexinoids that selectively bind RXR receptors. Based on the effects of these molecularly targeted agents, we hypothesized that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population. Methods: We performed in vivo and in vitro studies to address our hypothesis. Cell proliferation assays were used to determine whether rexinoids can inhibit the growth of normal, pre-malignant and malignant breast cell lines (HMECs, MCF10A, DCIS.COM, SUM225, HCC1937 and HCC38 cell lines). To determine whether these agents affect stem cells, we performed mammosphere assays and FACS analysis after treatment with vehicle or rexiniods. We next conducted in vivo studies using MMTV-Wnt transgenic mice treated with LG100268 (50mg/kg) or vehicle, and studied the stem cell properties of mammary epithelial cells from these mice. The stem cell population in mammary glands was quantified by staining of CD24, CD49f and using FACS analysis and mammosphere assays. We also performed limiting dilution assay to demonstrate the in vivo effect of LG100268 on stem cell population. Fvb mice injected with 1,10,100,1000 wnt tumor cells were treated with vehicle or LG100268 and tumor outgrowth rate is recorded in each group. Results: Our results showed that the rexionid LG100268 inhibited the growth of breast cell lines only at high concentrations (IC50>10uM) in most of the tested cell lines. Results of in vitro mammosphere assays showed that a dose of LG100268 (100nM) much lower than IC50 reduced mammosphere formation efficiency by more than 40% in all breast cancer cell lines. Results from our in vivo experiments showed that LG100268 reduced the mammary epithelial stem cell population by 57% as defined by the CD24+/CD49f++ population identified by FACS and by 47% as measured by in vitro mammosphere assays. In addition in vivo limiting dilution assay studies demonstrate slower tumor outgrowth rate and lower repopulation frequency in groups with LG100268 treatment. Conclusion: These results from in vitro and in vivo studies demonstrate that the rexinoid LG100268 is a potent mammary stem cell inhibitor. This study shows that rexinoids suppress stem cell expansion and suggest that mammary stem cell inhibitors will be useful breast cancer preventive agents. This work is supported by a grant from the Breast Cancer Research Foundation (to PHB) and from an NIH/NCI RO1 grant (CA078480) (to PHB). This abstract is also presented as Poster B30. Citation Format: Jing Zhao, Yun Zhang, Jamal L. Hill, Abhijit Mazumdar, Ivan Uray, Reid P. Bissonnette, Powel H. Brown. Targeting breast cancer stem cells using cancer preventive rexinoids. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr PR-06.

Synthetic rexinoids effectively suppress both estrogen receptor-positive and estrogen receptor-negative mammary tumors in animal models, which makes them prime candidates for a novel class of cancer-preventive agents. When used in combination with chemotherapy for non-small-cell lung cancer, the rexinoid bexarotene was most effective for patients who developed hypertriglyceridemia as a side effect. Although serum triglycerides originate from the liver, the effect of bexarotene on lipogenesis in breast epithelial cells is not known. Gene expression studies with normal mammary epithelial cells indicated that rexinoids modulate lipid metabolism, particularly enzymes involved in triglyceride synthesis. High-content analysis revealed dose-dependent accumulation of neutral lipids within adipocyte differentiation-related protein-associated cytoplasmic lipid droplets after long-term bexarotene treatment. Bexarotene also induced mRNA and protein levels for peroxisome proliferator-activated receptor (PPAR) γ, whereas selective knockdown of PPARγ attenuated the induction of both lipid droplets and adipocyte differentiation-related protein. Pharmacological activation of PPARγ, but not PPARα or retinoic acid receptors, effectively induced lipid accumulation. Furthermore, the combination of the PPARγ agonist rosiglitazone with bexarotene synergistically suppressed the growth of human mammary epithelial cells and revealed a strong, nonlinear, inverse correlation of cell growth with lipid droplet accumulation in the cell population. These findings indicate that rexinoids activate a lipogenic program in mammary epithelial cells through a retinoid X receptor/PPARγ-mediated mechanism. It is noteworthy that combining low doses of bexarotene with the PPARγ agonist rosiglitazone provides effective growth suppression of mammary epithelial cells, potentially dissociating systemic adverse effects associated with standard bexarotene treatment from the antiproliferative effects on mammary epithelium.

Abstract Introduction: Commonly used biomarkers to assess the cancer preventive activity of cancer preventive agents include markers of cell proliferation or apoptosis. However, the expression of proliferation or apoptosis markers is highly variable, and measurement is often done using semi-quantitative assays. In addition, cancer preventive agents may act through mechanisms that do not depend on proliferation or apoptosis. Therefore, identification of new biomarkers indicative of the cancer preventive activity of chemopreventive agents is essential for the development of new cancer preventive drugs. Methods: We applied high-content microscopic analysis to assess the phenotypic changes occurring in normal mammary epithelial cells (HMECs) after treatment with cancer preventive drugs by measuring cell proliferation, DNA and neutral lipid content and localization, as well as cell cycle distribution of the cells. Gene specific knock-down was achieved by transfections with siRNA duplexes. Measurement of transcript levels was performed using quantitative real-time RT-PCR. Results: Multiparametric analysis of high-throughput image based studies have revealed that anti-proliferative activity of cancer preventive rexinoids is associated with increased formation of cytoplasmic lipid droplets. Accumulation of cytoplasmic neutral lipids was restricted to cell types that undergo significant growth suppression by bexarotene (HMECs, SUM225, and ZR75.1), but not observed in retinoid-resistant cells (MCF10A, HCC1937, and HCC1143). Other growth suppressive drugs including the kinase inhibitor lapatinib, vitamin D, and certain natural compounds, including epicatechine monogallate, duartin, actinonin and homoisoflavonoids, were also found to increase cellular neutral lipid content in breast cells. The addition of external fatty acids, such as oleic and palmitoleic acids did not result in dose-dependent growth suppression or accumulation of neutral esterified lipid droplets. Lipid accumulation and growth suppression were associated with the up-regulation of genes involved in regulating the formation of neutral lipids, triglycerides and phospholipids, but not genes involved in de novo fatty acid synthesis. Conversely, knock-down of key proteins controlling cellular triglyceride synthesis and storage, diacylglycerol acyltransferase 1 (DGAT1) and adipophyllin (Adfp) increased cell proliferation whereas their induction was associated with growth suppression in HMECs. Conclusion: These results suggest that increased accumulation of cellular triglycerides may be a useful biomarker to assess the preventive effect of putative cancer preventive agents. These results support the systematic testing of cellular neutral lipid levels in preclinical and clinical models testing novel chemopreventive agents. This work was supported by the grant RO1 CA 78480 (to P.H.B.). Citation Information: Cancer Prev Res 2011;4(10 Suppl):B69.

Abstract 3391 Granulocyte colony stimulating factor (GCSF) is the essential cytokine for the regulation of neutrophilic granulocytes. Binding of GCSF to its receptor (GCSFR) triggers receptor dimerization, leading to activation of JAK1 and JAK2, phosphorylation of GCSFR, STAT3, STAT5, and Ras/mitogen-activated protein kinase (MAPK), and results in proliferation and differentiation of granulocytic cells. Recombinant human GCSF (rhGCSF) is used successfully to alleviate chemotherapy-induced neutropenia, neutropenia associated with hematopoietic stem cell transplantation, and severe chronic neutropenia. A small molecule oral GCSFR agonist may offer a safer and more convenient alternative to the current injectable rhGCSF therapy. Whereas a previous effort to identify small-molecule mimetics of GCSF found SB-247464 that selectively activated the murine GCSFR, no small-molecule human GCSF mimetics have been developed. Recently, we have discovered a series of novel non-peptidyl small molecules that selectively activate human GCSFR (hGCSFR) function, and may provide a significant innovation in the treatment of neutropenia. In cells transiently transfected with an hGCSFR expression vector and a STAT3-responsive luciferase reporter, a lead compound, LG7455, activates luciferase expression with an efficacy of 50% relative to rhGCSF, and potency (EC50) of 100 nM. LG7455 also activates luciferase expression in cells transfected with hGCSFR and a STAT5-responsive luciferase reporter (65%, 40 nM EC50). The activity of LG7455 is dependent on the expression of hGCSFR, and LG7455 is not active in luciferase assays when human thrombopoietin receptor (hTPOR) or erythropoietin receptor (hEPOR) is expressed. In UT-7 cells made responsive to GCSF by stable transfection of hGCSFR (UTP-hGCSFR), LG7455 stimulated cell growth and increased the phosphorylation of STAT3 and STAT5. LG7455 did not increase growth of TPO- or EPO-responsive UT-7 cells. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), LG7455, increased the percentage of cells positive for the granulocyte-specific marker CD15 (FUT4). The effect of LG7455 in BM-HCs was additive to the effect of rhGCSF. LG7455 is active in luciferase assays with expressed cynomolgus monkey GCSFR, but not mouse, guinea pig or rabbit GCSFR. Similar to what has been demonstrated for small-molecule human TPOR agonists such as eltrombopag, the activity of LG7455 is dependent on a specific residue in the hGCSFR transmembrane domain. When histidine 627 (His-627) in hGCSFR is changed to asparagine present at a similar location in the mouse GCSFR (Asp-602), unlike rhGCSF, LG7455 is no longer active. LG7455 is active, however, on mouse GCSFR with Asp-602 replaced by His. In radioligand-binding experiments using UTP-hGCSFR cells, LG7455 did not displace [125I]rhGCSF, however binding of [125I]rhGCSF was augmented in a concentration dependent manner consistent with allosteric receptor modulation. These data demonstrate that LG7455 is a novel small-molecule selective hGCSFR agonist that activates the receptor in a manner distinct from GCSF and similar to the mechanism of small-molecule hTPOR agonists. Further optimization of the LG7455 chemical series should provide orally-available molecules to treat neutropenia with improved safety and convenience compared to current injectable rhGCSF.Disclosures: Marschke: Ligand Pharmaceuticals: Employment. Rungta: Ligand Pharmaceuticals: Employment. Slavin: Ligand Pharmaceuticals: Employment. Sanders: Ligand Pharmaceuticals: Employment. Roach: Ligand Pharmaceuticals: Employment. Pickens: Ligand Pharmaceuticals: Employment. Shen: Ligand Pharmaceuticals: Employment. van Oeveren: Ligand Pharmaceuticals: Employment. Hong: Ligand Pharmaceuticals: Employment. Sun: Ligand Pharmaceuticals: Employment. Bissonnette: Ligand Pharmaceuticals: Employment. Syka: Ligand Pharmaceuticals: Employment. Zhi: Ligand Pharmaceuticals: Employment.

Abstract Background: Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR), play an important role in regulating cell proliferation and differentiation. Our previous studies demonstrated that related molecules, rexinoids (Bexarotene (LGD1069) and LGD100268), that bind the RXR receptor, suppress but do not totally prevent ER-negative mammary tumorigenesis. Using a transgenic mouse model of ER-negative breast cancer (MMTV-erbB2 mice), our laboratory demonstrated that bexarotene delays but does not totally prevent ER-negative mammary tumorigenesis with minimal toxicity. We hypothesized that bexarotene in combination with the anti-estrogen drug tamoxifen would more effectively prevent the development breast cancer. To test this hypothesis we treated MMTV-erbB2 mice with bexarotene, tamoxifen, or the combination from the age of 3 months to 9 months and monitored the mice for mammary tumors. Methods: Virgin MMTV-erbB2 mice at age of 3 months were used for these studies. A defined diet was used (AIN-76A). Mice were randomized into 4 groups (10 mice per group): 1) Tamoxifen control (Sham pellets) and bexarotene control (sesame oil), 2) Tamoxifen control (Sham pellets) and bexarotene (50mg/kg), 2) Tamoxifen pellets (2.5mg/pellet, 90-day-release) and bexarotene control (sesame oil), 4) Tamoxifen pellets (2.5mg/pellet, 90-day-release) and bexarotene (50mg/kg). Tamoxifen or sham pellets were replaced once after 90 days to deliver 180 days of tamoxifen (from age 3 months to 9 months). Bexarotene or sesame oil control was administered by gastric gavage daily (5 days per week) for 180 days (from age 3 months to 9 months). The mice were observed daily for tumor formation, weights were recorded weekly, and tumors were measured twice a week. Mice were sacrificed when tumor sizes exceeded 1500mm3. Analysis of time to tumor formation (defined as the time to develop a 100mm3 tumor) was the primary endpoint of these studies. Analysis of the expression of mammary tissue biomarkers is ongoing. Results: Bexarotene and tamoxifen as single agents slightly delayed the development of mammary tumors in these mice. However, the combination of bexarotene and tamoxifen was much more effective. By 350 days of age, 100% of the control animals developed mammary tumors, while only 10% of the animals treated with bexarotene and tamoxifen developed mammary tumors. This difference in time to tumor development is highly statistically significant (p <0.05; Generalized Wilcoxon test). These mice are still being followed, and updated results will be presented. We are now conducting biomarker studies to measure the effect of tamoxifen, bexarotene, and the combination on the expression of the erbB2 transgene, the estrogen receptor, and on markers of proliferation (Ki67 and cyclin D1) and apoptosis (cleaved caspase 3). Conclusions: The combination of bexarotene and tamoxifen is highly effective at preventing ER-negative mammary tumors in MMTV-erbB2 mice. These preclinical results suggest that combining a rexinoid with an anti-estrogen drug may be useful to prevent both ER-positive and ER-negative breast cancer in humans. These studies were supported by NIH R01 grants (CA101211) and by a Breast Cancer SPORE grant (P50 CA58183). Citation Information: Cancer Prev Res 2010;3(1 Suppl):A54.

Nuclear hormone receptors, including RXR and PPARγ, represent novel therapeutic targets in melanoma. We have previously shown that the DRO subline of the amelanotic melanoma A375 responds to rexinoid and thiazolidinedione (TZD) treatment in vitro and in vivo. We performed microarray analysis of A375(DRO) after TZD and combination rexinoid/TZD treatment in which the calcium binding protein S100A2 had increased expression after rexinoid or TZD treatment and a synergistic increase to combination treatment. Increased S100A2 expression is dependent on an intact PPARγ receptor, but it is not sufficient to mediate the antiproliferative effects of rexinoid/TZD treatment. Over expression of S100A2 enhanced the effect of rexinoid and TZD treatment while inhibition of S100A2 expression attenuated the response to rexinoid/TZD treatment, suggesting that S100A2 is necessary for optimal response to RXR and PPARγ activation by respective ligands. In summary, we have identified potential downstream mediators of rexinoid and TZD treatment in a poorly differentiated melanoma and found that alterations in S100A2 expression affect RXR and PPARγ signaling in A375(DRO) cells. These studies provide insight into potential mechanisms of tumor response or resistance to these novel therapies.

Bexarotene is an RXR-selective vitamin A analog that has been shown to prevent ER-negative mammary tumorigenesis in animal models. While investigating the mechanism by which bexarotene prevents ER-negative breast cancer development, we found that the expression of cyclin D1, a critical cell cycle promoter, was repressed by bexarotene in vitro and in vivo. Time course and cycloheximide experiments show that repression of cyclin D1 is a late effect and requires new protein synthesis. Previously we discovered that DEC2 (differentially expressed in chondrocytes-2), a helix–loop–helix transcription repressor, was induced by bexarotene in human mammary epithelial cells. Therefore, we hypothesized that bexarotene represses the transcription of cyclin D1 through induction of DEC2. Luciferase reporter studies demonstrated that either bexarotene treatment or forced expression of DEC2 can repress the transcription of a cyclin D1 promoter reporter by affecting the basal transcriptional activity. Results from chromatin immunoprecipitation experiments showed that bexarotene treatment causes the recruitment of DEC2 and HDAC1 (histone deacetylase 1) to the cyclin D1 promoter. Co-immunoprecipitation confirms the interaction between DEC2 and HDAC1, suggesting that the recruitment of HDAC1 to the cyclin D1 promoter is through DEC2. Trichostatin A, a HDAC inhibitor, reverses the cyclin D1 repression by bexarotene, suggesting that repression of cyclin D1 involves histone deacetylation. Knock-down of DEC2 by siRNA abolishes the cyclin D1 repression, further supporting our hypothesis. Finally, we demonstrated that overexpression of DEC2 dramatically inhibited cell proliferation and repressed the expression of cyclin D1 in human mammary epithelial cells. These results suggest that bexarotene down-regulates cyclin D1 through induction of DEC2, followed by recruitment of HDAC1 to the cyclin D1 promoter causing transcriptional repression. By elucidating the mechanism by which rexinoids inhibit cell proliferation, it will be possible to develop more effective and less toxic drugs to prevent ER-negative breast cancers.

Abstract Abstract 1565 Erythropoietin (EPO) acts on the homodimeric EPO receptor (EPOR) to stimulate proliferation of erythroid progenitor cells and induce their survival and differentiation into red blood cells. Various recombinant human EPO derivatives, also known as erythropoiesis-stimulating agents (ESAs), are marketed or in clinical development for the treatment of anemia due to renal failure or cancer chemotherapy. However, ESA treatment is associated with an increased risk of adverse cardiovascular complications in patients with kidney disease, and may be related to an increase in mortality in cancer patients, when it is used to increase hemoglobin levels above 13.0 g/dl. We have identified a series of novel non-peptidyl small molecules that selectively activate EPOR function, which may provide a unique therapeutic opportunity in the treatment of anemia. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), a representative analog, LG5640, potently (2 nM EC50) increased the percentage of cells positive for the erythrocyte-specific marker CD235a (glycophorin A) with an efficacy partial (42%) to the maximal effect of EPO (3 U/ml), but greater than the efficacy of the normal serum EPO concentration (∼0.01 U/ml). The erythropoietic effect of LG5640 in BM-HCs was additive to the effect of EPO. LG5640 stimulated the expression of several EPO responsive genes in CD34 positive BM-HCs, including hemoglobin α, EPOR and the anti-apoptotic protein BCL2L1. In addition, LG5640 stimulated the formation of BFUe colonies with partial efficacy (30%) when incubated with BM-HCs for 14 days. The effect of LG5640 on BM-HCs was specific for the erythroid lineage. LG5640 did not increase the percentage of BM-HCs positive for the megakaryocyte marker CD41 or the granulocyte marker CD15. Using human cell lines, we have determined that the action of LG5640 is dependent on EPOR and involves the selective activation of the PI3K/AKT-GATA1 signaling pathway. In the human EPO-dependent cell line UT7EPO, LG5640 blocked apoptosis induced by EPO withdrawal (10 nM EC50), and stimulated the expression of BCL2L1 with an efficacy comparable to EPO. LG5640 stimulated the phosphorylation of EPOR, PI3K, and GATA1, and induced the binding of GATA1 to DNA. Incubation of UT7EPO cells with the PI3K inhibitor LY294002 blocked the effect of LG5640 on cell survival. However, LG5640 did not stimulate phosphorylation of STAT5 or ERK/MAPK, or induce STAT5 DNA binding. LG5640 did not block apoptosis or stimulate BCL2L1 expression in the GM-CSF- and TPO-responsive human leukemia Mo7e cells that lack EPOR. Furthermore, transfection of UT7EPO cells with EPOR- and GATA1-specific siRNAs blocked the activity of LG5640. These data demonstrate that LG5640 is a novel small molecule selective EPOR agonist that unlike other ESAs selectively activates the EPOR/PI3K/GATA1 signal transduction pathway resulting in survival and differentiation of BM-HCs into erythrocytes, possibly through uniquely altering the conformation of the homodimeric EPOR. The selective agonists display an efficacy partial to the maximal effect induced by EPO, and lack excessive erythropoietic stimulation that may possibly contribute to the adverse effects of ESAs. Based on the novel profile of the series, several lead compounds that increase the percentage of CD235a positive BM-HCs with nanomolar potency, and display oral bioavailability in the rat and monkey, have been identified as potential preclinical development candidates. Disclosures: Bissonnette: Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Rungta:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hudson:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Roach:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hong:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Sun:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hu:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Ward:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Luo:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Sanders:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Syka:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Slavin:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Zhi:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Marschke:Ligand Pharmaceuticals Inc: Employment, Equity Ownership.