Natalie C. Twine

Population Pharmacokinetics of CCI-779: Correlations to Safety and Pharmacogenomic Responses in Patients with Advanced Renal Cancer

Our objective was to estimate the pharmacokinetic parameters of CCI‐779 and its metabolite, sirolimus, and evaluate associations of exposure parameters with safety and clinical activity. Exposure parameters were also correlated with pharmacogenomic responses in peripheral blood mononuclear cells (PBMCs).

Potent in vitro and in vivo anticancer activities of des-methyl, des-amino pateamine A, a synthetic analogue of marine natural product pateamine A

We report here that des-methyl, des-amino pateamine A (DMDA-PatA), a structurally simplified analogue of the marine natural product pateamine A, has potent antiproliferative activity against a wide variety of human cancer cell lines while showing relatively low cytotoxicity against nonproliferating, quiescent human fibroblasts. DMDA-PatA retains almost full in vitro potency in P-glycoprotein-overexpressing MES-SA/Dx5-Rx1 human uterine sarcoma cells that are significantly resistant to paclitaxel, suggesting that DMDA-PatA is not a substrate for P-glycoprotein-mediated drug efflux. Treatment of proliferating cells with DMDA-PatA leads to rapid shutdown of DNA synthesis in the S phase of the cell cycle. Cell-free studies show that DMDA-PatA directly inhibits DNA polymerases α and γ in vitro albeit at concentrations considerably higher than those that inhibit cell proliferation. DMDA-PatA shows potent anticancer activity in several human cancer xenograft models in nude mice, including significant regressions observed in the LOX and MDA-MB-435 melanoma models. DMDA-PatA thus represents a promising natural product-based anticancer agent that warrants further investigation.[Mol Cancer Ther 2009;8(5):1250–60]

Novel small molecule inhibitors of TLR7 and TLR9: mechanism of action and efficacy in vivo

The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-[4-(6-(3-(dimethylamino)propoxy)benzo[d]oxazol-2-yl)phenoxy]-N,N-dimethylpropan-1-amine} and E6446 {6-[3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenyl]benzo[d]oxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)–containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti–double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.

Gene Expression Analyses Support Fallopian Tube Epithelium as the Cell of Origin of Epithelial Ovarian Cancer

Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.

E7449: A dual inhibitor of PARP1/2 and tankyrase1/2 inhibits growth of DNA repair deficient tumors and antagonizes Wnt signaling.

Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/β-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/β-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in β-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/β-catenin signaling addicted tumors. E7449 is currently in early clinical development.

EP4 Antagonism by E7046 diminishes Myeloid immunosuppression and synergizes with Treg-reducing IL-2-Diphtheria toxin fusion protein in restoring anti-tumor immunity

ABSTRACT Reprogramming of immunosuppressive tumor microenvironment (TME) by targeting alternatively activated tumor associated macrophages (M2TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Tregs), represents a promising strategy for developing novel cancer immunotherapy. Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite and mediator of chronic inflammation, has emerged as a powerful immunosuppressor in the TME through engagement with one or more of its 4 receptors (EP1-EP4). We have developed E7046, an orally bioavailable EP4-specific antagonist and show here that E7046 has specific and potent inhibitory activity on PGE2-mediated pro-tumor myeloid cell differentiation and activation. E7046 treatment reduced the growth or even rejected established tumors in vivo in a manner dependent on both myeloid and CD8+ T cells. Furthermore, co-administration of E7046 and E7777, an IL-2-diphtheria toxin fusion protein that preferentially kills Tregs, synergistically disrupted the myeloid and Treg immunosuppressive networks, resulting in effective and durable anti-tumor immune responses in mouse tumor models. In the TME, E7046 and E7777 markedly increased ratios of CD8+granzymeB+ cytotoxic T cells (CTLs)/live Tregs and of M1-like/M2TAM, and converted a chronic inflammation phenotype into acute inflammation, shown by substantial induction of STAT1/IRF-1 and IFNγ-controlled genes. Notably, E7046 also showed synergistic anti-tumor activity when combined with anti-CTLA-4 antibodies, which have been reported to diminish intratumoral Tregs. Our studies thus reveal a specific myeloid cell differentiation-modifying activity by EP4 blockade and a novel combination of E7046 and E7777 as a means to synergistically mitigate both myeloid and Treg-derived immunosuppression for cancer treatment in preclinical models.

Combination of EP4 antagonist and checkpoint inhibitors promotes anti-tumor effector T cells in preclinical tumor models

Immunotherapies targeting the immune checkpoint receptors have shown great promise for a subset of cancer patients. However, robust and safe combination therapies are still needed to increase the benefit of cancer immunotherapy and bring it to broader patient populations. We have recently shown that E7046, a specific EP4 antagonist, possesses significant anti-tumor growth activity in multiple preclinical tumor models through modulating myeloid cells including tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) (AACR 2015, poster #275). Here we evaluated the anti-tumor activities of E7046 in combination with the checkpoint inhibitors anti-CTLA4 and anti-PD1 antibodies, and also with E7777, a recombinant IL-2/diphtheria toxin fusion protein, in immunogenic CT26 and poorly immunogenic 4T1 tumor models. In the CT26 model, concomitant treatment of E7046 and anti-PD1 led to pronounced tumor growth inhibition, with 40% of the mice rendered stably tumor free, while either single agent produced mostly tumor growth inhibitory activity and only an occasional tumor-free animal. In the same model, markedly improved anti-tumor activity was observed for the combination of E7046 and E7777, with up to 20% of animals rendered stably tumor free, compared with only modest anti-tumor activity of either single agent treatment alone. In the 4T1 model, combining E7046 and anti-CTLA4 resulted in a nearly complete tumor growth inhibition; either agent alone had only modest growth inhibitory activity. In the tumor microenvironment of both models, an effective anti-tumor immune response was induced by the combination treatments including E7046 as indicated by the robust accumulation and activation of CD8 cytotoxic T cells (CTL) and/or significantly improved ratio of activated GZMB+CD8+ CTL vs CD4+CD25+Foxp3+ Treg cells. In addition, combination treatments including E7046 in tumor-bearing mice showed no additional gross toxicity compared with immune checkpoint inhibitors alone or E7777 alone. Taken together, these results demonstrated a superior activity of combinational therapies including E7046 over immune checkpoint inhibitors or E7777 alone with acceptable toxicity in preclinical models, and therefore candidates for combination trials in patients. IND filing number of E7046 is 125272.

Abstract P5-06-03: Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer

Introduction: In a small neoadjuvant study in patients with triple negative breast cancer (TNBC) the combination of eribulin plus carboplatin was effective, with a pathologic complete response rate of 43% following 4 cycles of treatment. Significant numbers of sporadic TNBC tumors are deficient in DNA repair capacity and share clinical and pathological features with hereditary BRCA1 mutant disease. PARP inhibitors have demonstrated synthetic lethality in cancer cells with defective DNA repair and have therapeutic potential for TNBC. In this study we describe the combination of PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of TNBC. Methods: E7449, an orally available PARP inhibitor, was administered in combination with eribulin +/- carboplatin to 4 s.c. xenograft models of TNBC: MDA-MB-436 (BRCA1 mutant, PTEN deficient), MDA-MB-468 (BRCA wild type, PTEN deficient), HCC1806 and MDA-MB-231 (BRCA and PTEN wild type). Results and Discussion: Addition of E7449 to eribulin significantly delayed tumor progression in PTEN deficient MDA-MB-468 xenografts. In the BRCA1 mutant and PTEN deficient MDA-MB-436 xenograft model, combination of E7449 with eribulin enhanced antitumor activity versus eribulin alone. Similar potentiation was observed for carboplatin upon combination with E7449. Treatment of MDA-MB-436 xenografts with the triple combination of E7449 + eribulin + carboplatin was more efficacious than any double combination and was well tolerated at the doses examined. In contrast, no significant combination activity was observed for E7449 plus eribulin in the BRCA and PTEN wild type xenografts HCC1806 and MDA-MB-231, and similarly no potentiation of carboplatin was observed in an MDA-MB-231 xenograft. Notably, combination activity was observed in the BRCA1 mutant (MDA-MB-436) and PTEN deficient (MDA-MB-436 and MDA-MB-468) xenografts and not in the BRCA and PTEN wild-type models (HCC1806 and MDA-MB-231). Data from ongoing studies to evaluate the combination activity of E7449 + eribulin in patient-derived xenograft (PDx) models of TNBC will be presented at the meeting. Potential biomarkers of sensitivity to the combination are under investigation in both cell line xenograft and PDx models and will be described. Conclusion: The addition of E7449 to eribulin +/- carboplatin increased antitumor activity in a subset of TNBC models. Biomarker studies aimed at a better understanding of the underlying cause of sensitivity are underway. The preclinical data support assessment of E7449 + eribulin + carboplatin combination therapy in the current phase I/II clinical trial. Citation Format: Sharon McGonigle, Jiayi Wu, Donna Kolber-Simonds, Natalie C Twine, Jue-lon Shie, Noel Taylor, Sergei Agoulnik, Zoltan Dezso, Shannon McGrath, Mark Matijevic, Shanqin Xu, Galina Kuznetsov, Mary Woodall-Jappe, Kenichi Nomoto. Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-03.

Abstract 2733: Antitumor activity of the PARP inhibitor E7449 in Ewing's sarcoma

Ewing9s sarcoma affects mostly adolescents and young adults with tumors that predominate in bone, characterized by the presence of fusion proteins created by chromosomal translocations (EWS-FLI1, EWS-ERG etc.). Recent studies demonstrated an interaction between EWS fusion proteins and PARP1; increased DNA damage and elevated PARP levels upon fusion protein expression as well as sensitivity to PARP inhibition have been reported. E7449, a potent, orally available PARP inhibitor was evaluated in various xenograft models of Ewing9s sarcoma as a single agent and in combination with TMZ or irinotecan, chemotherapies often used in relapsed patients. In an RD-ES (ATCC® HTB166™) Ewing9s sarcoma (EWS-FLI1) s.c. xenograft model, treatment with single agent E7449 or TMZ resulted in no antitumor activity. However, when combined exquisite synergy that resulted in tumor regression and tumor-free mice was observed, even at the low E7449 combination dose of 1 mg/kg. E7449 dose responsive re-growth of tumors was observed and 8/8 mice remained tumor-free in the highest dose group (30 mg/kg) at study termination. Enhanced toxicity as measured by body weight loss was observed in combination treatment groups but mice generally recovered well. Significant antitumor activity was observed for irinotecan alone in the RD-ES model; combination with E7449 enhanced that activity. Combining TMZ and irinotecan was also efficacious in the RD-ES model. Antitumor activity was further enhanced by the addition of E7449 which also potentiated combination toxicity. Studies are ongoing to optimize dosing of E7449 and chemotherapy and to identify a treatment schedule that maximizes activity and tolerability of the various combinations. Additionally, the activity of E7449 alone and in combination was evaluated in 2 Ewing9s sarcoma patient-derived xenograft (PDx) models. In one model (CTG-0143) neither TMZ nor E7449 as single agents had significant antitumor activity. However, the combination of E7449 + TMZ resulted in striking synergy and tumor regression. Although tumors were large when treatment started, significant antitumor activity was also observed with single agent irinotecan and combination treatment. The second model (CTG-0142) was sensitive to E7449 alone at high dose and to both chemotherapies (TMZ and irinotecan) as single agents. Addition of E7449 to either TMZ or irinotecan resulted in enhanced anti-tumor activity as measured by significantly delayed tumor re-growth following regression. Biomarker analysis is ongoing, including gene expression profiling and assessment of the DNA damage response in tumors from RD-ES and PDx models following drug treatments. In conclusion, the combination of E7449 with TMZ and irinotecan resulted in highly potent anti-cancer activity against cell line and PDx models of Ewing9s sarcoma. These data support the assessment of E7449 as a combination therapy for Ewing9s sarcoma in clinical trials. Citation Format: Sharon McGonigle, Zhihong Chen, Jingzang Tao Miu, Kuan-Chun Huang, Donna Kolber-Simonds, Nanding Zhao, Natalie C. Twine, Qiongfang Cao, Galina Kuznetsov, Shanqin Xu, Kenichi Nomoto. Antitumor activity of the PARP inhibitor E7449 in Ewing9s sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2014-2733

Abstract 899: Characterization and establishment of a drug-resistant human lung cancer model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction In 2012, lung cancer is expected to be the leading cause of cancer death in the United States, accounting for about 28% of all cancer deaths. Eribulin mesylate (Halaven®) is a microtubule-dynamics inhibitor approved for third line clinical use in patients with heavily pretreated metastatic breast cancer based upon statistically significant increase in median overall survival (OS) compared to treatment of physicians choice. Methods H522-T1 human non-small cell lung cancer subcutaneous xenograft tumors were established in nude mice that were subsequently treated with a sub-optimal dose of eribulin (half MTD). Two to three days after final drug administration, residual tumors were harvested and implanted again into naive nude mice. These tumors were treated again with the same regimen and the residual tumors were again collected and re-implanted into naive nude mice for another cycle of selection. A cell line named H522-T5er was established from the 4th cycle of residual tumor. Results In vitro, H522-T5er showed strikingly increased resistance to eribulin compared to the parental cell line (82-fold). In vivo, H522-T5er xenograft tumors also showed high resistance to eribulin dosed at MTD. Further flow cytometry analysis identified enrichment of a CD44+/CD24- subpopulation of H522-T5er cells, suggesting stem/progenitor cell properties of H522-T5er. Expression profiling analysis of parental tumors and drug-selected residual tumors, as well as H522-T1 and H522-T5er cells were conducted. A total of 71 genes with more than 2-fold changes were identified in the comparison between parental tumor and drug-selected residual tumors, while 187 genes showed more than 2-fold changes between H522-T1 and H522-T5er cells. Twelve genes were identified in both comparison and only 5 of them were changed in the same direction. Protein levels of several drug pumps reported to be frequently involved in drug resistance were also determined by Western blot analysis. No significant increase of Pgp (1.3-fold) was identified in H522-T5er cells. Both BCRP and LRP showed about a 2-fold increase in H522-T5er cells, while a slight decrease of MRP1 (20%) was observed in H522-T5er cells. Conclusions An eribulin-resistant cell line, not characterized by significant increase of Pgp expression, has been generated. It provides a valuable tool to develop and test agents which can target residual tumor after eribulin treatment. Citation Format: Kuan-Chun Huang, Judith Oestreicher, Natalie Twine, Winnie Lee, Xingfeng Bao, Sergei Agoulnik, Bruce A. Littlefield, Mary Woodall-Jappe, Kenichi Nomoto. Characterization and establishment of a drug-resistant human lung cancer model. [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 899. doi:10.1158/1538-7445.AM2013-899