Xingfeng Bao

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.

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.

Abstract 275: ER-886046, an antagonist of PGE2 receptor type-4, induces an effective antitumor immune response in mice by attenuating intratumoral MDSCs and TAMs

One of the hallmarks of an immunosuppressive tumor microenvironment is the presence of myeloid-derived suppressor cells (MDSCs) and type 2 tumor-associated macrophages (TAMs). These myeloid cells derive from immature monocytes and their differentiation is highly regulated by the engagement of prostaglandin E2 (PGE2) receptor type 4 (EP4) with PGE2 in the tumor microenvironment. To understand the importance of EP4 for tumor support, we implanted mouse tumors in EP4 inducible knockout mice and found that these tumors grew significantly more slowly than in wild type mice, indicating an important role of host cell EP4 signaling for tumor progression. Here we report the development and evaluation of ER-886046, a novel and specific EP4 antagonist, for cancer treatment. Daily oral administration of ER-886046 inhibited the growth of multiple mouse syngeneic tumor models with an inhibitory activity up to 100%. The anti-tumor activity of ER-886046 was T cell-dependent since it was not observed in T cell deficient mice. Furthermore, we found in vitro and in vivo mechanistic evidence that ER-886046 interferes with tumor-induced monocyte differentiation into immunosuppressive type 2 macrophages and MDSCs, instead supporting monocyte differentiation into APCs and facilitating intratumoral T cell accumulation. Importantly, ER-886046 has a desirable pharmacokinetic and metabolism profile in mice, rats and dogs which qualifies it as a good candidate for clinical studies in humans. Thus, the preclinical data support further investigation of targeting EP4 signaling by ER-886046 as a novel immune therapy for cancer treatment in clinical setting. Citation Format: Diana I. Albu, Zichun Wang, Jiayi Wu, Kuan-chun Huang, Wei Li, Diana Liu, Galina Kuznetsov, Qian Chen, Xingfeng Bao, Mary Woodall-Jappe. ER-886046, an antagonist of PGE2 receptor type-4, induces an effective antitumor immune response in mice by attenuating intratumoral MDSCs and TAMs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 275. doi:10.1158/1538-7445.AM2015-275

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 4586: Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models

T regulatory (Treg) cells play an important role in maintaining immunological tolerance to self-antigens, thus limiting immune responses to tumor antigens. Therefore, depleting or suppressing Tregs is one strategy by which anti-tumor immunity can be restored. The immunotoxin ONTAK® is an IL-2-diphteria toxin fusion protein that has been shown to diminish Tregs in patients and animal models of cancer in peripheral blood using a systemic intravenous (i.v.) administration route. E7777 is a new version of ONTAK®. In this study we tested the hypothesis that locally-diminished Tregs by intratumoral (i.t.) administration of E7777 generate effective anti-tumor immune response at both local and systemic levels. First, we showed superior anti-tumor activity and safety of E7777 i.t. over E7777 i.v., where i.t. administration resulted in complete tumor regressions in both moderately immunogenic CT26 and non-immunogenic B16F10 tumors with minimal animal body weight loss. In contrast, only tumor growth delay was observed for E7777 i.v. with dose-limiting animal body weight reduction in the same models. Immune phenotyping showed a 4 fold reduction of intratumoral Tregs in treated CT-26 tumors without significant change of Tregs in the spleens of treated animals, confirming a local Treg-depleting effect of E7777 i.t. In contrast, intratumoral CD8+ T cells were not reduced. Second, E7777 i.t. enhanced overall anti-tumor immune response, manifested by significantly increased numbers of CD45+ hematopoietic cells, Granzyme B+ CD8+ cytotoxic T cells, and ratios of cytotoxic T cells/Tregs in the treated tumors. Importantly, E7777 i.t. also resulted in distant effects in the spleen characterized by increased ratio of T lymphocytes to myeloid cells and increased frequencies of both effector memory CD8+ T cells (CD8+CD62L-CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+) indicative of systemic immune activation. Consistent with the generation of immunological memory, 60% of the tumor-free animals treated with E7777 i.t. rejected completely and 40% displayed delayed tumor growth of B16F10 cell challenge while all naive control animals grew tumors. Taken together, our results demonstrate that intratumoral Treg depletion by local administration of E7777 leads to an effective local and memory anti-tumor response in preclinical models and support further evaluation of local E7777 delivery as a cancer immunotherapy. Citation Format: Diana I. Albu, Christy Ingersoll, Kuan-Chun Huang, Mary Woodall-Jappe, Xingfeng Bao. Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4586. doi:10.1158/1538-7445.AM2017-4586

Abstract 4607: Specific inhibition of PGE2-EP4 signaling by E7046 promotes anti-tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity

Purpose: Immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of cancer patients; however, robust and safe combination therapies are still needed. In the tumor microenvironment, prostaglandin E2 receptor type 4 (EP4) signaling has been implicated in both protumoral myeloid cell differentiation and cytotoxic T cell exhaustion. We evaluated the combination of the EP4 antagonist E7046 (clinical trial NCT02540291) with anti-PD1 or anti-CTLA4 in preclinical tumor models, and also interrogated the relationship between PGE2 pathway activation and cancer patient survival. Materials/Methods: Mouse syngeneic tumor models CT-26 and 4T1 were used for pharmacological investigation. GMP grade E7046 was administered to tumor-bearing animals by oral gavage. Co-culture of EG7-OVA and OT1 cells in an antigen-specific cytotoxic T cell (CTL) activation assay provided mechanistic insights. For translational validation, transcripts of five major genes involved in PGE2 synthesis, transport and degradation were compared between malignant and normal tissues across all TCGA tumor types, and correlation of their expression with overall survival was assessed. Results: In the CT26 tumor model, the combination of E7046 and anti-PD1 resulted in significantly more tumor-free animals compared with either agent alone. In the 4T1 tumor model, the combination of E7046 and anti-CTLA4 was also more effective in suppressing tumor growth and tumor rejection compared with anti-CTLA4 alone, and was accompanied by a markedly increased accumulation of GZMB+CD8T+ CTLs in the treated tumors. Consistent with those findings, addition of anti-PD1 antibody promoted OVA-specific CTL activation in vitro while addition of PGE2 strongly inhibited it, as measured by IFNγ secretion. Inclusion of E7046 dose-dependently reversed the PGE2-induced suppressive activity in the presence of anti-PD1 antibody. Among major human PGE2 pathway genes, TCGA analysis showed that PTGES1 was upregulated and HPGD downregulated across a broad range of tumor types. In contrast, COX1, COX2 and PGT showed less difference between malignant and normal tissues. Importantly, these differences of one or multiple PGE2 pathway genes were strongly associated with patient survival in certain cancer types. Conclusions: A subset of human cancer types displays upregulated PGE2 pathway that is associated with a poorer prognosis. PGE2-EP4 signaling potently suppresses antigen-specific CTL activation in the presence of PD1 signaling blockade. The combination of EP4 antagonist E7046 with either anti-PD1 or anti-CTLA4 demonstrated superior anti-tumor activity compared with anti-PD1 or anti-CTLA4 alone. This increased activity was accompanied by increased CTL activation. Citation Format: Diana I. Albu, David Verbel, Yuan Huang, Donna Kolber-Simonds, Zichun Wang, Xulong Wang, Zoltan Dezso, Christy Ingersoll, Kuan-Chun Huang, Janna Hutz, Mary Woodall-Jappe, Xingfeng Bao. Specific inhibition of PGE2-EP4 signaling by E7046 promotes anti-tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2017-4607

Abstract B034: Preclinical immune antitumor activity of myeloid-targeting E7046 and Treg depleting E7777

Purpose: To assess the combined activity of E7046 and E7777 against multiple murine syngeneic tumor models. Background: Both regulatory T cells (Tregs)and myeloid cells are significant constituents in the tumor microenvironment in many types of cancer, where they help to maintain an immunosuppressive milieu that inhibits cytotoxic T cell function, which favors tumor persistence. Prostaglandin E2 (PGE2) engages the EP4 receptor on monocytes to direct myeloid cell activities away from antigen presentation and toward immunosuppression (wound healing). PGE2-driven myeloid responses have been reported to enhance Treg immunosuppressive activity, while Tregs with high expression of the FoxP3 transcription factor have been reported to up-regulate cyclooxygenase expression and PGE2 secretion. E7046 is a highly selective small molecule that potently competes with PGE2 binding to EP4. Single agent E7046 slows the growth of multiple syngeneic murine tumor types, in a mechanism dependent upon the presence of T cells. Denileukin diftitox (ONTAK®), an IL-2/diphtheria toxin fusion protein, was originally approved by the US FDA for treating patients with relapsed/refractory cutaneous T cell lymphoma. E7777 is a new version of denileukin diftitox developed using an improved manufacturing process. We and others have shown that denileukin diftitox selectively depletes Tregs. Neither E7046 nor E7777 affects tumor cell viability in vitro. Here we assessed their combined activity against syngeneic tumor models and the mechanisms driving their activity. Methods: Tumors were implanted subcutaneously in groups of 5-10 female BALB/c mice. When tumors were > 50 mm 3 , mice were treated with E7046 (150 mg/kg, QD x 21, p.o.) and/or E7777 (2.5 or 3 μg/head, Q7D x 2 or 3, i.v). Tumors, spleens, and tumor-draining lymph nodes were excised and analyzed by flow cytometry for immune cell composition and function. Results: As single agents, both E7046 and E7777 delayed the growth of established tumors. When combined, their anti-tumor activities were significantly enhanced, with up to 20% of animals rendered stably tumor-free. Activity was significantly reduced if animals were treated with antibody to deplete CD8 + T cells. In the tumors, the combination treatment dramatically increased the ratio of CD8 T cells to CD4 + CD25 + Foxp3 + Tregs, and also decreased the frequency of immunosuppressive myeloid cells. Importantly, the CD8 + T cells in the treated tumors, but not in the control tumors, were found to express granzyme B. Spleens from the treated animals also showed an increased ratio of CD8 + T cells vs the highly active CD4 + CD25 + Foxp3 + ICOS + Tregs. Conclusions: The combination of E7046 and E7777 showed promising preclinical anti-tumor activity via an immune-mediated mechanism. Citation Format: Diana I. Albu, Kuan-Chun Huang, Jiayi Wu, Xingfeng Bao, Kenichi Nomoto, Mary Woodall-Jappe. Preclinical immune antitumor activity of myeloid-targeting E7046 and Treg depleting E7777. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B034.

Abstract B198: Pharmacological profile of the PGE2 EP4 receptor antagonist E7046

E7046 is a potent and selective small molecule antagonist of the type 4 prostaglandin E2 (PGE2) receptor EP4. The discovery and evaluation of this novel EP4 antagonist for cancer treatment was recently presented at the AACR 2015 annual conference (abstract # 275). Here we show new evidence of E7046 in vivo activity against a series of syngeneic tumor models as well as its pharmacological effect on APCMin/+ mice, which possess a mutation in the adenomatous polyposis coli (APC) tumor suppressor gene. We found that daily oral administration of E7046 was able to both slow down the growth of established subcutaneous tumors and significantly delay the recurrence of tumors after surgical resection. These activities of E7046 correlated with differences in the immune cell composition of the microenvironments of each type of tumor. In this context, cyclooxygenase 2 - positive (COX-2+) tumors that were rich in myeloid cells showed an enhanced response to E7046 regardless of their T lymphocyte infiltration status. In addition, E7046 was superior to the COX-2 inhibitor celecoxib against mutant APC-driven neoplastic polyp formation in the intestines of APCMin/+ mice exposed to dextran sodium sulfate. Using the APCMin/+ mouse model, we found that E7046 significantly reduced the combined colon polyp area and the size of individual polyps without influencing the total polyp number. For each of these parameters, E7046 activity against colon and small intestine tumors was greater than that of a comparable dose of celecoxib. Quantification of cyclin D1 staining in colon and small intestine polyps further indicated that both compounds led to a significant reduction in the proliferation of tumor cells, with a greater effect for E7046 compared to celecoxib. Overall, these preclinical results suggest that this agent should be further investigated in patients with COX-2+ tumors infiltrated with myeloid cells, including patients with APC-mutated colon cancer. First-In-Human study of E7046 is currently enrolling patients (IND 125272). Citation Format: Diana I. Albu, Jiayi Wu, Kuan-chung Huang, Renee Wright-Michaud, Shanqin Xu, Galina Kuznetsov, Xingfeng Bao, Mary Woodall-Jappe. Pharmacological profile of the PGE2 EP4 receptor antagonist E7046. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B198.

Abstract 2788: Antiproliferative schweinfurthins reveal a role of the trans-Golgi-network in mTOR-AKT signaling and cancer cell survival

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The schweinfurthin class of natural compounds showed selective and potent anti-proliferative activities in the NCI-60 cancer cell panel1,2). Recent work by Burgett et al3) suggested that schweinfurthins may bind to oxysterol-binding protein-related proteins (ORPs) for their anti-proliferative activity. However, the mechanism of the selectivity and potency is still unknown. Here we show that schweinfurthins preferentially inhibited cell proliferation of PTEN-deficient cancer cells across different cancer types, and specifically inhibited AKT phosphorylation in both PTEN wild-type and mutant cancer cells. Intracellularly, a fluorescent schweinfurthin probe localized within the endosome/lysosome structure. Treatment of cells with active schweinfurthins caused fragmentation of the trans-Golgi-network (TGN), a vesicular trafficking system, in a dose- and time-dependent manner, without impacting the cis, med or trans-Golgi matrix structures. An inactive trimethylated analog of schweinfurthins did not display this activity. Using biotinylated probes, we found that schweinfurthins bind to the sterol-binding domain of ORPs; the capacity of binding to ORPs was positively correlated with both TGN fragmentation activity and anti-proliferative activity. These results are consistent with the recent observation that knockdown of specific ORP proteins leads to TGN trafficking disruption4). The fragmentation of TGN by schweinfurthins resulted in specific inhibition of mTOR-AKT pathway signaling in two ways: 1) disruption of lipid raft organization at the plasma membrane, evidenced by diminished trafficking of ceramide, GM1 and sphingomyelin, which inhibited PDK1 phosphorylation; and 2) dislocation of mTOR from its active sites at endosomes and/or lysosomes. Furthermore, we found that PTEN-deficient DLBCL cancer cells were highly sensitive to schweinfurthin treatment in vitro (IC50 values < 10 nM). Normal human fibroblasts were 1000 times less sensitive than the DLBCL cells. Taken together, our results disclosed an important role of the TGN, which was specifically targeted by schweinfurthins, in mTOR-AKT signaling and in cancer cell survival, suggesting that analogs of schweinfurthin might offer promising novel therapeutics for treating mTOR-AKT signaling-addicted cancer cells such as PTEN-deficient DLBCL. 1. Beutler JA, et al. Cytotoxic geranyl stilbenes from Macaranga schweinfurthii. J Nat Prod., 1998, 61:1509-1512. 2. Yoder BJ, et al. Antiproliferative prenylated stilbenes and flavonoids from Macaranga alnifolia from the Madagascar rainforest. J Nat Prod., 2007, 70:342-346. 3. Burgett AW, et al. Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nat Chem Biol., 2011, 7:639-647. 4. Du X, et al. A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J Cell Biol., 2011, 192:121-135. Citation Format: Xingfeng Bao, Naoko Hata, Kishan Agarwala, Zichun Wang, Winnie Lee, Lana Parent, Hongsheng Cheng, Dayong Qiu, Yongchun Shen, Bingfan Du, Wanjun Zheng, Nanding Zhao, Maarten Postema, Mary Woodall Jappe, Yasutaka Takase, Toshimitsu Uenaka, Kenichi Nomoto. Antiproliferative schweinfurthins reveal a role of the trans-Golgi-network in mTOR-AKT signaling and cancer cell survival. [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 2788. doi:10.1158/1538-7445.AM2014-2788

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