Preclinical Xenograft Models Research Articles

Abstract 3575: Ferroptosis: A novel nonapoptotic cell death overcomes head and neck cancer resistance to cisplatin

Abstract Ferroptosis was recently identified as a novel form of iron-dependent cell death different from apoptosis or necrosis. Inhibition of key molecules related to ferroptosis, cystine-glutamate antiporter (xCT) and glutathione peroxidase (GPX4), may induce the eradication of cancer cells resistant to conventional chemotherapy or radiotherapy. The present study investigated whether ferroptosis overcome head and neck cancer (HNC) resistant to cisplatin treatment. Three cisplatin-resistant HNC cell lines (AMC-HN3R, -HN4R and -HN9R), their parental lines, and other human HNC lines were used. The effects of cysteine and glutamate alteration and pharmacological and genetic inhibition of xCT were assess by measuring viability, death, total and cytoplasmic lipid reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), protein expression, and preclinical mouse tumor xenograft models. Conditioned media with no cysteine or high glutamate supplement induced ferroptosis of both cisplatin-sensitive and -resistant HNC cells without apparent change of necrosis and apoptosis markers, RIP1 and RIP3 and casplase-3 expression, and ΔΨm. This was blocked by antioxidants trolox or N-acetyl cysteine pretreatment. Sulfasalazine, artensunate, and erastin inhibited the growth of HNC cells and accumulated cytoplasmic lipid ROS via induction of ferroptosis and inhibition of xCT protein expression. Genetic silencing of xCT with siRNA or shRNA treatment also induced the effective cell death (ferroptosis) of resistant HNC cells. These effects were blocked with trolox or ferrostatin-1 pretreatment. Pharmacological and genetic inhibition of xCT significantly sensitized resistant HNC cells to cisplatin in vitro and in vivo. Cystine deficiency or glutamate excess induces ferroptosis in HNC cells. Further, pharmacological and genetic inhibition of xCT overcomes the cisplatin resistance of HNC cells by inducing ferroptosis. Citation Format: Hye Jin Jang, Jin Young Park, Eun Hye Kim, Ji Won Kim, Minsu Kwon, Jong-Lyel Roh. Ferroptosis: A novel nonapoptotic cell death overcomes head and neck cancer resistance to cisplatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3575.

Abstract 3264: Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models

Abstract Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; the platelet-derived growth factor (PDGF) receptor PDGFRα; KIT; and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis, and is currently marketed for the treatment of patients with radioactive iodine-refractory differentiated thyroid cancer. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in Phase 2 study. The aim of this study is to elucidate the activity of the combination of lenvatinib and everolimus in preclinical human RCC xenograft models. We examined antitumor activity in two human RCC (A-498 and Caki-1) xenograft models orally treated with lenvatinib (10 mg/kg), everolimus (30 mg/kg), and the combination of lenvatinib and everolimus for 1 or 2 weeks. The antitumor proliferation and antiangiogenic effects were evaluated by immunohistochemistry (IHC) using anti Ki67 antibody and anti CD31 antibody, respectively. The induction of apoptosis was detected by TUNEL assay. To analyze the gene expression profile of tumor samples, microarray analysis were also conducted. The antitumor activity of the combination of lenvatinib and everolimus was greater than that of either agent administered alone in A-498 and Caki-1 xenograft models. The combination caused tumor regression and had no remarkable body weight loss. IHC analysis revealed decrease of microvessel density in lenvatinib and combination groups, and also decrease in the proportion of proliferative cells in everolimus treated and combination-treated group in A-498 model. In TUNEL assay, significant induction of apoptosis was observed only in the combination-treatment group. The analysis of gene expression profile in A-498 xenograft tumors also supported these results: lenvatinib alone upregulated hypoxia-related genes and everolimus decreased proliferation-related genes. The combination of these 2 drugs induced blends of the gene expression changes caused by each single treatment. Our results indicate that treatment of lenvatinib in combination with everolimus caused significant antitumor effect by combining the potent antiangiogenic activity of lenvatinib as well as direct antitumor activity of everolimus in A-498 model. These preclinical results provide one of the mechanisms to support the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus. Citation Format: Yusuke Adachi, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Kiyochi Okamoto, Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Yasuhiro Funahashi, Junji Matsui. Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3264.

Abstract 2966: Preclinical combinations of the antibody-drug conjugate SGN-LIV1A with chemotherapies show increased activity

Abstract SGN-LIV1A is an antibody-drug conjugate (ADC) currently being evaluated in a phase 1 clinical trial for metastatic breast cancer. SGN-LIV1A consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), conjugated to the anti-LIV-1 humanized monoclonal antibody hLIV22. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. We have previously shown that as a single agent, SGN-LIV1A displays target specific internalization and cytotoxic activity against a breast cancer cell line in vitro and also demonstrates antitumor activity in in vivo preclinical xenograft models with significant delay of tumor growth. We report here additive and synergistic effects when combining SGN-LIV1A with current chemotherapeutic modalities used in the treatment of metastatic breast cancer. Specifically, we show synergy between SGN-LIV1A in combination with either doxorubicin or Abraxane in MCF-7 breast cancer tumor model. In addition, we show additive effects when carboplatin or protein kinase inhibitors are dosed in combination with SGN-LIV1A in this tumor model. These findings support further evaluation and development of SGN-LIV1A in combination with standard of care chemotherapeutic agents for the treatment of breast cancer. Citation Format: Fu Li, Martha Anderson, Joshua Hunter, Jaime Miyamoto, Michelle Ulrich, Ana Kostic, Che-Leung Law, Django Sussman. Preclinical combinations of the antibody-drug conjugate SGN-LIV1A with chemotherapies show increased activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2966.

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

Antibody-Targeted Chemotherapy for the Treatment of Melanoma.

Antibody-directed chemotherapy (ADC) offers an advantage over conventional chemotherapy because it provides antibody-directed targeting, with resultant improvement in therapeutic efficacy and reduced toxicity. Despite extensive research, with notable exceptions, broad clinical application of ADC remains elusive; major hurdles include the instability of antibody-chemotherapy linkers and reduced tumor toxicity of the chemotherapy when bound to the antibody. To address these challenges, we have developed a platform technology that utilizes the nab-paclitaxel formulation of paclitaxel, Abraxane, in which hydrophobic paclitaxel is suspended in 130-nm albumin nanoparticles and thus made water-soluble. We have developed a method to noncovalently coat the Abraxane nanoparticle with recombinant mAbs (anti-VEGF, bevacizumab) and guide Abraxane delivery into tumors in a preclinical model of human A375 melanoma. Here, we define the binding characteristics of bevacizumab and Abraxane, demonstrate that the chemotherapy agent retains its cytotoxic effect, while the antibody maintains the ability to bind its ligand when the two are present in a single nanoparticle (AB160), and show that the nanoparticle yields improved antitumor efficacy in a preclinical human melanoma xenograft model. Further data suggest that numerous therapeutic monoclonal IgG1 antibodies may be utilized in this platform, which has implications for many solid and hematologic malignancies. Cancer Res; 76(13); 3954-64. ©2016 AACR.

Identifying novel therapeutic agents using xenograft models of pediatric cancer.

In the USA, the overall cure rate for all childhood cancers is seventy percent, and in many patients that ultimately fail curative therapy, initial responses to current multimodality treatments (surgery, radiation therapy and chemotherapy) is good, with overall 5-year event-free survival approaching 80%. However, current approaches to curative therapy result in significant morbidity and long-term sequelae, including cardiac dysfunction and cognitive impairment. Furthermore, dose-intensive chemotherapy with conventional agents has not significantly improved outcomes for patients that present with advanced or metastatic disease. Classical cytotoxic agents remain the backbone for curative therapy of both hematologic and solid tumors of childhood. While 'molecularly' targeted agents have shown some clinical activity, responses are often modest and of short duration; hence, there is a need to identify new classes of cytotoxic agent that are effective in patients at relapse and that have reduced or different toxicity profiles to normal tissues. Here we review the pediatric preclinical testing program experience of testing novel agents, and the value and limitations of preclinical xenograft models and genetically engineered mouse models for developing novel agents for treatment of childhood cancer.

A Supplemented High-Fat Low-Carbohydrate Diet for the Treatment of Glioblastoma

Dysregulated energetics coupled with uncontrolled proliferation has become a hallmark of cancer, leading to increased interest in metabolic therapies. Glioblastoma (GB) is highly malignant, very metabolically active, and typically resistant to current therapies. Dietary treatment options based on glucose deprivation have been explored using a restrictive ketogenic diet (KD), with positive anticancer reports. However, negative side effects and a lack of palatability make the KD difficult to implement in an adult population. Hence, we developed a less stringent, supplemented high-fat low-carbohydrate (sHFLC) diet that mimics the metabolic and antitumor effects of the KD, maintains a stable nutritional profile, and presents an alternative clinical option for diverse patient populations. The dietary paradigm was tested in vitro and in vivo, utilizing multiple patient-derived gliomasphere lines. Cellular proliferation, clonogenic frequency, and tumor stem cell population effects were determined in vitro using the neurosphere assay (NSA). Antitumor efficacy was tested in vivo in preclinical xenograft models and mechanistic regulation via the mTOR pathway was explored. Reducing glucose in vitro to physiologic levels, coupled with ketone supplementation, inhibits proliferation of GB cells and reduces tumor stem cell expansion. In vivo, while maintaining animal health, the sHFLC diet significantly reduces the growth of tumor cells in a subcutaneous model of tumor progression and increases survival in an orthotopic xenograft model. Dietary-mediated anticancer effects correlate with the reduction of mTOR effector expression. We demonstrate that the sHFLC diet is a viable treatment alternative to the KD, and should be considered for clinical testing. Clin Cancer Res; 22(10); 2482-95. ©2015 AACR.

A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer.

Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro, and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo. In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo, including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.

Mesothelin's minimal MUC16 binding moiety converts TR3 into a potent cancer therapeutic via hierarchical binding events at the plasma membrane.

TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies.

Abstract B19: Inactivation of KLF4 in T cell acute lymphoblastic leukemia promotes the expansion of leukemia cells by activating the Map2k7/Jnk pathway.

Abstract Acute lymphoblastic leukemia is the most common hematological malignancy in pediatric patients, and disease relapse is the leading cause of cancer-associated death in children. Despite steadily improved outcomes in patients with newly diagnosed disease, little progress has been made to treat relapse leukemia and to reduce the incidence of relapse by increasing the cure rates of frontline therapy. Targeted therapy is currently not available for T-ALL and the development of novel agents requires a better understanding of how leukemia-initiating cells (LIC) are maintained. In contrast to normal blood cells, we found that the transcription factor KLF4 is significantly downregulated in pediatric T-ALL. Next-generation bisulfite sequencing revealed that the KLF4 promoter is hypermethylated in lymphoblasts from T-ALL patients but not in normal bone marrow cells, T cells, and pediatric B-ALL. Deletion of the Klf4-floxed gene with Vav-iCre transgene and transplantation of Klf4Δ/Δ bone marrow cells transduced with the Notch1-L1601P-ΔP mutant resulted in a significant acceleration of T-ALL due to a 9-fold expansion of LIC and increased G1-S transition in leukemic T cells. A combined analysis of global gene expression and genome-wide binding revealed that KLF4 directly represses the dual specificity mitogen-activated protein kinase 7 (Map2k7) and thus Klf4Δ/Δ T-ALL mice exhibit increased Map2k7 expression. Most remarkably, T-ALL cells from both mouse model and pediatric patients showed elevated levels of total and phosphorylated Map2k7 and subsequent activation of downstream targets JNK, c-Jun, and ATF2. Finally, we tested several JNK inhibitors in T-ALL cell lines, primary patient samples, and pre-clinical xenograft models of T-ALL and confirmed that JNK inhibition blocks ATF2 activation and controls expansion of leukemia cells, validating the Map2k7/Jnk pathway as a selective target for T-ALL therapy. Thus, KLF4 emerges as a novel tumor suppressor in T-ALL and Jnk inhibition as a novel therapeutic approach for adjunctive therapy in T-ALL patients with refractory or relapse disease. Citation Format: Ye Shen, Koramit Suppipat, Chun Shik Park, Toni-Ann Mistretta, Terzah Horton, Karen Rabin, Daniel Lacorazza. Inactivation of KLF4 in T cell acute lymphoblastic leukemia promotes the expansion of leukemia cells by activating the Map2k7/Jnk pathway.. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B19.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Comparison of hypoxia-activated prodrug evofosfamide (TH-302) and ifosfamide in preclinical non-small cell lung cancer models

ABSTRACTEvofosfamide (TH-302) is a hypoxia-activated prodrug of the cytotoxin bromo-isophosphoramide. In hypoxic conditions Br-IPM is released and alkylates DNA. Ifosfamide is a chloro-isophosphoramide prodrug activated by hepatic Cytochrome P450 enzymes. Both compounds are used for the treatment of cancer. Ifosfamide has been approved by the FDA while evofosfamide is currently in the late stage of clinical development. The purpose of this study is to compare efficacy and safety profile of evofosfamide and ifosfamide in preclinical non-small cell lung cancer H460 xenograft models. Immunocompetent CD-1 mice and H460 tumor-bearing immunocompromised nude mice were used to investigate the safety profile. The efficacy of evofosfamide or ifosfamide, alone, and in combination with docetaxel or sunitinib was compared in ectopic and intrapleural othortopic H460 xenograft models in animals exposed to ambient air or different oxygen concentration breathing conditions. At an equal body weight loss level, evofosfamide showed greater or comparable efficacy in both ectopic and orthotopic H460 xenograft models. Evofosfamide, but not ifosfamide, exhibited controlled oxygen concentration breathing condition-dependent antitumor activity. However, at an equal body weight loss level, ifosfamide yielded severe hematologic toxicity when compared to evofosfamide, both in monotherapy and in combination with docetaxel. At an equal hematoxicity level, evofosfamide showed superior antitumor activity. These results indicate that evofosfamide shows superior or comparable efficacy and a favorable safety profile when compared to ifosfamide in preclinical human lung carcinoma models. This finding is consistent with multiple clinical trials of evofosfamide as a single agent, or in combination therapy, which demonstrated both anti-tumor activity and safety profile without severe myelosuppression.

TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun.

Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown promising clinical efficacy in non-squamous non-small cell lung cancer (NSCLC); however, resistance is frequently observed in malignant cells, operating through a mechanism that remains largely unknown. The present study shows that T-lymphokine-activated killer cell-originated protein kinase (TOPK) is upregulated in NSCLC and excessively activated in TKI-refractory cells. TOPK dictates the responsiveness of lung cancers to the EGFR-targeted TKI gefitinib through the transcription factor AP-1 component c-Jun. TOPK binds directly to and phosphorylates c-Jun, which consequently activates the transcription of AP-1 target genes, including CCND1 and CDC2. TOPK silencing sensitizes EGFR-TKI-resistant lung cancer cells to gefitinib and increases gefitinib efficacy in preclinical lung adenocarcinoma xenograft models. These findings represent a novel mechanism of lung cancer resistance to TKIs and suggest that TOPK may have value both as a predictive biomarker and as a therapeutic target: TOPK-targeted therapy may synergize with EGFR-targeted therapy in lung cancers.

Preclinical Study for the Use of Abatacept to Prevent Rejection of Allogeneic CD34+ Cells in a Xenograft Model

The aims of this study were to analyze the role of T cells on the engraftment of allogeneic CD34+ cells after transplantation in immunodeficient nonobese diabetic/ltsz-scid/scid (NOD/SCID) IL2 receptor gamma chain knockout (NSG) mice and to test the in-vivo ability of abatacept (CTLA4-Ig) in preventing graft failure. Human CD34+ cells (0.2x106 cells/animal) were co- transplanted with allogeneic CD3+ T cells into sublethally irradiated NSG mice at ratios ranging from 1:50 to 1:0.5, or without T cells as a control. The engraftment of huCD45+ cell subsets in the bone marrow and spleen was measured by flow cytometry after 4-8 weeks. An expansion of T cells without engraftment of CD34+ cells was detected in each group of mice transplanted with CD34:T cells at ratios ≥ 1:0.5. To test whether T cells prevented any engraftment of CD34+ cells, or caused rejection after initial CD34+ cell homing in the bone marrow, kinetics experiments were performed by analyzing the marrow and spleen of mice at 1,2 or 4 weeks after transplant of CD34+ and T cells at 1:1 ratio. These experiments showed that at two weeks after transplant, CD34+ cells had repopulated the bone marrow but not the spleen, while T cells were found primarily in the spleen. Instead, in mice sacrificed at 4 weeks after transplant the marrow and the spleen contained only T cells and the CD34+ cells had been rejected. Based on our previous in-vitro studies showing that CD34+ cell immunogenicity is mainly dependent on B7:CD28 costimulatory signaling, we then tested whether costimulatory blockade with abatacept (CTLA4-Ig, Bristol Myers Squibb) would block stem cell rejection. Three groups of mice were transplanted with CD34+ and allo-T cells at 1:1 ratio and injected with Abatacept at 250 ug i.p. every other day from: a) day -1 to +28, b) day -1 to day +14 or c) day +14 to +28, then the animals were sacrificed at day +56 (8 weeks) after transplant to assess the engraftment. In Group a) the overall engraftment of huCD45+ cells was only <10%, but Abatacept completely prevented T cell-mediated stem cell rejection with >98% huCD45+ cells of myeloid or B cell lineage and <1% T cells in the marrow and spleen. In Group b) 70% of huCD45+ cells both in the marrow and spleen were T cells, and the remaining fraction of myeloid or B cells were derived from CD34+ cells. In Group c), instead, 100% of huCD45+ cells were T cells, with complete rejection of CD34+ cells. T cells recovered from the spleen of mice in groups b) and c) were also tested as responders in MLC stimulated with the original CD34+ cells and showed a brisk proliferation, consistent with lack of tolerance. Finally, another group of mice that received Abatacept from day -1 to + 28 was rechallenged with a boost of CD34+ cells on day +28 to test whether the low CD34+ cell engraftment was secondary to a latent rejection or partial stem cell exhaustion. The CD34+ cell boost resulted in a full hematopoietic recovery with 37% huCD45+CD3- cells, including myeloid and B cells, as well as CD34+ cells in the bone marrow and spleen.In this preclinical xenograft model we demonstrated that costimulatory blockade with Abatacept at the time of allogeneic transplant of human CD34+ cells can prevent T cell mediated rejection provide the basis for the future non-myeloablative protocols for incompatible stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Abstract A85: Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors

Abstract Background: AMG 211 is a novel, bispecific single-chain antibody of the bispecific T-cell engager (BiTE® antibody construct) class. AMG 211 targets carcinoembryonic antigen (CEA) on tumor cells and the CD3 epsilon subunit of the human T-cell receptor complex on T-cells. CEA is a glycosylated human oncofetal antigen and is abundantly expressed by a variety of tumors; especially those in the gastrointestinal tract. We radiolabeled AMG 211 with zirconium-89 (89Zr) to study the tumor targeting and tissue distribution of AMG 211 in preclinical mouse tumor xenograft models. Material and Methods: AMG 211 was conjugated with desferal for 89Zr labeling and quality control was performed. A tracer amount of 10 μg (5 MBq) 89Zr-AMG 211 was administered to mice bearing subcutaneously-implanted CEA-expressing LS174T human colorectal adenocarcinoma. MicroPET imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after tracer injection. A dose-escalation biodistribution study was performed 6 h after injection of 2, 10, 50, 100 or 500 μg 89Zr-AMG 211. 89Zr-AMG 211 was also evaluated in a CEA-negative HL60 promyelocytic leukemia xenograft. Furthermore, the non-CEA-binding BiTE® antibody construct, MEC 14, served as a negative control in LS174T tumors. Ex vivo gel electrophoresis and autoradiography was used to determine 89Zr-AMG 211 integrity in plasma and tumor lysate. Results: In vitro analysis of desferal-conjugated AMG 211 showed similar CEA binding as unlabeled AMG 211. Radiochemical purity of 89Zr-AMG 211 exceeded 95%, with a maximum specific activity of 500 MBq/mg. Size-exclusion high-performance liquid chromatography showed no degradation products and less than 5% of aggregates. MicroPET imaging revealed time-dependent tumor uptake of 89Zr-AMG 211 in LS174T bearing mice with the highest uptake 3 h after injection and a prolonged imageable tumor retention up to 48 h. 89Zr-AMG 211 rapidly decreased in blood (T1/2 = 1 h). Dose-escalation showed a dose-dependent tumor uptake 6 hours after injection with the highest tumor uptake observed with 2 μg (7.5 ± 1.5%ID/g) and the lowest tumor uptake with 500 μg (3.9 ± 0.1%ID/g). Mice bearing CEA-positive LS174T versus CEA-negative HL60 tumors showed higher tumor uptake (6.0 ± 1.3 vs 0.5 ± 0.1%ID/g) and tumor-to-blood ratios (21.0 ± 4.0 vs 1.6 ± 0.2) after injection of 10 μg 89Zr-AMG 211. In addition, the non-specific 89Zr-MEC 14 showed low tumor (0.5 ± 0.2%ID/g) uptake and tumor-to-blood ratio (5.5 ± 2.1) that is similar to tissue background in the LS174T model. Ex vivo autoradiography showed intact 89Zr-AMG 211 in all plasma samples. Moreover, in line with biodistribution data, intact 89Zr-AMG 211 was present only in the LS174T xenograft lysates and not detected in HL60 lysate. 89Zr-MEC14 did not bind CEA and was not detected in LS174T lysates. Conclusions: This study showed dose-dependent CEA-specific targeting of 89Zr-AMG 211. In addition, intact labeled AMG 211 was present in plasma and LS174T tumor lysates indicating in vivo tracer integrity. Our data support the use of 89Zr-ImmunoPET in the clinical setting to assess the distribution and tumor-targeting properties of AMG 211 and BiTE® antibody constructs. Citation Format: S.J.H. Waaijer, F.J. Warnders, M.N. Lub-de Hooge, S. Stienen, M. Friedrich, A. Sternjak, P.C. Pieslor, K. Cheung, A.G.T. Terwisscha van Scheltinga, C.P. Schröder, E.G.E. de Vries. Preclinical evaluation of the radiolabeled bispecific T-cell engager 89Zr-AMG 211 targeting CEA-positive tumors. [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 A85.

Abstract B6: Advanced image analysis of H3K27 trimethylation in skin from subjects dosed with the EZH2 inhibitor tazemetostat

Abstract Tazemetostat (EPZ-6438 or E7438) is a selective small molecule inhibitor of the enhancer of zeste homolog 2 (EZH2) histone methyltransferase (HMT). EZH2 is the catalytic subunit of the multi-protein HMT complex known as polycomb repressive complex 2 (PRC2), which is responsible for mono-, di-, and trimethylation (me3) of histone H3 lysine 27 (H3K27). An exploratory objective for the phase 1 study of tazemetostat in subjects with advanced solid tumors or B-cell lymphomas was to explore the pharmacokinetic: pharmacodynamic (PK: PD) relationships such as correlation of H3K27me3 inhibition in patient tissue with systemic drug exposure. Pre-clinical xenograft models of EZH2 inhibition have previously shown tumor regression correlating with dose-dependent H3K27me3 decreases in both tumor tissue and skin epidermis (Knutson 2014). H3K27me3 reduction in the epidermis was calculated as a change in percentage of H3K27me3 positive cell employing an immunohistochemistry (IHC) assay with subsequent image analysis. These studies and the low feasibility of obtaining matched pre- and post-treatment tumor biopsies, led to the selection of skin as a surrogate tissue for evaluation in the phase 1 study at screening and 28 days post dose and the use of IHC as a PD biomarker of target inhibition. Examination of phase 1 skin biopsies exhibited dose and exposure-dependent decreases in the percentage of H3K27me3 positive cells, however, the magnitude of reduction (60%) was less than observed in pre-clinical models (80%). Further investigation of the IHC images revealed a difference of H3K27me3 levels in distinct layers or strata of the skin. The stratum spinosum exhibited a distinct dose and exposure-dependent loss of H3K27me3 staining while the stratum basale showed a concomitant minimal loss of H3K27me3. These observations gave rise to the possibility of a stratum-specific PD response in skin to tazemetostat exposure. To fully evaluate this notion, a more sophisticated, unbiased, image analysis algorithm was employed to measure H3K27me3 levels across the strata of the epidermis. The image analysis algorithm was designed to identify three distinct regions: the full thickness epidermis, stratum basale to stratum corneum; the stratum basale alone; and the stratum spinosum. Iterative rounds of optimization were performed to identify the basale layer while excluding follicles and glands. Because skin has inherent challenges related to the chromogenic similarity between DAB and melanin, careful attention was paid to the negative control to ensure that endogenous melanin was not included in the analysis. Segregation of the stratum basale layer from the remainder of the epidermis led to a refined H3K27me3 analysis and resulted in an improved correlation of H3K27me3 inhibition with drug exposure. This analysis highlights that the zonal layers of the epidermis may require separate evaluation when analyzing skin as a surrogate tissue for proof of mechanism studies. Although more accessible for repeat biopsy, the use of skin as a surrogate tissue does not preclude the need to understand the PD effects in the target tumor tissue. To that end, optional paired tumor biopsies are being collected in the Phase 2 tazemetostat trial and will be evaluated for PD endpoints including H3K27me3 in addition to other biomarkers. Citation Format: Alice A. McDonald, Galina Kuznetsov, Namita Kumar, Amy Weaver, Trevor Do, Jeff Dzubay, Heike Keilhack, Nigel waters, Nathalie Rioux, Stephen J. Blakemore. Advanced image analysis of H3K27 trimethylation in skin from subjects dosed with the EZH2 inhibitor tazemetostat. [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 B6.

Abstract LB-B18: Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy

Abstract Epithelial-mesenchymal transition (EMT) is a dynamic process whereby epithelial cells acquire mesenchymal properties. Despite the clinical significance of the acquired mesenchymal properties for metastasis and drug resistance, histopathological evidence of transitional cells in patient samples is lacking and EMT remains an unproven clinical hypothesis. We previously developed and validated a multiplex immunofluorescence assay (IFA) that quantifies the levels of EMT biomarkers (E-Cadherin, Vimentin, β-catenin) in snap-frozen, formalin-fixed, paraffin-embedded (FFPE) tumor tissue (Navas et al, AACR 2013). Building upon that method, we now report a precise, quantitative and unbiased IFA method of tissue analysis (EMT-IFA) using Definiens® software to quantify co-expression of the epithelial marker E-cadherin (E) and mesenchymal marker Vimentin (V) at the cellular level in FFPE clinical biopsies. FFPE human tumor xenografts and cell lines serve as calibrators and reference materials for establishing initial image acquisition parameters for segmented tumor regions of interest. Flanking H&amp;E sections of the clinical biopsies are initially annotated by an anatomic pathologist to identify regions of tumor tissue, non-tumor related areas (including normal or stromal tissues), inflammatory and/or necrotic regions, and freeze artifacts. Adjacent sections are used for the EMT-IFA, and further tumor segmentation from stroma is applied to extracted regions of interests (fields) using the β-catenin layer. Within segmented tumor regions, E-cadherin (E) and Vimentin (V) sub-cellular pixel areas are measured using predetermined thresholds for each biomarker and reported as Log10 (V:E). High resolution images confirmed the co-localization of plasma membrane E-cadherin and cytoplasmic Vimentin to individual transitional tumor cells, and the co-localization of E and V within individual tumor cells of the segmented regions of interest was key for distinguishing EMT from tumor heterogeneity in which adjacent tumor cells can be exclusively E+ or V+. Quantitative analysis of a clinical biopsy series of various histologies revealed all possible phenotypes: epithelial (E+V- colorectal carcinoma), mesenchymal (E-V+ sarcomas), heterogeneous mixtures of E+V- and E-V+ subpopulations, and EMT. Importantly, using the EMT-IFA, we discovered that pharmacological targeting of VEGFR/FGFR/PDGFR signaling with the multi-kinase inhibitor pazopanib stimulated EMT in a preclinical xenograft model of gastric carcinoma (MKN45), as revealed by the significant increase in the V:E ratio (P = 0.0023) coupled with an increase in the EMT phenotype (co-localized V+E+ at the individual cell level) (P = 0.0070) in biopsy specimens. Thus, the EMT-IFA has potential value for investigating EMT in the clinic and its ramifications for drug response and other clinical endpoints. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Tony Navas, Robert J. Kinders, Scott M. Lawrence, Katherine Ferry-Galow, Thomas D. Pfister, Apurva K. Srivastava, Sergio Y. Alcoser, Melinda G. Hollingshead, Lindsay M. Dutko, Brad A. Gouker, Donna O. Butcher, Hala Makhlouf, Rodrigo Chuaqui, Donald P. Bottaro, Shivaani Kummar, Alice Chen, James H. Doroshow, Ralph E. Parchment. Epithelial to mesenchymal transition in human tumor biopsies: Quantitative, histopathological proof of the existence of EMT in vivo by immunofluorescence microscopy. [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 LB-B18.

Leptin, BMI, and a Metabolic Gene Expression Signature Associated with Clinical Outcome to VEGF Inhibition in Colorectal Cancer

VEGF (vascular endothelial growth factor) signaling inhibitors are widely used in different cancer types; however, patient selection remains a challenge. Analyses of samples from a phase III clinical trial in metastatic colorectal cancer testing chemotherapy versus chemotherapy with the small molecule VEGF receptors inhibitor cediranib identified circulating leptin levels, BMI, and a tumor metabolic and angiogenic gene expression signature associated with improved clinical outcome in patients treated with cediranib. Patients with a glycolytic and hypoxic/angiogenic profile were associated with increased benefit from cediranib, whereas patients with a high lipogenic, oxidative phosphorylation and serine biosynthesis signature did not gain benefit. These findings translated to pre-clinical tumor xenograft models where the same metabolic gene expression profiles were associated with in vivo sensitivity to cediranib as monotherapy. These findings suggest a link between patient physiology, tumor biology, and response to antiangiogenics, which may guide patient selection for VEGF therapy in the future.

Activation of mitochondrial oxidation by PDK2 inhibition reverses cisplatin resistance in head and neck cancer

Dichloroacetate (DCA), an orphan drug that promotes a shift from glycolysis to oxidative phosphorylation, has been repurposed for cancer therapy. The present study investigated whether DCA may overcome cisplatin resistance in head and neck cancer (HNC). Two cisplatin-resistant HNC cell lines (AMC-HN4R and -HN9R), their parental lines, and other human HNC lines were used. The effect of DCA, alone and in combination with cisplatin, was assessed by measuring cell cycle, viability, death, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), and protein expression in preclinical mouse tumor xenograft models. Increased glycolysis correlated with decreased sensitivity to cisplatin and was reduced by DCA. Cisplatin-resistant cells overexpressed pyruvate dehydrogenase kinase 2 (PDK2). DCA induced HNC cell death by decreasing ΔΨm and promoting mitochondrial ROS production. This effect was decreased by the antioxidant N-acetyl-l-cysteine or by inhibition of caspase-mediated apoptosis. Activation of mitochondrial glucose oxidation by DCA eventually activated downstream mitochondrial apoptotic signaling, leading to the death of chemoresistant cancer cells. Therefore, DCA significantly sensitized resistant HNC cells to cisplatin in vitro and in vivo. High glycolysis and PDK2 overexpression are closely linked to cisplatin resistance in HNC cells; the latter can be overcome by DCA.

Targeting the miR-221-222/PUMA/BAK/BAX Pathway Abrogates Dexamethasone Resistance in Multiple Myeloma.

Despite recent therapeutic advances that have doubled the median survival time of patients with multiple myeloma, intratumor genetic heterogeneity contributes to disease progression and emergence of drug resistance. miRNAs are noncoding small RNAs that play important roles in the regulation of gene expression and have been implicated in cancer progression and drug resistance. We investigated the role of the miR-221-222 family in dexamethasone-induced drug resistance in multiple myeloma using the isogenic cell lines MM1R and MM1S, which represent models of resistance and sensitivity, respectively. Analysis of array comparative genome hybridization data revealed gain of chromosome X regions at band p11.3, wherein the miR-221-222 resides, in resistant MM1R cells but not in sensitive MM1S cells. DNA copy number gains in MM1R cells were associated with increased miR-221-222 expression and downregulation of p53-upregulated modulator of apoptosis (PUMA) as a likely proapoptotic target. We confirmed PUMA mRNA as a direct target of miR-221-222 in MM1S and MM1R cells by both gain-of-function and loss-of-function studies. In addition, miR-221-222 treatment rendered MM1S cells resistant to dexamethasone, whereas anti-miR-221-222 partially restored the dexamethasone sensitivity of MM1R cells. These studies have uncovered a role for miR-221-222 in multiple myeloma drug resistance and suggest a potential therapeutic role for inhibitors of miR-221-222 binding to PUMA mRNA as a means of overcoming dexamethasone resistance in patients. The clinical utility of this approach is predicated on the ability of antisense miR-221-222 to increase survival while reducing tumor burden and is strongly supported by the metastatic propensity of MM1R cells in preclinical mouse xenograft models of multiple myeloma. Moreover, our observation of increased levels of miR-221-222 with decreased PUMA expression in multiple myeloma cells from patients at relapse versus untreated controls suggests an even broader role for miR-221-222 in drug resistance and provides a rationale for the targeting of miR-221-222 as a means of improving patient outcomes.