Significant Tumor Growth Delay Research Articles

Peptide-Drug Conjugate GnRH-Sunitinib Targets Angiogenesis Selectively at the Site of Action to Inhibit Tumor Growth.

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [d-Lys(6)]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphorylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site (∼4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.

Poxvirus-Based Active Immunotherapy with PD-1 and LAG-3 Dual Immune Checkpoint Inhibition Overcomes Compensatory Immune Regulation, Yielding Complete Tumor Regression in Mice.

Poxvirus-based active immunotherapies mediate anti-tumor efficacy by triggering broad and durable Th1 dominated T cell responses against the tumor. While monotherapy significantly delays tumor growth, it often does not lead to complete tumor regression. It was hypothesized that the induced robust infiltration of IFNγ-producing T cells into the tumor could provoke an adaptive immune evasive response by the tumor through the upregulation of PD-L1 expression. In therapeutic CT26-HER-2 tumor models, MVA-BN-HER2 poxvirus immunotherapy resulted in significant tumor growth delay accompanied by a robust, tumor-infiltrating T cell response that was characterized by low to mid-levels of PD-1 expression on T cells. As hypothesized, this response was countered by significantly increased PD-L1 expression on the tumor and, unexpectedly, also on infiltrating T cells. Synergistic benefit of anti-tumor therapy was observed when MVA-BN-HER2 immunotherapy was combined with PD-1 immune checkpoint blockade. Interestingly, PD-1 blockade stimulated a second immune checkpoint molecule, LAG-3, to be expressed on T cells. Combining MVA-BN-HER2 immunotherapy with dual PD-1 plus LAG-3 blockade resulted in comprehensive tumor regression in all mice treated with the triple combination therapy. Subsequent rejection of tumors lacking the HER-2 antigen by treatment-responsive mice without further therapy six months after the original challenge demonstrated long lasting memory and suggested that effective T cell immunity to novel, non-targeted tumor antigens (antigen spread) had occurred. These data support the clinical investigation of this triple therapy regimen, especially in cancer patients harboring PD-L1neg/low tumors unlikely to benefit from immune checkpoint blockade alone.

Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model

Despite the use of ionizing radiation (IR) and temozolomide (TMZ), outcome for glioblastoma (GBM) patients remains dismal. Poly (ADP-ribose) polymerase (PARP) is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM.

Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH‐302) is a prodrug created by the conjugation of 2‐nitroimidazole to bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross‐linking effector, Br‐IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA‐MB‐231‐TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer‐induced bone destruction was measured using micro‐CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

Baculoviral vector loaded mesenchymal stem cells as efficient gene therapy tools for cancer treatment

Insufficient targeting of the therapeutic genes to tumor cells is one of the major reasons for failure in cancer gene therapy. Mesenchymal stem cells (MSCs) seem to be a good candidate as a carrier for gene therapy because of its selective tumor tissue-homing properties. In the current study, we constructed baculoviral vectors (BVs) carrying cytosine deaminase (CD) (BV-CD) or green fluorescence protein (GFP) genes (BV-GFP) and tested the transduction efficiency of the vectors in tumor and mesenchymal stem cells. We also tested the in vivo efficacy of the BV-CD vector in a colon cancer model. Our results showed that the recombinant baculoviral vectors can efficiently transduce mammalian cells and express genes of interest. The BV-CD vector treatment caused significant in vitro cytotoxicity when used with 5-fluorocytosine. MSCs loaded with the BV-CD vector caused a significant delay in tumor growth and increased survival when compared to control and MSC alone treated groups bearing colon cancer.

In vivo Biocompatibility, Biodistribution and Therapeutic Efficiency of Titania Coated Upconversion Nanoparticles for Photodynamic Therapy of Solid Oral Cancers.

Despite the advantages of using photodynamic therapy (PDT) for the treatment of head and neck tumors, it can only be used to treat early stage flat lesions due to the limited tissue penetration ability of the visible light. Here, we developed near-infrared (NIR) excitable upconversion nanoparticle (UCN) based PDT agent that can specifically target epithelial growth factor receptor (EGFR) overexpressing oral cancer cells, in a bid to widen the application of PDT against thick and solid advanced or recurrent head and neck cancers. In vivo studies using the synthesized anti-EGFR-PEG-TiO2-UCNs following systemic administration displayed no major sub-acute or long term toxic effects in terms of blood biochemical, hematological or histopathological changes at a concentration of 50 mg/kg. NIR-PDT even in the presence of a 10 mm tissue phantom placed over the xenograft tumor, showed significant delay in tumor growth and improved survival rate compared to conventional chlorin-e6 (Ce6) PDT using 665 nm red light. Our work, one of the longest study till date in terms of safety (120 d), PDT efficacy (35 d) and survival (60 d), demonstrates the usefulness of UCN based PDT technology for targeted treatment of thick and bulky head and neck tumors.

Abstract PR03: P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD)

Abstract Fluorouracil (5-FU) a widely used chemotherapeutic drug whose unpredictable pharmacokinetics is controlled by the pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD), that has recently also been shown to be a gatekeeper of the epithelial-to-mesenchymal transition (EMT) in breast cancer. Relatively little is known about the transcriptional control of DPYD and here we show for the first time an interaction between p53 and DPYD (involved in catabolism of pyrimidines as well as 5-FU) where p53 represses both the base-line expression of DPYD and that following 5-FU administration in vitro and in vivo. This mechanism affects the catabolic conversion of 5-FU to 5-FUH2 in mice in vivo. Using an in-silico approach we also identified several putative p53 binding sites (P53DBS) in and around ~20Kb upstream and downstream of the mouse DPYD gene. In-vivo ChIP from mice livers identified a key p53DBS binding site downstream (chr3: 119451237-11941257) of the gene to which p53 binds to at about 1.8 ± 0.05 fold over untreated control following a single IV bolus of 5-FU (150 mg/kg bw). Interestingly DPYD mRNA and protein levels were decreased by 1.8 and 1.5 fold respectively (P< 0.0005) in a p53-dependent manner in the liver. Similarly DPYD-repression was documented at both the mRNA and protein levels in the wild-type (WT) p53-expressing cancer cell lines H460, HCT-116, and A569. To further characterize the functional effect of DPYD repression in vivo, we utilized mice with a liver specific deletion of p53 (Albcre p53Δ/Δ) which showed ~2-fold (p≤0.03 wilcoxon rank-sum test) decrease plasma ratio of 5-FUH2/5-FU to mice with an intact allele of p53 in their livers (Albcre p53Δ/+). Increased plasma concentrations of 5-FU caused a significant tumor growth delay (TGD) in mouse colon cancer cells implanted in syngeneic Albcre p53Δ/+ versus Albcre p53Δ/Δ mice (TGD 11 vs 8.93 days). Our data also suggest a possible feedback mechanism of DPYD repression by p53 due to thymidylate synthase (TS) inhibition, a key mediator for supplying thymidine for DNA replication and itself a target for regulation by 5-FU. The addition of exogenous thymidine abrogated the p53-dependent repression of DPYD expression following siRNA-mediated targeting of TS. Further cross-validation experiments using the specific TS inhibitors raltitrexed and methotrexate showed similar effects. Furthermore treatment with the ATM (KU-55933) or DNA-PK (NU7026) inhibitors reversed the p53-dependent repression of DPYD-expression indicating a specific DNA damage feedback mechanism following TS inhibition. Taken together our data suggest that p53 plays an important role in controlling pyrimidine catabolism, particularly following metabolic stress imposed by nucleotide imbalances. In light of more recent discoveries of the role of DPYD in malignant progression, it is plausible that the relationship between p53 and DPYD can have implications beyond influencing the pharmacokinetics of 5-FU and may extend to control of EMT and metastasis. Thus our current efforts are focused on exploring short- and long-term consequences of p53-dependent DPYD-repression as part of p53⊠s tumor suppressor function as well as understanding cellular context for the observed link in vitro and in vivo. Citation Format: Prashanth Ravishankar Gokare, Niklas Finnberg, Jenny Dai, Wafik El-Deiry. P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD). [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR03.

The MEK1/2 Inhibitor Pimasertib Enhances Gemcitabine Efficacy in Pancreatic Cancer Models by Altering Ribonucleotide Reductase Subunit-1 (RRM1).

Gemcitabine, a nucleoside analogue, is an important treatment for locally advanced and metastatic pancreatic ductal adenocarcinoma (PDAC) but provides only modest survival benefit. Targeting downstream effectors of the RAS/ERK signaling pathway by direct inhibition of MEK1/2 proteins is a promising therapeutic strategy, as aberrant activation of this pathway occurs frequently in PDAC. In this study, the ability of pimasertib, a selective allosteric MEK1/2 inhibitor, to enhance gemcitabine efficacy was tested and the molecular mechanism of their interaction was investigated. Cell survival and apoptosis were assessed by MTT and Caspase 3/7 Glo assays in human pancreatic cancer cell lines. Protein expression was detected by immunoblotting. The in vivo sensitivity of gemcitabine with pimasertib was evaluated in an orthotopic model of pancreatic tumor. Synergistic activity was observed when gemcitabine was combined sequentially with pimasertib, in human pancreatic cancer cells. In particular, pimasertib reduced ribonucleotide reductase subunit 1 (RRM1) protein, and this was associated with sensitivity to gemcitabine. Pretreatment with MG132 impaired reduction of RRM1 protein induced by pimasertib, suggesting that RRM1 is degraded posttranslationally. Immunoprecipitation indicated enhanced MDM2-mediated polyubiquitination of RRM1 through Lys-48-mediated linkage following pimasertib treatment, an effect mediated, in part, by AKT. Finally, the combination treatment with pimasertib and gemcitabine caused significant tumor growth delays in an orthotopic pancreatic cancer model, with RRM1 downregulation in pimasertib-treated mice. These results confirm an important role of RRM1 in gemcitabine response and indicate MEK as a potential target to sensitize gemcitabine therapy for PDAC. Clin Cancer Res; 21(24); 5563-77. ©2015 AACR.

Activation of NK and CD8+ T-Cells with a Novel IL-15 and TGF-Beta Receptor Fusion Protein Confers Anti-Tumor Immunity

The use of cytokines as agents to augment immune responses against malignancies have been dealt setbacks due to immune selection of tumors, resulting in subpopulations that elaborate tumor-derived soluble factors, such as transforming growth factor-beta (TGF-β), which suppress immune effector functions. TGF-β is overexpressed by many solid and hematological malignancies and is well known to inhibit the proliferation and anti-tumor functions of lymphomyeloid cells.In order to maximize cytokine-based immunotherapy against tumors, we have designed a novel fusion protein consisting of proinflammatory murine interleukin-15 (IL-15) linked to the sushi domain of the IL-15Rα chain (IL15Rαsushi +IL15) fused in frame to the C'-terminus of a dimeric murine TGF-β-receptor (type II, TβRII) ectodomain-based ligand trap, termed FIST-15 (Fusion of Interleukin 15 with Sushi to TGF-β receptor). The rationale for the design of this protein is to prevent tumor-derived TGF-β from suppressing the immune response via the TGF-β ligand trap moiety, while simultaneously providing a potent stimulus for the activation of anti-tumor responses by an IL-15R agonist (IL-15Rαsushi +IL15).FIST-15 can neutralize TGF-β induced Smad signaling, and induce STAT3 and STAT5 phosphorylation by immunoblot and intracellular flow cytometric analysis of lymphocytes, suggesting that both protein domains are biochemically active. Functionally, FIST-15 is able to induce CD8+ T-cell proliferation at rates greater than IL-15 alone (CD8+ T-cell replicative index or fold-expansion of responding cells: 40, FIST-15, vs. 10, IL-15; p-value of unpaired T-test <0.05). The mitogenic effects of IL-15 are abrogated in CD8+ T-cells and NK cells in the presence of TGF-β. However, FIST-15 can overcome TGF-β mediated inhibition in both these cellular subsets (CD8+ T-cell replicative index: 20, FIST-15, vs. 5, IL-15, and NK cell replicative index: 40, FIST-15 vs. 5, IL-15; p-value <0.05).Rapid proliferation of the CD8+ central memory phenotype (CD62L+, CD44+) T-cells are seen with FIST-15 treatment. Compared to IL-15 expanded CD8+ T-cells, FIST-15 treatment also produced more IFN-γ, TNF-α, and IL-2 secreting CD8+ T-cells upon PMA/ionomycin stimulation. In addition to cytokines, production of anti-tumor effector molecules such as granzyme B is known to be inhibited by TGF-β. FIST-15 treated NK cells were superior to IL-15 treated NK cells in granzyme B production, even in the presence of TGF-β, as assayed by flow cytometric analysis (86.8% vs. 30.7% granzyme B expressing cells). Functionally, FIST-15 treated NK cells were also significantly more cytolytic against TGF-β secreting B16 murine melanoma cells in vitro compared to IL-15 treated NK cells (83.5% killing, FIST-15, vs. 24.4% killing, IL-15).C57Bl/6 mice with pre-established, syngeneic B16 melanoma tumors were treated with FIST-15 to assay the anti-tumor effects of the fusion protein in vivo. Mice receiving FIST-15 showed a significant delay in tumor growth (mean tumor volume: 345mm3) compared to control mice receiving conditioned media (mean tumor volume: 814.12mm3; p-value of paired T-test = 0.02) by day 21 post-tumor implantation. Furthermore, FIST-15 treated mice showed a significant survival advantage compared to control treated mice (80% vs 0%; p-value of log rank test = 0.0019) by day 27 post-tumor implantation. Mice immunized with B16 tumors transduced to express FIST-15 were also protected against subsequent wildtype B16 tumor challenge, suggesting that FIST-15 can trigger an adaptive immune response against tumor.Ongoing work utilizing FIST-15 in murine models of hematological malignancies, such as EL-4 lymphoma and C1498 AML, is currently underway. These models were selected due to their known overexpression of TGF-β isoforms that systemically inhibit endogenous anti-tumor responses, as well as the efficacy of immunotherapeutic agents. Indeed, many hematological malignancies acquire mutations that render them insensitive to the growth-inhibitory effects of TGF-β, where it may then be overexpressed as an oncogene to promote further tumor growth by inhibiting the immune system's anti-tumor capabilities (Dong et al Blood 2006). FIST-15 may present a viable immunotherapeutic strategy for hematological malignancies by combining the immune activating effects of IL-15 with the neutralization of immunosuppressive TGF-β. DisclosuresNg:Emory University: Patents & Royalties. Galipeau:Emory University: Patents & Royalties.

Abstract B58: BIM, a key player in apoptosis induction and acquired venetoclax resistance in follicular lymphoma cells

Abstract The chromosomal translocation t(14;18) in follicular lymphoma (FL) cells is a primary oncogenic event resulting in BCL-2 over-expression. BCL-2 is not directly responsible for growth of tumor cells, but it protects them against death by binding pro-apoptotic proteins. This study investigates in vitro and in vivo activity of the BH3 mimetic venetoclax (ABT-199) and mechanisms of acquiring resistance in FL. Primary FL cells with similar expression of anti-apoptotic proteins, such as BCL-2 and MCL-1, showed variable sensitivity to treatment, which correlated with expression of BIM. To further study this correlation we used two t(14;18)+ cell lines, WSU-FSCCL and FC-TxFL2. As with primary cells, FC-TxFL2 cells with higher expression of BIM protein were more sensitive to venetoclax-induced apoptosis. The treatment caused dissociation of BCL-2/ BIM complexes, decreased mitochondrial potential, activation of caspase-3 and caspase-7, and PARP cleavage. Interestingly the population of cells that survived venetoclax treatment showed an increase of phospho-ERK1/2 and phospho-BIM (S69), as well as a decrease of total BIM levels. Venetoclax treatment of murine xenografts of FC-TxFL2 cells resulted in significant delay in tumor growth. However, viable cells isolated from treated tumors showed a decrease of sensitivity to venetoclax exposure (IC50 increased almost 10 times) in comparison with the cells from the mice treated with vehicle only. These cells contained higher levels of MCL-1 and lower levels of BIM protein compared to control cells. BCL-2 levels remained unchanged. Venetoclax resistant cells prepared in vitro by continuous treatment with increasing concentrations of venetoclax initially showed elevated levels of p-AKT, p-FOX01/3a, MCL-1, dissociation of BIM/BCL-2/BECLIN1 complex, decrease of p62 level (a sign of an increased autophagy) together with a slight decline of BIM expression. After a stable resistant cell line was established, a significant reduction of BCL-2 levels and almost total absence of BIMEL, BIML and BIMS isoforms was observed. Detailed knowledge of venetoclax action and the mechanisms for acquired resistance in t(14;18)+ cells may help inform biomarker strategies and dosing schedules in future clinical trials. Inhibition of pathways activated in order to protect the FL cells (e.g. by down-regulating BIM (ERK or AKT pathway) or by autophagy) can significantly augment venetoclax potency and decrease incidence of acquired resistance. Citation Format: Juraj Bodo, Xiaoxian Zhao, Andrew J. Souers, Darren C. Phillips, Mitchell R. Smith, Eric D. Hsi. BIM, a key player in apoptosis induction and acquired venetoclax resistance in follicular lymphoma cells. [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 B58.

ATPS-07NEO214, ROLIPRAM CONJUGATED TO PERILLYL ALCOHOL, IS A NOVEL DRUG CYTOTOXIC FOR GLIOMAS BY TARGETING THE DEATH RECEPTOR MEDIATED TRAIL PATHWAY

Glioblastoma multiforme (GBM) is a primary brain tumor with a poor prognosis. Treatment with temozolomide (TMZ) usually results in drug resistance and tumor recurrence. This study shows that NEO214, rolipram conjugated to perillyl alcohol, is cytotoxic for a wide range of different TMZ-resistant glioma cells. This conjugate is significantly more effective than either rolipram, perillyl alcohol or the mixture of these agents, demonstrating that NEO214 is a novel entity. NEO214 is effective for drug resistant tumor cells, but not toxic to normal astrocytes or endothelial cells. To identify the mechanism of cell death, effects of NEO214 on death receptor (DR)5 expression were examined. The results show that NEO214 significantly increased the expression of DR5 in both TMZ-sensitive and TMZ-resistant glioma cells. In addition, tumor cells treated with NEO214 and immunostained for DR5 demonstrated over 90% positivity. To show that DR5 receptors were functional, the interaction with the receptor ligand, TRAIL, with NEO214 was tested. The results showed that NEO214 plus TRAIL induced a dramatic increase in cell death. TRAIL alone was not cytotoxic to cells. The combination of NEO214 plus TRAIL resulted in a 60–80% decrease in cell survival. NEO214-treated glioma cells also exhibited an increase in CHOP, marker for ER stress activation. Downregulation of CHOP expression correlated with DR5 expression suggesting that ER stress may be involved in stimulating DR5 expression. To determine effects of NEO214 in vivo, glioma cells were implanted subcutaneously into animals, and treated with NEO214 or vehicle. Results showed that NEO214 caused a significant delay in tumor growth. These results demonstrate that NEO214 is an effective drug against a wide range of different TMZ- resistant gliomas, the combination of NEO214 and TRAIL is very effective in enhancing tumor cytotoxicity, and NEO214 is active in vivo causing significant delays in tumor progression.

Dual Targeting of Protein Degradation Pathways with the Selective HDAC6 Inhibitor ACY-1215 and Bortezomib Is Synergistic in Lymphoma.

Pan-class histone deacetylase (HDAC) inhibitors are effective treatments for select lymphomas. Isoform-selective HDAC inhibitors are emerging as potentially more targeted agents. HDAC6 is a class IIb deacetylase that facilitates misfolded protein transport to the aggresome for degradation. We investigated the mechanism and therapeutic impact of the selective HDAC6 inhibitor ACY-1215 alone and in combination with bortezomib in preclinical models of lymphoma. Concentration-effect relationships were defined for ACY-1215 across 16 lymphoma cell lines and for synergy with bortezomib. Mechanism was interrogated by immunoblot and flow cytometry. An in vivo xenograft model of DLBCL was used to confirm in vitro findings. A collection of primary lymphoma samples were surveyed for markers of the unfolded protein response (UPR). Concentration-effect relationships defined maximal cytotoxicity at 48 hours with IC50 values ranging from 0.9 to 4.7 μmol/L. Strong synergy was observed in combination with bortezomib. Treatment with ACY-1215 led to inhibition of the aggresome evidenced by acetylated α-tubulin and accumulated polyubiquitinated proteins and upregulation of the UPR. All pharmacodynamic effects were enhanced with the addition of bortezomib. Findings were validated in vivo where mice treated with the combination demonstrated significant tumor growth delay and prolonged overall survival. Evaluation of a collection of primary lymphoma samples for markers of the UPR revealed increased HDAC6, GRP78, and XBP-1 expression as compared with reactive lymphoid tissue. These data are the first results to demonstrate that dual targeting of protein degradation pathways represents an innovative and rational approach for the treatment of lymphoma.

ID: 99: Identification, function and clinical development of the heterodimeric IL-15 cytokine (hetIL-15)

We have shown that the physiologically relevant Interleukin-15 molecule in vivo is a heterodimeric molecule consisting of the IL-15 polypeptide chain and the so-called IL-15 Receptor alpha chain (IL-15Ra), a membrane protein. There are two forms of IL-15 in the body: membrane bound heterodimer of IL-15 and IL-15Ra and soluble heterodimeric IL-15 produced by a specific proteolytic cleavage of the heterodimer at the extracellular domain of IL-15Ra. Our results indicate that IL-15Ra polypeptide is not a receptor but the other half of the heterodimeric IL-15 cytokine. We have characterized the soluble heterodimeric IL-15 (hetIL-15) and developed methods for its efficient production and purification. Injection of hetIL-15 resulted in a significant delay in tumor growth, in preclinical models of tumor-bearing mice. Intratumoral delivery of hetIL-15 induced regression of established tumors. hetIL-15 has the unique property to increase intratumoral CD8 cells and to also increase dramatically the ratio of CD8 to Treg. We dissect the mechanisms leading to tumor control in hetIL-15-treated mice, revealing a novel interplay between IL-15 and the cognate cytokine interleukin-2 (IL-2). Repeated subcutaneous administration of purified hetIL-15 in macaques resulted in sustained plasma IL-15 levels and in dose-dependent expansion of NK and T cells in blood and tissues, demonstrating pharmacokinetics and in vivo bioactivity superior to monomeric IL-15. On the basis of these data, we have produced and characterized a cGMP preparation of hetIL-15 and we have initiated a Phase 1 dose escalation clinical trial in metastatic cancer to assess safety and activity of this cytokine.

Zoledronic acid prevents the tumor-promoting effects of mesenchymal stem cells via MCP-1 dependent recruitment of macrophages.

Zoledronic acid (ZA) has been tested in clinical trials as an additive therapy for early-stage breast cancer. However, the mechanism by which ZA exerts its antitumor activity is still unclear. The aim of this study is to investigate whether the prevention of tumor growth by ZA is through regulating the mesenchymal stem cells (MSC)-monocyte chemotactic protein 1 (MCP-1)-macrophages axis in the tumor microenvironment. To address this issue, MDA-MB-231-FLUC human breast cancer cells were cultured and injected either alone, or coupled with MSC into the mammary fat pads of nude mice. MSC were treated with either ZA or untreated. Tumor growth was determined by using an in vivo bioluminescence imaging (BLI) and the tumor-associated macrophages (TAMs) in tumor tissues were immunohistochemically analyzed by using CD206 antibody. The effects of ZA on the cytokine related gene expression of MSC were assessed by using real-time PCR. In this study, we found that ZA-treated mice showed a significant delay in tumor growth. In addition, our data revealed that ZA weakened the ability of MSC to promote tumor growth by impairing TAMs recruitment and tumor vascularization. Furthermore, it was found that ZA decreased MCP-1 expression of MSC, and therefore reduced the recruitment of TAMs to the tumor sites and hence inhibited the tumor growth. Altogether, our study demonstrated ZA can prevent the tumor-promoting effects of MSC. The antitumor effects of ZA were caused by decreasing the MCP-1 expression of MSC, which further decreased the infiltration of TAMs into tumor sites, and therefore inhibited the tumor growth.

Abstract 774: Cabozantinib, a multi-target receptor tyrosine kinase inhibitor, decreases tumor growth and angiogenesis in a patient-derived dedifferentiated liposarcoma xenograft

Abstract Background: Upregulation of receptor tyrosine kinase signaling pathways, including vascular endothelial growth factor receptor (VEGFR) and MET (also known as hepatocyte growth factor receptor), is found in different subtypes of soft tissue sarcoma. Cabozantinib (CABO) is a multi-target inhibitor of receptor tyrosine kinases including VEGFR, MET, RET, and KIT. We aimed to evaluate the anti-angiogenic and anti-tumor effect of CABO alone and in combination with the standard-of-care cytotoxic agent doxorubicin (DOX) in a patient-derived xenograft model of dedifferentiated liposarcoma (DDLPS). Methods: NMRI nu/nu mice were bilaterally transplanted with human UZLX-STS3 DDLPS tumors, a model originating from a patient with DDLPS. Twenty eight tumor-bearing mice were assigned to 4 groups and treated for three weeks with either vehicle (control), DOX (i.p., 1.2mg/kg/twice per week), CABO (p.o., 30mg/kg/QD) or CABO+DOX (same dose/schedule as for the single agents). Tumor volume was measured three times a week. Mitotic and apoptotic activity were assessed on hematoxylin and eosin (H&amp;E) staining and with phospho-histone H3 (pHH3) and cleaved-caspase 3 (CC3) immunostainings. Microvascular density (MVD) was evaluated by counting CD34 positive vessels. Statistics were calculated using the Mann-Whitney U pairs tests. p &amp;lt; 0.05 was considered as statistically significant. Results: A significant delay in tumor growth was observed in CABO and CABO+DOX treated groups as compared to controls (p&amp;lt;0.05) or DOX single agent (p&amp;lt;0.05). Both CABO and CABO+DOX reduced the mitotic activity in comparison with control (38% and 66% decrease, respectively, p&amp;lt;0.005). CABO+DOX showed better antimitotic activity than either single agent DOX or CABO (p&amp;lt;0.005 for both). The CABO-based treatments induced higher apoptotic activity as compared to control (2.1 fold in CABO and 2.4 fold in CABO+DOX, p&amp;lt;0.005). Results were confirmed by pHH3 and CC3 stainings. Furthermore, significantly decreased MVD was observed in the CABO and the combination treated tumors as compared to control (64% and 67% decrease, respectively, p&amp;lt;0.005) and as compared to DOX alone (57% and 61% decrease, respectively, p&amp;lt;0.005). Conclusions: CABO has anti-tumor activity in the UZLX-STS3 DDLPS xenograft model by inhibiting proliferation, inducing apoptosis and suppressing angiogenesis. These in vivo results support ongoing and planned clinical trials with cabozantinib in mesenchymal malignancies. Citation Format: Haifu Li, Agnieszka Wozniak, Karel Van Den Bossche, Thomas Van Looy, Jasmien Wellens, Daphne Hompes, Dana Aftab, Maria Debiec-Rychter, Raf Sciot, Patrick Schöffski. Cabozantinib, a multi-target receptor tyrosine kinase inhibitor, decreases tumor growth and angiogenesis in a patient-derived dedifferentiated liposarcoma xenograft. [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 774. doi:10.1158/1538-7445.AM2015-774

Abstract 5527: Oral administration of a nano-enabled form of Met-Enkephalin peptide controls pancreatic cancer growth

Abstract Introduction Peptides are a potential source of highly specific bioactive molecules useful for the treatment of various conditions, including cancer. Their wider application is limited by their poor oral bioavailability due to e.g. rapid degradation in the gut or in the blood. Unfavorable physical-chemical properties can further limit their applicability. For example, Met-enkephalin (MENK) is an endogenous opioid peptide having anti-tumor activity against a diverse range of cancers. While previous reports describe anti-cancer effects of MENK after parenteral administration in humans this approach has significant limitations because only low drug concentrations are achievable due to the peptide's poor aqueous solubility. Here, we demonstrate that a nano-enabled form of MENK created by combining MENK with the amphiphilic polymer GCPQ to form ‘molecular envelopes’, is orally bioavailable and active in murine models of pancreatic cancer. Method Nano-enabled MENK (10 mg/ml) formulation was prepared by encapsulating the peptide in GCPQ nanoparticles (50 mg/ml). A MENK solution was prepared in water (10 mg/ml). Swiss nude mice bearing tumor xenografts of pancreatic cancer cells (MIA PaCa-2) were randomized in to three groups (n = 5), each receiving MENK (50 mg/kg), nano-enabled MENK formulation (50 mg/kg) or water. All formulations were orally administered every two days over a period of 29 days once tumors were fully established. Throughout the study the tumor volumes were measured using calipers and the body weight of the mice monitored. Results For mice treated with the nano-enabled MENK formulation at a dose of 50 mg/kg, a complete suppression in the tumor growth was observed within two weeks of the start of treatment which continued while treatment continued and for two weeks after withdrawal of the treatment on 4th week. In contrast, no statistically significant delay in tumor growth was observed for mice treated with the MENK solution (50 mg/kg) compared with the untreated control. Overall the treatment was well tolerated as suggested by the limited impact of therapy on animal body weight. In vivo pharmacodynamics studies demonstrated significantly higher anti tumor efficacy for nano-enabled MENK formulation compared to free MENK in established MIA PaCa-2 tumor xenograft model. Conclusion The current study demonstrates that the MENK peptide when administered orally in the form of nano-enabled ‘molecular envelopes’ is able to control the growth of MIA PaCa-2 pancreatic tumour xenograft tumors over a period of four weeks. The above results suggest that a nano-enabled oral form of this endogenous peptide could have wider application in cancer therapy. Furthermore, it suggests that other peptides with anti-cancer activity could be utilized as oral peptide pill for cancer therapy. Citation Format: Preethi Marimuthu, Ijeoma F. Uchegbu, Andreas G. Schätzlein. Oral administration of a nano-enabled form of Met-Enkephalin peptide controls pancreatic cancer growth. [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 5527. doi:10.1158/1538-7445.AM2015-5527

Abstract 3136: Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma

Abstract Bone marrow (BM) serves as a reservoir for a unique population of memory T cells. Tumor-specific T cells have been identified in the bone marrow of cancer patients. However, few direct functional analyses of tumor-specific responses in T cells isolated from BM have been performed. In this study, we investigated the ability of bone marrow T cells to induce anti-tumor immunity in mice bearing neuro2A neuroblastoma or B16 melanoma. To demonstrate the presence of antigen-specific T cells in BM, we first examined the percent of antigen-specific CD8+ T cells in vaccinated mice. The overall percent of antigen-specific CD8+ T cells was much higher in BM compared to spleen and lymph nodes of vaccinated mice. To investigate the ability of antigen-specific BM T cells to induce cytotoxic function, BM T cells were isolated from mice vaccinated with OVASIINFEKL peptide pulsed dendritic cells (DC). Expanded BM T cells from DC vaccinated mice co-cultured with M05 or irrelevant tumor cells led to tumor-specific cytotoxicity (p&amp;lt;0.001). In B16 tumor-bearing mice, T cells isolated from BM produced IFN-gamma in response to gp100 peptide (1400+210 pg/ml). Adoptive transfer of BM T cells isolated from B16-bearing mice led to a delay in tumor growth compared to control groups (p&amp;lt;0.001). In the neuroblastoma model, T cells isolated from BM of neuro2A-bearing mice produced higher levels of IFN-gamma and were tumor-specific (250+90 pg/ml, p&amp;lt;0.01 compared to T cells cultured with irrelevant tumor cells). Adoptive transfer of expanded BM T cells and DC vaccination in the setting of BM transplant led to a significant delay in tumor growth and enhanced survival in neuro2A bearing mice compared to BM transplant + DC vaccination alone group (p&amp;lt;0.02). These studies demonstrate that tumor-specific T cells can be isolated from the BM of tumor-bearing mice. Adoptive transfer of T cells expanded from the BM can delay tumor growth and improve survival. These studies support the isolation and expansion of T cells from the BM for use in adoptive cell therapy strategies in patients with neuroblastoma and melanoma. Citation Format: Krithika N. Kodumudi, Amy Weber, Ellen Moore, Amod Sarnaik, Shari Pilon-Thomas. Adoptive transfer of bone marrow T cells induces antitumor immune responses in murine models of melanoma and neuroblastoma. [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 3136. doi:10.1158/1538-7445.AM2015-3136

Abstract 491: Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. The proto-oncogene MYCN is amplified in 25% of NB, and is associated with enhanced angiogenesis and poor survival. Vascular endothelial growth factor (VEGF) is the most potent angiogenic growth factor and, with the hepatocyte growth factor (HGF)/c-MET signalling pathway, is implicated in the progression of human NB. Cabozantinib (CBZ) is a small-molecule kinase inhibitor with potent activity against VEGFR2 and MET, currently in clinical trials against NB. In this study, the efficacy of CBZ was evaluated in tumors spontaneously arising in the TH-MYCN GEM model of high-risk NB, previously shown to faithfully recapitulate the radiological and hypervascular characteristics of childhood disease. TH-MYCN mice with spontaneous abdominal tumors were treated daily with either vehicle or 30mg/kg CBZ. Non-invasive T2-weighted anatomical MRI revealed that CBZ induced significant (p&amp;lt;0.001) tumor growth arrest after 48 hours compared to control. Quantitative functional MRI showed this volumetric response was associated with significant decreases in both the native longitudinal MRI relaxation time T1 (p&amp;lt;0.01) a measure of the ratio of bound to free water in tissue reflecting the increased extracellular space in tumors, and transverse MRI relaxation rate R2* (p&amp;lt;0.05), sensitive to paramagnetic deoxyhemoglobin. Histopathological analysis revealed a significant increase in necrosis (p&amp;lt;0.01), and significant decrease in microvessel density (p&amp;lt;0.01) and VEGFR2 scoring (p&amp;lt;0.02) in CBZ-treated tumors compared to control. Significant (p&amp;lt;0.01) tumor growth delay was maintained after 7 days treatment with CBZ compared to control, which translated into a significant improvement in survival rate (p&amp;lt;0.0001). This study demonstrates that CBZ is efficacious against, and conveys a survival benefit for the TH-MYCN GEM model of NB. The data reinforces native T1 as a biomarker of response to anti-vascular therapy due to its sensitivity to therapy-induced damage to erythrocytes and/or cell death-mediated increase in macromolecules into the extracellular space. Relatively fast R2* rates in NB arising in the TH-MYCN model are strongly associated with vascular instability, characterised by numerous regions of aggregated paramagnetic erythrocytes (“blood lakes”), and treatment-induced thrombosis with the pan-VEGFR inhibitor cediranib which increased tumor R2*. The CBZ-induced decrease in R2* is clearly associated with vascular disruption, hence precluding the extravasation of deoxygenated red blood cells. Citation Format: Gilberto S. Almeida, Philippa King, Yann Jamin, Albert Hallsworth, Hannah Webber, Sergey Popov, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Tumor response to cabozantinib in the TH-MYCN GEM model of neuroblastoma. [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 491. doi:10.1158/1538-7445.AM2015-491

Abstract 796: Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression

Abstract Alofanib (formerly known as RPT835) is a novel allosteric FGFR2 inhibitor with activity in FGFR2-expressing triple-negative breast cancer [Tjulandin S, et al. San Antonio Breast Cancer Symposium 2014]. Early data suggest that combining FGFR2 inhibitors with platinum-containing cytotoxic agents for the treatment of epithelial ovarian cancer may yield increased antitumor activity [Cole C, et al. Cancer Biol Ther. 2010]. We investigated antitumor activity of alofanib in ovarian cancer in vitro and in vivo. To assess the efficacy of alofanib on FGF-mediated cell proliferation, ovarian cancer (SKOV-3) FGFR2-expressing cells were incubated in a 96-well microculture plate and were treated with serially diluted RPT835. Basic FGF was added at a concentration of 25 ng/ml. Control wells were left untreated. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Immunocompromised mice were used for xenotransplantation of FGFR2 high-expressing ovarian cancer cells (SCOV-3). Seventy animals with measurable tumors were selected on day 10 and randomized into control groups (no treatment or chemotherapy alone (paclitaxel + carboplatin) and treatment groups (alofanib orally or intravenously (different dose levels) in combination with chemotherapy). Measurements of tumor volume (mm3) were performed by digital calipers every 3 days during 30 days after tumor inoculation. Basic FGF significantly increased proliferation of the ovarian cancer cells in untreated control group (P = 0.001). Alofanib treatment resulted in growth inhibition of SKOV-3 cell line in vitro. Treatment of alofanib in combination with paclitaxel/carboplatin demonstrated significant tumor growth delay phenotype in all treatment groups compared to control non-treatment groups. Alofanib exhibited a dose-dependent effect on tumor growth. Daily intravenous regimen of alofanib (total maximum dose per week was 350 mg/kg) demonstrated dramatic effect (inhibiting growth by 80% and by 53% in comparison with vehicle and chemotherapy group alone, respectively (P&amp;lt;0.001). Alofanib decreased number of vessels in tumor on 49% compared with chemotherapy alone (P&amp;lt;0.05). There were tumor necrosis and cell degeneration in alofanib group. These results provide strong rationale for evaluation of alofanib in combination with paclitaxel and carboplatin in patients with ovarian cancer. Citation Format: Sergei Tjulandin, Mikhail Byakhov, Evgenia Stepanova, Dmitry Khochenkov, Daniel Harrison, Ilya Tsimafeyeu. Alofanib, a novel allosteric FGFR2 inhibitor, shows potent antitumor activity in ovarian cancer with FGFR2 expression. [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 796. doi:10.1158/1538-7445.AM2015-796

Abstract 2592: Anti-tumor activity of icotinib (BPI-2009H) and BPI-403B in the BxPC-3 pancreatic cancer cell line

Abstract Introduction: The purpose of this research was to investigate the anti-tumor effect of BPI-2009H and BPI-403B, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) in the human pancreatic cancer cell lines BxPC-3 and Panc-1, in vitro and in vivo and determine the maximum tolerated dose (MTD) of the compound in mouse. Experimental Design: In vitro, human pancreatic cancer cell lines, BxPC-3 and Panc-1 were exposed to varying concentrations of BPI-2009H and BPI-403B, the concentration of compound required to inhibit growth by fifty percent (IC50) was estimated. In vivo, tumor growth inhibition of low dose BPI-2009H was tested in xenografted nude mice with BxPC-3 pancreatic cancer tumors. The maximum tolerated dose (MTD) was established via oral administration of low, mid and high doses to Harlan mice of both BPI-2009H and BPI-403B. Upon determination of the MTD, the in vivo efficacy of high dose BPI-2009H was evaluated via tumor growth inhibition study on xenografted nude mice with BxPC-3 pancreatic cancer tumors. Results: BPI-2009H, as a single agent, inhibited the growth of BxPC-3 cells in a dose dependent manner, with a relative IC50 value of 25.8 μM. BPI-403B, as a single agent, inhibited growth of BxPC-3 cells in a dose dependent manner as well, with a relative IC50 value of 2.7 μM. BPI-2009H and BPI-403B were tested up to 50μM in Panc-1 cells with no apparent effect at the upper limit of the study for either of the compounds. In vivo, the mice treated with BPI-403B demonstrated a statistically significant tumor growth delay, 14.3 days. 20% of the mice experienced tumor regression. The compound was well tolerated with minimal body weight loss. At low doses, BPI-2009H did not show any statistically significant tumor growth delay, but upon increasing the dose to the MTD, significant signs of anticancer activity was demonstrated, with an increase in tumor growth delay. Conclusions: The in vitro and in vivo findings show that both BPI-2009H and BPI-403B have antiproliferative effects against BxPC-3 pancreatic cancer cells. These compounds are both well tolerated and may prove to be an alternative option of EGFR-TKI to erlotinib treatment in pancreatic cancer cases. Citation Format: Victoria Lynn Wilde, Don Zhang, Jirong Peng, Michael A. Green, Michael N. Greco, Michael Costanzo. Anti-tumor activity of icotinib (BPI-2009H) and BPI-403B in the BxPC-3 pancreatic cancer cell line. [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 2592. doi:10.1158/1538-7445.AM2015-2592