Substantial Tumor Growth Inhibition Research Articles

Abstract 3608: RAD140, an orally available selective androgen receptor modulator, inhibits the growth of AR/ER positive breast cancer cell line- and patient-derived xenograft models

Abstract Breast cancer is the most frequently diagnosed malignancy for women in the US with 246,660 new cases predicted in 2016. Histological and molecular characterization of breast cancer has led to improved understanding of the heterogeneity of this disease and development of therapeutic modalities tailored for each disease subtype. Hormone receptor positive breast cancers, which represent 75% of breast cancer cases, are routinely treated with therapies targeting the estrogen receptor (ER) axis, including aromatase inhibitors or anti-estrogens, either alone or in combination with other agents. Recent studies have shown that the androgen receptor (AR) is widely expressed across all subtypes of breast cancer, with AR detected in 75-95% of ER positive tumors. Mounting preclinical evidence from has demonstrated that AR agonists can suppress AR and ER positive (AR/ER+) breast cancer cell growth. Furthermore, prior to the development of ER-targeted therapy, androgens were used to treat breast cancers with favorable clinical responses. RAD140 is an oral, nonsteroidal, nonaromatizable selective androgen receptor modulator (SARM) with a distinct tissue selectivity profile and mechanism of action. Here we examined the molecular activity of RAD140 in breast cancer cells and evaluated its efficacy using AR/ER+ breast cancer xenograft models. The tissue-specific activity of RAD140 was examined using androgen response element (ARE)-driven reporter assay in AR/ER+ ZR75 breast cancer cells and AR+ LNCaP prostate cancer cells. RAD140 was shown to be a potent AR agonist, comparable to dihydrotestosterone (DHT), in breast cancer cells, but did not induce AR activity in prostate cancer cells, while DHT demonstrated full AR agonist activity. In vivo anti-tumor efficacy of RAD140 as monotherapy, compared with fulvestrant, or in combination with a CDK4/6 inhibitor (palbociclib) or mTOR inhibitor (everolimus) was evaluated in AR/ER+ patient derived xenograft (PDX) models and cell line-derived xenograft (CDX) models. Tumor-bearing mice were randomized into treatment groups and received vehicle, RAD140, fulvestrant, palbociclib, everolimus or combinations of RAD140 with palbociclib or everolimus. Tumor volume and growth was measured to evaluate efficacy. At the end of the study, plasma and tumor samples were collected for pharmacokinetic and pharmacodynamic analysis. In AR/ER+ breast cancer PDX models, RAD140 treatment led to substantial tumor growth inhibition that was greater than that seen with fulvestrant. Furthermore, combination of RAD140 with palbociclib or everolimus produced further enhanced anti-tumor efficacy in these models. In summary, RAD140 demonstrated tissue-selective AR agonism with marked anti-tumor activity in AR/ER+ breast cancer CDX and PDX models, together with enhanced anti-tumor activity in combination with a CDK4/6 inhibitor or mTOR inhibitor. Citation Format: Ziyang Yu, Suqin He, Jeffrey Brown, Chris Miller, Jamal Saeh, Gary Hattersley. RAD140, an orally available selective androgen receptor modulator, inhibits the growth of AR/ER positive breast cancer cell line- and patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3608. doi:10.1158/1538-7445.AM2017-3608

Abstract 3609: RAD140, a selective androgen receptor modulator, has a differentiated mechanism of action in AR/ER positive breast cancers

Abstract Recent studies have shown that the androgen receptor (AR) is widely expressed across all subtypes of breast cancer, with AR detected in 75-95% of estrogen receptor (ER) positive tumors. Prior to the development of ER-targeted therapy, androgens were used to treat breast cancers with favorable clinical responses. The lack of tissue-selectivity and understanding of mechanism of action of classic androgens led to its declined use in this disease. Selective androgen receptor modulators (SARMs) are a class of molecules developed as tissue selective non-steroidal androgens that modulate AR signaling. However, the effect of SARMs on AR signaling in breast cancer cells is not well understood. RAD140 is an orally available SARM with potent anabolic activity in muscle and bone but a highly attenuated effect in seminal vesicles and prostate. Its efficacy is being evaluated in in vivo xenograft models of AR/ER+ breast cancer. To further our understanding of the mechanism of action of RAD140 in breast cancer cells, we examined the effects of RAD140 on AR and ER signaling pathways. The specificity and selectivity of RAD140 were examined using competitive binding assays for AR, ER, glucocorticoid receptor (GR) and progesterone receptor (PR), and using an AR reporter assay in AR/ER+ ZR-75 breast cancer cells and LNCaP prostate cancer cells. RAD140 was found to bind AR with high affinity and specificity, with 250-fold target selectivity over the next nuclear receptor PR. The AR reporter assay results demonstrated a potent AR agonist activity of RAD140 in ZR-75 breast cancer cells but not in LNCaP prostate cancer cells, in contrast to the full agonist activity of DHT seen in both cell types. RAD140 treatment led to substantial tumor growth inhibition in AR/ER+ breast cancer xenografts. Samples of RAD140-treated xenografts were analyzed by immunocytochemistry, western blotting and quantitative real-time PCR to evaluate the modulation of AR and ER pathways. It was found that the expression of known AR target genes including KLK2, FKBP5 and the tumor suppressor gene, ZBTB16, were potently induced in RAD140-treated tumors. Furthermore, RAD140 led to substantial suppression of known ER target genes including progesterone receptor (PR), TFF1 and GREB1. In conclusion, RAD140 is a potent tissue-selective AR agonist in AR/ER+ breast cancer cells with robust activation of AR targets genes, and a unique mechanism of action that leads to the suppression of ER signaling, and marked anti-tumor activity. Citation Format: Ziyang Yu, Suqin He, Dannie Wang, Jeffrey Brown, Jamal Saeh, Gary Hattersley. RAD140, a selective androgen receptor modulator, has a differentiated mechanism of action in AR/ER positive breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3609. doi:10.1158/1538-7445.AM2017-3609

Redox- and light-responsive alginate nanoparticles as effective drug carriers for combinational anticancer therapy.

Nanoparticles have been extensively explored as effective means to deliver chemotherapeutic agents or photosensitizers for chemotherapy or photodynamic therapy (PDT) against cancer. In the present work, pheophorbide A (PheoA), a hydrophobic photosensitizer, was conjugated via a redox-sensitive disulfide linkage to alginate (PheoA-ALG). Anticancer agent, doxorubicin (DOX), was also loaded within the PheoA-ALG nanoparticles (DOX/PheoA-ALG NPs) and used as drug carriers for combinational antitumor treatment. The DOX/PheoA-ALG NPs were spherical in shape with a uniform diameter of approximately 210 nm. Redox-responsive drug releasing properties were shown by the DOX/PheoA-ALG NPs, with an accelerated amount of DOX and PheoA release observed in the presence of a high glutathione level (10 mM). Cellular uptake results showed that DOX/PheoA-ALG NPs were readily taken up by B16 tumor cells (murine melanoma) and enhanced DOX and PheoA uptake were detectable in the DOX/PheoA-ALG NPs-treated B16 cells in comparison to carrier free drugs. DOX/PheoA-ALG NPs also elicited intracellular ROS generation, which leads to enhanced toxicity in B16 cells. In vivo studies using B16 tumor-bearing mice further demonstrated that DOX/PheoA-ALG NPs were preferentially accumulated in tumor tissues, resulting in substantial inhibition of B16 tumor growth by chemotherapy and photodynamic therapy, which is also attributable to DOX/PheoA-ALG NP-elicited increase of serum INF-λ levels. Our results demonstrate a major potential of DOX/PheoA-ALG NPs for combinational cancer therapy.

AKT activation controls cell survival in response to HDAC6 inhibition.

HDAC6 is emerging as an important therapeutic target for cancer. We investigated mechanisms responsible for survival of tumor cells treated with a HDAC6 inhibitor. Expression of the 20 000 genes examined did not change following HDAC6 treatment in vivo. We found that HDAC6 inhibition led to an increase of AKT activation (P-AKT) in vitro, and genetic knockdown of HDAC6 phenocopied drug-induced AKT activation. The activation of AKT was not observed in PTEN null cells; otherwise, PTEN/PIK3CA expression per se did not predict HDAC6 inhibitor sensitivity. Interestingly, HDAC6 inhibitor treatment led to inactivating phosphorylation of PTEN (P-PTEN Ser380), which likely led to the increased P-AKT in cells that express PTEN. Synergy was observed with phosphatidylinositol 3'-kinases (PI3K) inhibitor treatment in vitro, accompanied by increased caspase 3/7 activity. Furthermore, combination of HDAC6 inhibitor with a PI3K inhibitor caused substantial tumor growth inhibition in vivo compared with either treatment alone, also detectable by Ki-67 immunostaining and 18F-FLT positron emission tomography (PET). In aggregate AKT activation appears to be a key survival mechanism for HDAC6 inhibitor treatment. Our findings indicate that dual inhibition of HDAC6 and P-AKT may be necessary to substantially inhibit growth of solid tumors.

Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma.

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors.

Abstract 2679: Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma

Abstract Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. The most prevalent mechanisms of acquired resistance appear to reactivate MAPK pathway. Furthermore, relatively little is known about the determinants of de novo resistance. Here, we establish such a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: Neel M. Fofaria, Dennie T. Frederick, Ryan J. Sullivan, Keith T. Flaherty, Sanjay K. Srivastava. Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma. [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 2679. doi:10.1158/1538-7445.AM2015-2679

Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy.

Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS.

Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery.

IntroductionTumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death.MethodsThe superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice.ResultsAll nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H = 15.4 kA/m, f = 435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced.ConclusionThe therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorally.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0576-1) contains supplementary material, which is available to authorized users.

A polymeric conjugate foreignizing tumor cells for targeted immunotherapy in vivo

Antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) are key elements of immunological rejection in transplantation as well as cancer immunotherapy. Most tumors, however, are not immunologically rejected because they have self antigens, which are not recognized as the foreigner by CTLs. In this study, we hypothesized that “foreignizing” tumor cells by delivering non-self foreign antigens into the tumors would result in rejection by foreign antigen-reactive CTLs. As the model system to foreignize the tumors, we prepared a polymeric conjugate consisting of hyaluronic acid as the CD44+ tumor-targeting ligand and ovalbumin (OVA) as a foreign antigen. When the conjugate was treated with CD44high TC-1 tumor cells, it was effectively taken up and allowed for displaying of antigenic OVA257–264 peptide at MHC class I on the surface of the cells. In addition, the conjugate was effectively accumulated into tumor tissue after its systemic administration to mice which are immunized with a vaccine for a vaccinia virus expressing OVA to generate OVA257–264 specific CTLs, resulting in substantial inhibition of tumor growth. Overall, these results suggest that the polymeric conjugates bearing foreign antigens may be innovative and promising cancer immunotherapeutic agents by foreignizing tumor cells, leading to immunological rejection.

NT113, a pan-ERBB inhibitor with high brain penetrance, inhibits the growth of glioblastoma xenografts with EGFR amplification.

This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM.

Liposome-coated lipoplex–based carrier for antisense oligonucleotides

The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL‑2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

Abstract 2668: RG7116, a novel humanized anti-HER3 antibody with superior preclinical in vitro and in vivo efficacy in combination with, everolimus and other anti-cancer agents

Abstract HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. HER3 is a kinase dead receptor, but by forming heterodimers with other HER family receptors, HER3 works as amplifier for PI3 kinase driven tumorigenesis. It has been reported that tumors treated with EGFR-, HER2-, cMET-or mTOR targeted therapies can escape via HER3 activation or upregulation. HER3 is expressed in a large variety of tumors for example in non-small cell lung cancer (NSCLC), head and neck, colorectal, gastric, pancreatic, breast, ovarian, thyroid and prostate cancer. Anti-HER3 antibodies can work via various mechanisms including: (1) blocking ligand (HRGs) binding to the receptor, (2) blocking heterodimerization with other HER family members (HER1, 2 and 4), (3) downregulation of the receptor from the cell surface, and (4) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). The first three mechanisms lead to inhibition of HER3 phosphorylation and downstream signaling thereby resulting in tumor cell growth inhibition, while ADCC is a mechanism of direct target cell killing triggered by cross-linking of Fc receptors on immune effector cells (e.g. NK cells, macrophages). RG7116 is a novel humanized and glycoengineered IgG1 antibody currently in clinical development, that binds to HER3 with high affinity. This antibody prevents ligand binding and receptor heterodimerization, thereby blocking receptor phosphorylation. In various tumor xenograft models monotherapy treatment with this antibody leads to substantial tumor growth inhibition. Only in a few cases monotherapy treatment resulted in complete tumor remission. Most of the models initially show convincing efficacy, but tumors regrow after a while. Combination therapy with other HER targeted therapies such as pertuzumab, cetuximab or erlotinib, lead to complete remission in preclinical models. The combination of RG7116 with downstream signaling inhibitors like: everolimus (mTOR) andand other anti-cancer agents leads to increased efficacy. Citation Format: Birgit Bossenmaier, Thomas Friess, Martin Weisser, Stefanie Lechner, Esther Abraham, Monika Hoch, Christian Mirschberger. RG7116, a novel humanized anti-HER3 antibody with superior preclinical in vitro and in vivo efficacy in combination with, everolimus and other anti-cancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2668. doi:10.1158/1538-7445.AM2014-2668

A cell-surface β-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis as a result of widespread intra- and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(asparaginyl)-β-hydroxylase (ASPH) is a cell-surface enzyme that generates enhanced cell motility, migration, invasion, and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer-assisted drug design. Candidate compounds were tested for inhibition of β-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion, and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biological effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor, MO-I-1100, was selected because it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion, and anchorage-independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. These studies suggest that the enzymatic activity of ASPH is important for hepatic oncogenesis. Reduced β-hydroxylase activity generated by the SMI MO-I-1100 leads to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease.

Bortezomib and IL-12 produce synergetic anti-multiple myeloma effects with reduced toxicity to natural killer cells

The aim of this study was to examine the hypothesis that a combination of proteasome inhibition by bortezomib and immune therapy with interleukin-12 (IL-12) can produce enhanced antitumor efficacy relative to the effects of either of these agents alone. A mouse xenograft model of myeloma was developed. The mice were randomly divided into saline control (NS), IL-12 (0.4 µg/animal; intraperitoneal), bortezomib (0.75 mg/kg; intravenous), and bortezomib+IL-12 groups. Effects of treatments on tumor growth were assessed by before and after treatment comparisons and group comparisons. The effects of various treatments on the number of peripheral blood lymphocytes and natural killer (NK) cells were assessed by complete blood count and flow cytometry analysis. The cell-killing function of NK cells in splenocytes was evaluated using the lactate dehydrogenase release assay. IL-12 treatment alone produced a mild decrease in tumor volume compared with control (P>0.05). Bortezomib alone resulted in substantial inhibition of tumor growth at varying time points, reaching ~65 and ~60% reduction in tumor volume after 15 and 21 days of therapy, respectively. At the same time points, the combination therapy produced ~75 and ~84% decreases in tumor growth, respectively, which were significantly greater than the reduction produced by bortezomib monotherapy. Tumors resumed growth upon termination of bortezomib treatment at 2 weeks, although the tumor volume was still significantly smaller than that in the time-matched NS and IL-12 animals. This rebound of tumor growth was completely prevented with the combination therapy, and tumor volume continued to decrease throughout the time course. The percentage and total number of NK cells were significantly decreased after bortezomib monotherapy and combination therapy; however, they remained unaltered after IL-12 treatment compared with no treatment. Further, combination therapy significantly restored the bortezomib-induced functional impairment of the cell-killing capability of NK cells, relative to bortezomib alone. We conclude that the bortezomib-IL-12 combination therapy offers superior antitumor efficacy over monotherapy with either bortezomib or IL-12 in a mouse model of myeloma. Restoration of bortezomib-induced functional impairment of NK cells by IL-12 may be a mechanism for the synergetic effects of the two agents. Therefore, a combination of the two agents may represent a more rational therapeutic approach for myeloma.

The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4), E2F1 and an increase of p21waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK) and suppressed mammalian target of rapamycin (mTOR), the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.

Myeloid Leukemia Cells With MLL partial Tandem Duplication Are Sensitive To Pharmacological Inhibition Of The H3K79 Methyltransferase DOT1L

Genetic alterations of the mixed-lineage leukemia (MLL) gene are commonly identified in acute leukemias. In acute myeloid leukemia (AML), a partial tandem duplication (PTD) of MLL occurs in about 5-10% of AML patients and is associated with adverse prognosis. The mutation leads to an in-frame duplication of exons 5 to 11 resulting in the production of an aberrant MLL protein. Unlike chromosomal rearrangements of MLL, this mutation does not affect the functional histone 3 lysine 4 (H3K4) methyltransferase domain. However, AMLs carrying a MLL-PTD and MLL-rearranged leukemias share some common characteristics, such as overexpression of HOXA-cluster genes and dysregulated epigenetic functions. Recently, leukemias with various MLL-translocations have been shown to be dependent on the histone 3 lysine 79 (H3K79) methyltransferase, DOT1L, and are sensitive to EPZ004777, a recently described selective small-molecule DOT1L inhibitor. EPZ-5676, a DOT1L-inhibitor with improved potency and drug-like properties, has recently been identified and is currently under clinical investigation.To evaluate the therapeutic potential of DOT1L-inhibition in MLL-PTD positive leukemia cells, we assessed the effect of EPZ004777 on the MLL-PTD containing leukemia cell lines MUTZ-11 and EOL-1. In vitro treatment with EPZ004777 over a 14-day period resulted in dramatic reduction of cell proliferation compared to DMSO vehicle control in both cell lines beginning 7 days after the start of treatment. Similar results were obtained for MOLM-13, a leukemia cell line harboring a MLL-translocation, but not for HL-60, a non-MLL-rearranged leukemia cell line. To further investigate whether these findings reflect a selective response to EPZ004777 or non-specific drug toxicity, we first explored the genome-wide H3K79 dimethylation (H3K79me2) profile using chromatin immunoprecipitation (ChIP) followed by next generation sequencing in untreated MUTZ-11 cells. Across the HOXA-cluster locus, we detected a similar H3K79me2 distribution pattern as previously reported in MLL-rearranged leukemias. Further analysis of H3K79me2 in MUTZ-11 and EOL-1 cells after treatment with the inhibitor showed profound suppression of those marks as assessed by western blot and ChIP-PCR. Subsequent global gene expression analysis revealed concurrent downregulation of HOXA-cluster and other MLL-target genes after 7 days of DOT1L inhibition. To investigate the effect of EPZ004777 on the MLL-PTD positive cells in more detail, we analyzed cell differentiation and apoptosis upon a 10-day exposure to the compound. As previously described for EPZ004777-sensitive MLL-rearranged leukemias, drug treatment resulted in increased expression of CD11b and morphological changes consistent with myeloid cell differentiation. We further observed apoptotic cell death after EPZ004777 treatment as measured by an increase in the percentage of Annexin V positive cells and cleaved Caspase 3 protein compared to vehicle controls. In order to determine the effect of DOT1L inhibition in vivo, we tested the recently identified DOT1L-inhibitor, EPZ-5676, for its ability to inhibit leukemia growth in a subcutaneous EOL-1 xenograft model in immunocompromised rats. Similar to what we observed in vitro, continuous intravenous administration over 21 days led to substantial dose-dependent inhibition of tumor growth, abrogation of H3K79me2, and concurrent downregulation of selected MLL-target genes.Thus, we demonstrate unexpected sensitivity of MLL-PTD containing leukemia cell lines to the DOT1L inhibitors EPZ004777 in vitro and EPZ-5676 in vivo. These data suggest that patients with myeloid malignancies carrying this particular mutation might benefit from treatment with therapeutic approaches that target DOT1L. Disclosures:Daigle:Epizyme, Inc: Employment, Equity Ownership. Olhava:Epizyme Inc.: Employment. Pollock:Epizyme Inc.: Employment, Equity Ownership, Patents & Royalties, Stock Options Other. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Epigallocatechin-3 gallate inhibits cancer invasion by repressing functional invadopodia formation in oral squamous cell carcinoma

Although the polyphenol EGCG has various beneficial biological effects, its effect on cytoskeletal activities during cancer invasion is not well defined, and the precise molecular mechanisms are largely unknown. Here, we provide molecular evidence on the anti-invasion effect of EGCG in OSCC cells using an in vitro 3-D culture system and in vivo athymic mouse model. Briefly, EGCG exerted an inhibitory effect on the Matrigel-based Transwell invasion and migration of OSCC cells. These effects were not due to decreased cell viability or adhesion capacity to ECM. EGCG-treated OSCC cells possessed fully extended actin fibers without invadopodia, indicating a loss of ECM degradation capacity. Decreased phosphorylation of Src, CTTN, and FAK also followed EGCG treatment. Additionally, EGCG reduced activation of RhoA in dominant-negative RhoA N19 and constitutively active RhoA Q63E cells, and inhibited the invasive capability of these cells in the 3-D cell growth model. Furthermore, the administration of EGCG led to substantial inhibition of tumor growth and activation of invadopodial proteins in the tumor tissues of mice inoculated with OSCC cells. The data indicate the potential value of EGCG as an invadopodia-targeted anti-invasive agent in cancer therapy.

Abstract 2016: A subset of small cell lung cancer (SCLC) cell lines is Mcl-1-dependent and responds to cyclin-dependent kinase (cdk)9 inhibition in vitro and in vivo

Abstract Small cell lung cancer (SCLC) accounts for approximately 13% of lung cancer cases, and current treatment options are limited to radiation and/or chemotherapy, which frequently lead to drug resistance. Recently, multiple SCLC cell lines have been found to be responsive to the Bcl-2/Bcl-xL inhibitor, ABT-737, both in vitro and in vivo. Clinical trials of the related orally-bioavailable compound ABT-263 have shown promising activity in SCLC patients. However, a subset of SCLC cell lines that express Mcl-1, another anti-apoptotic protein, are resistant to ABT-737. Therefore, drugs targeting Mcl-1 may be particularly useful in this disease. Here, we have investigated dependence of a panel of SCLC cell lines on Mcl-1 for survival. A subset of Mcl-1-expressing SCLC cell lines, including NCI-H82, NCI-N417 and SHP-77, demonstrate apoptosis upon Mcl-1 knockdown. However, other SCLC cell lines, including NCI-H69, NCI-H196 and SW1271, are not responsive to Mcl-1 knockdown. In addition, the dependence of SCLC cell lines on Mcl-1 for survival can be predicted by BH3 profiling, a methodology that measures dependence of a cell on a variety of anti-apoptotic proteins. We have also investigated the efficacy of Flavopiridol, a potent inhibitor of the transcriptional cyclin-dependent kinase (cdk)9, against the panel of SCLC cell lines. Flavopiridol acutely down-regulates Mcl-1 expression in all SCLC cell lines tested; however, only Mcl-1-dependent cells, including NCI-H82, NCI-N417 and SHP-77, undergo abrupt apoptosis. In contrast, cells that were not found to be Mcl-1 dependent are not effectively killed by Flavopiridol. We have further investigated the activity of Flavopiridol against Mcl-1-dependent SCLC in vivo utilizing nude mice bearing NCI-H82 xenografts. Flavopiridol treatment results in substantial tumor growth inhibition in this aggressive and chemotherapy-refractory model. To potentially stratify patients who would be predicted to be responsive to Flavopiridol treatment, we have performed MCL-1 gene fluorescence in situ hybridization in SCLC cell lines and deidentified patient specimens. MCL-1 gene copy number gains exist in both patient and cell line samples, and copy number gain of MCL-1 or MYC in cell lines correlates with Mcl-1 dependence. Strategies for targeting MCL-1 and MYC copy number gains will be discussed further. Finally, we have demonstrated that the combination of Flavopiridol and ABT-737 significantly reduces viability in Mcl-1-independent SW1271 cells compared to treatment with either drug alone. Taken together, our studies suggest that Flavopiridol-mediated cdk9 inhibition may effectively target Mcl-1-dependent SCLCs, including those resistant to Bcl-2/Bcl-xL inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2016. doi:1538-7445.AM2012-2016

Abstract 1776: Antitumor activity of Chk1 inhibitor LY2606368 as a single agent in SW1990 human pancreas orthotopic tumor model

Abstract ChK1 is a key kinase in the DNA damage response signaling network, including cell cycle checkpoints, DNA repair, apoptosis and transcription, thus emerging as an attractive target in anti-cancer therapy. LY2606368, a potent and selective ATP competitive inhibitor of the Chk1 protein kinase, is currently in clinical development. LY2606368 has been reported to inhibit Chk1 auto-phosphorylation activated by DNA damaging agents, and induce phosphorylation of H2AX, a DNA damage maker in multiple cancer cell lines in vitro. In addition, LY2606368 has demonstrated potent single agent activity and potentiates the anti-tumor activity of DNA damaging agents in vivo. Pancreatic cancer is one of the least curable cancers, with an approximate 5% overall 5-year survival for all patients. In the current study, we evaluated the efficacy of LY2606368 (as its methanesulfonate salt, hydrate; LY2606368.MeSO3H.H2O = LY2940930) in pancreatic cancer cell lines in vitro and xenograft tumors (including subcutaneous and orthotopic models) in vivo. LY2606368 alone significantly inhibited the cell proliferation in a variety of pancreatic cell lines (SW1990, SU86.86, Bx-PC3, AsPC-1, CFPAC-1, Capan-2, HPAF-II) with SW1990 being the most sensitive (IC50=1.5 nM). In SW1990 subcutaneous xenograft model, LY2606368 showed substantial dose-dependent inhibition of tumor growth. In SW1990 pancreas orthotopic model, which represents the local and metastatic growth pattern seen in pancreas cancer patients, LY2606368 treatment resulted in over 92% inhibition of primary tumor growth as well as 100% inhibition of metastasis to lymph node, spleen and intestine. The anti-tumor effect of LY2606368 treatment was further demonstrated in comparing with gemcitabine (the standard of care for pancreas cancer patient) in SW1990 orthotopic model. The anti-tumor and/or anti-metastasis mechanism of LY2606368 was also investigated, including phosphorylation of H2AX, cell proliferation, cell survival, and soft agar anchorage-independent growth. Together, our findings demonstrate the therapeutic potential of LY2606368 as a single agent in the treatment of pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1776. doi:1538-7445.AM2012-1776

Abstract 2508: GE-huMab-HER3, a novel humanized, glycoengineered HER3 antibody with enhanced ADCC and superior preclinical in vitro and in vivo efficacy

Abstract HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. HER3 is a kinase dead receptor, but by forming heterodimers with other HER family receptors, HER3 works as amplifier for PI3 kinase driven tumorigenesis. It has been reported that tumors treated with EGFR-, HER2- or cMET-targeted therapies can escape via HER3 activation or upregulation. HER3 is expressed in a large variety of tumors for example in non-small cell lung cancer (NSCLC), head and neck, colorectal, gastric, pancreatic, breast, ovarian and prostate cancer. Anti-HER3 antibodies can work via various mechanisms including: (1) blocking ligand (HRGs) binding to the receptor, (2) blocking heterodimerization with other HER family members (HER1, 2 and 4), (3) downregulation of the receptor from the cell surface, and (4) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). The first three mechanisms lead to inhibition of HER3 phosphorylation and downstream signaling thereby resulting in tumor cell growth inhibition, while ADCC is a mechanism of direct target cell killing triggered by cross-linking of Fc receptors on immune effector cells (e.g. NK cells, macrophages). GE-huMab-HER3 is a novel humanized and glycoengineered IgG1 antibody that binds to HER3 with high affinity. This antibody prevents ligand binding and receptor heterodimerization thereby blocking receptor phosphorylation. In various tumor xenograft models treatment with this antibody leads to substantial tumor growth inhibition. E.g. GE-huMab-HER3 treatment achieved >50% tumor growth inhibition in 10 out of 17 NSCLC models and in some cases even resulted in complete tumor remission. However, these xenograft experiments only reveal part of this antibody's therapeutic potential. A unique feature of GE-huMab-HER3 that differentiates it from other anti-HER3 antibodies, including AMG 888 and MM-121, is its ability to bind to human FcgRIIIa on immune effector cells with a 50-fold higher affinity than standard IgG1 antibodies, a property conferred by the engineered glycosylation of the antibody Fc region. Consequently, GE-huMab-HER3 exhibits superior potency and efficacy in ADCC, as shown in vitro using recombinant A549 cells and in vivo by its Fc mediated greater anti-tumor effect in A549 orthotopic mouse models compared to a non-glyco-engineered variant of the antibody, WT-huMab-HER3. The combination of strong signaling inhibition and enhanced ADCC capability renders GE-huMabHER3 a highly potent HER3-targeting agent. Phase I clinical testing of this promising novel compound is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2508. doi:1538-7445.AM2012-2508