Survival Of Tumor-bearing Animals Research Articles

A novel CPT1A covalent inhibitor modulates fatty acid oxidation and CPT1A-VDAC1 axis with therapeutic potential for colorectal cancer

Colorectal cancer (CRC) is a common and deadly disease of the digestive system, but its targeted therapy is hampered by the lack of reliable and specific biomarkers. Hence, discovering new therapeutic targets and agents for CRC is an urgent and challenging task. Here we report that carnitine palmitoyltransferase 1A (CPT1A), a mitochondrial enzyme that catalyzes fatty acid oxidation (FAO), is a potential target for CRC treatment. We show that CPT1A is overexpressed in CRC cells and that its inhibition by a secolignan-type compound, 2,6-dihydroxypeperomin B (DHP-B), isolated from the plant Peperomia dindygulensis, suppresses tumor cell growth and induces apoptosis. We demonstrate that DHP-B covalently binds to Cys96 of CPT1A, blocks FAO, and disrupts the mitochondrial CPT1A-VDAC1 interaction, leading to increased mitochondrial permeability and reduced oxygen consumption and energy metabolism in CRC cells. We also reveal that CPT1A expression correlates with the survival of tumor-bearing animals and that DHP-B exhibits anti-CRC activity in vitro and in vivo. Our study uncovers the molecular mechanism of DHP-B as a novel CPT1A inhibitor and provides a rationale for its preclinical development as well as a new strategy for CRC targeted therapy.

Oncolytic and protective effects of strain 100 of a new group of non-classified rotavirus of reoviridae family on B16/F10 melanoma growth in experiment.

e21587 Background: The purpose of this study was to assess the effect of a strain 100 of a new group of rotavirus of Reoviridae family on the growth of transplantable B16/F10 melanoma in mice and the survival of the animals. Methods: We studied the strain 100 of a new group of rotavirus of Reoviridae family (RVK) ( http://jbks.ru/archive/issue-10/article-6 ). RVK 100 is a live attenuated non-pathogenic virus growing on pig embryo kidney cell culture with concentration 5·109 per 1 ml. The dynamics of melanoma growth and the survival of tumor-bearing animals receiving RVK were studied in 25 male mice divided into 5 equal groups, each n = 5. RVK was administered intramuscularly and orally once a week 0.3 mL and 25 µL, respectively: to groups 1-2 21 days prior to the tumor inoculation, a total of 4 injections (“vaccination” regimen); to groups 3-4 – 7 days after tumor inoculation with the onset of tumor nodes, a total of 3 injections (“treatment” regimen); group 5 included controls receiving 0.85% NaCl solution. Groups 1 and 3 received live RVK, groups 2 and 4 - RVK inactivated by UV radiation. Results: Survival of mice in groups 1-2 receiving both live and inactivated RVK in the “vaccination” regimen was increased statistically significantly: in group 1, Me = 25 days after melanoma transplantation (LQ 24; UQ 30), in group 2 Me = 26 days (LQ 25; UQ 30), in controls Me = 14 days (LQ 10; UQ 22); p = 0.032 and p = 0.0079, respectively. RVK administration in the “treatment” regimen did not cause such changes. While mice of group 3 receiving the live strain 100 showed a tendency to higher survival, compared to controls (Me = 23 days, LQ 22; UQ 24, p = 0.056), the administration of inactivated RVK (group 4) did not significantly improve the survival of animals, compared to controls (Me = 22 days (LQ 21; UQ 33, p = 0.151). Conclusions: Improved survival of animals with B16/F10 melanoma after the RVK administration indicates the virus ability to slow down tumor growth and confirms our previously published data on the RVK strain 228, although, in contrast to it, the strain 100 is able to increase the survival rate of tumor-bearing mice only when used in the “vaccination” regimen, which refers mainly to the inactivated virus. Since the inactivation of the RVK strain 100 eliminates its effect when used in the “treatment”, but not “vaccination” regimen, it seems to be associated with an immunostimulatory effect but not with an oncolytic one.

Activation of the Hedgehog pathway mediates resistance to epidermal growth factor receptor inhibitors in non-small cell lung cancer.

The current study aimed to investigate the function of the Hedgehog pathway and its association with epithelial-mesenchymal transition (EMT) in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). Lung tumor tissue specimens from EGFR TKI-resistant patients, including those with brain metastases, had hyperactive Hedgehog signaling compared with those from TKI-sensitive patients. SHH stimulation promoted GLI1 activation as well as cell motility in parental PC9 cells while suppressing gefitinib-induced apoptosis in gefitinib-resistant cells. SHH also promoted EMT in parental PC9 cells via E-cadherin suppression and N-cadherin and vimentin upregulation. The knockdown of GLI1 exhibited the opposite effects. Besides, SHH induced, whereas GLI1 knockdown reversed gefitinib resistance in xenograft tumors. The Hedgehog pathway inhibitor GDC-0449 synergized with gefitinib to increase xenograft tumor sensitivity to chemotherapy and extend survival in tumor-bearing animals. These results suggest the Hedgehog pathway mediates EGFR TKI resistance and induces EMT in NSCLC, representing a potential therapeutic target to defeat TKI resistance.

Targeting extracellular matrix remodeling sensitizes glioblastoma to ionizing radiation.

BackgroundThe median survival of Glioblastoma multiforme (GBM) patients is 14+ months due to poor responses to surgery and chemoradiation. Means to counteract radiation resistance are therefore highly desirable. We demonstrate the membrane bound matrix metalloproteinase MT1-MMP promotes resistance of GBM to radiation, and that using a selective and brain permeable MT1-MMP inhibitor, (R)-ND336, improved tumor control can be achieved in preclinical studies.MethodsPublic microarray and RNA-sequencing data were used to determine MT1-MMP relevance in GBM patient survival. Glioma stem-like neurospheres (GSCs) were used for both in vitro and in vivo assays. An affinity resin coupled with proteomics was used to quantify active MT1-MMP in brain tissue of GBM patients. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP and inhibition via the MT1-MMP inhibitor (R)-ND336, were used to assess the role of MT1-MMP in radio-resistance.ResultsMT1-MMP expression inversely correlated with patient survival. Active MT1-MMP was present in brain tissue of GBM patients but not in normal brain. shRNA- or (R)-ND336-mediated inhibition of MT1-MMP sensitized GSCs to radiation leading to a significant increase in survival of tumor-bearing animals. MT1-MMP depletion reduced invasion via the effector protease MMP2; and increased the cytotoxic response to radiation via induction of replication fork stress and accumulation of double strand breaks (DSBs), making cells more susceptible to genotoxic insult.ConclusionsMT1-MMP is pivotal in maintaining replication fork stability. Disruption of MT1-MMP sensitizes cells to radiation and can counteract invasion. (R)-ND336, which efficiently penetrates the brain, is therefore a novel radio-sensitizer in GBM.

RARE-08. POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS IDENTIFIED THROUGH HIGH THROUGHPUT DRUG SCREENING

Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12-months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,570 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds - auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine - that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in vitro. To determine whether the in vitro efficacy could be replicated in vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide, auranofin and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different fenretinide formulations which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

To promote or inhibit glioma progression, that is the question for IL-33.

IL-33, a member of the IL-1 cytokine family has been shown to play a dual role within the body. First IL-33, similar to other IL-1 family members, is a secreted cytokine that binds to the cell surface receptor ST2 to induce a number of cell signaling pathways. Second, IL-33 enters the nucleus where it binds chromatin and directs transcriptional control of an array of growth factors and cytokines. Consistent with its complex cellular regulation, IL-33 mediates an array of biological functions by acting on a wide range of innate and adaptive immune cells. Recently, we found that IL-33 is expressed in a large number of human glioma patient specimens where its expression within the tumor correlates with the increased presence of Iba+ cells that include both resident microglia and recruited monocyte and macrophages. Strikingly, glioma derived expression of IL-33 correlates with a dramatic decrease in overall survival of tumor-bearing animals and thus supports its role as an influential factor in gliomagenesis. Notably however, when the nuclear localization function of IL-33 is crippled, the tumor microenvironment is programmed to be anti-tumorigenic and results in prolonged overall survival suggesting that when educated appropriately this could represent a novel therapeutic strategy for glioma (De Boeck et al. (2020), Nat Commun, doi: 10.1038/s41467-020-18569-4).

Loss of COPZ1 induces NCOA4 mediated autophagy and ferroptosis in glioblastoma cell lines

Dysregulated iron metabolism is a hallmark of many cancers, including glioblastoma (GBM). However, its role in tumor progression remains unclear. Herein, we identified coatomer protein complex subunit zeta 1 (COPZ1) as a therapeutic target candidate which significantly dysregulated iron metabolism in GBM cells. Overexpression of COPZ1 was associated with increasing tumor grade and poor prognosis in glioma patients based on analysis of expression data from the publicly available database The Cancer Genome Atlas (P < 0.001). Protein levels of COPZ1 were significantly increased in GBM compared to non-neoplastic brain tissue samples in immunohistochemistry and western blot analysis. SiRNA knockdown of COPZ1 suppressed proliferation of U87MG, U251 and P3#GBM in vitro. Stable expression of a COPZ1 shRNA construct in U87MG inhibited tumor growth in vivo by ~60% relative to controls at day 21 after implantation (P < 0.001). Kaplan–Meier analysis of the survival data demonstrated that the overall survival of tumor bearing animals increased from 20.8 days (control) to 27.8 days (knockdown, P < 0.05). COPZ1 knockdown also led to the increase in nuclear receptor coactivator 4 (NCOA4), resulting in the degradation of ferritin, and a subsequent increase in the intracellular levels of ferrous iron and ultimately ferroptosis. These data demonstrate that COPZ1 is a critical mediator in iron metabolism. The COPZ1/NCOA4/FTH1 axis is therefore a novel therapeutic target for the treatment of human GBM.

DIPG-07. HIGH THROUGHPUT DRUG SCREENING IDENTIFIES POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS (DIPGs)

DIPGs are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12 months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,500 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds- auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine- that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in-vitro. To determine whether the in-vitro efficacy could be replicated in-vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different Fenretinide formulations (LYM-X-Sorb and NanoMicelle) which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Antitumor Efficacy of PD-1 Blockade.

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade (ICB), responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of cotreatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit antitumor immunity, and limit the efficacy of ICB. In this study, we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow-derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the antitumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti-PD-1 monotherapy, increasing tumor-specific cytotoxic T cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that cotreatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in patients with cancer with resistant tumors.

P11.42 Magnetic targeting of the granzyme B functionalized nanoparticles for therapy of glioblastoma

Abstract BACKGROUND Magnetic drug targeting by employing external magnetic fields represents a promising approach for treatment of glioblastoma. To increase the anti-tumor therapeutic effect magnetic nanocarriers could be functionalized with tumor-targeting bioligands such as granzyme B (GrB). The serine protease GrB that is produced as an effector molecule by activated NK and T cells can induce specific tumor cell apoptosis. MATERIAL AND METHODS The targeting potential of the dextran-coated superparamagnetic iron oxide nanoparticles conjugated with GrB (GrB-SPIONs) was assessed in glioblastoma cells (U87, C6, GL261) by flow cytometry, confocal and electron microscopies. Magnetic field strength (320 mT) in the models of U87 glioblastoma in NMRI nu/nu mice and C6 glioma in Wistar rats was achieved by employing NdFeB cylindrical ferromagnet. The irradiation of the implanted tumors was performed using the CT-image guided Small Animal Radiation Research Platform (SARRP). Accumulation of the nanoparticles was assessed in paraffine-embedded specimens employing Prussian blues staining. Sections were additionally analyzed by IHC for apoptosis (caspase 3). RESULTS Synthesized GrB-SPIONs incorporated into the cytoplasm of tumor cells via the endolysosomal pathway and induced apoptosis in a dose-dependent manner. Intravenous injection of GrB-SPIONs resulted in the glioma-specific retention of the nanoparticles as was shown by high-field (11 T) magnetic resonance imaging and biodistribution studies (NLR-M2 measurements). Magnetic targeting of the nanoparticles in vivo drastically enhanced the accumulation of nanoparticles to the location of the magnet. Thus the local retention of nanoparticles was 12.3-fold higher as compared to application of GrB-SPIONs without external magnetic field. The presence of granzyme B on SPIONs has been shown to promote tumor cells death (IHC staining for caspase 3) and significantly increased overall survival. A combination of nanoparticle treatment with a single radiation dose (10 Gy) significantly prolonged the survival of rats as compared to animals treated only with magnetic targeting of GrB-SPIONs or radiotherapy alone (P&lt;0.001). CONCLUSION Single-agent therapy with GrB-SPIONs demonstrated an impressive increase in overall survival of tumor-bearing animals. Combinatorial regimen employing magnetic targeting and stereotactic radiotherapy further enhanced the therapeutic potential of magnetic conjugates.

Sequential combination of cisplatin with eugenol targets ovarian cancer stem cells through the Notch-Hes1 signalling pathway

BackgroundOvarian carcinomas are the deadliest gynecological malignancies owing to their high rate of recurrence and high resistance to platinum-based chemotherapy. Recent studies have shown platinum-dependent enrichment of ovarian tumors with side population as well as cancer stem cells, which are highly resistant to the treatment. To overcome this treatment-limiting factor, we sought to combine cisplatin with eugenol, a natural substance known to have anti-cancer effects.MethodsThe efficiency of combining cisplatin with eugenol was first tested in vitro on two ovarian cancer cell lines SKOV3 and OV2774 using the WST1 and the flow cytometry techniques. The effect of this combination on ovarian cancer stem cells was determined by the tumorsphere formation assay, while the implication of the Notch pathway was evaluated post-ectopic expression of the Hes1 gene. The resulting changes in the expression of several markers was assessed by immunoblotting, immunofluorescence as well as quantitative RT-PCR. Cell sorting was also used to isolate specific ovarian cancer sub-population of cells. Furthermore, tumor-bearing mouse models were utilized to prove the potential therapeutic value of the cisplatin/eugenol combination treatment in vivo.ResultsWe have shown that adding eugenol to cisplatin-treated ovarian cancer cells synergistically inhibited their growth and survival through induction of apoptosis. Importantly, this sequential inhibition strongly reduced the proportion of side population cells and suppressed cisplatin-dependent enrichment in ovarian cancer stem cells. Additionally, while increase in the level of Hes1 promoted stemness and enhanced resistance to cisplatin, cisplatin/eugenol cotreatment inhibited the Notch-Hes1 pathway and strongly downregulated the drug resistance ABC transporter genes. These findings were confirmed in vivo by showing that cisplatin/eugenol cotherapy inhibited tumor growth in animals, reduced the proportion and self-renewal capacities of cancer stem cells and significantly improved disease-free survival of tumor-bearing animals compared with either therapy alone.ConclusionsThese results indicate that cisplatin/eugenol sequential combination could be of great therapeutic value for ovarian cancer patients through targeting the Notch-Hes1 pathway and the consequent elimination of the resistant cancer stem cells.

Abstract 4377: Liver X receptor mediated lipotoxicity represents a treatment option for liver cancer

Abstract Solid tumors evolve significant changes in metabolic pathways during development by virtue of their specific biosynthetic demands, their particular microenvironment and the potential occurrence of toxic metabolites such as reactive oxygen species. However, the development of cancer treatment approaches that are based on the inhibition of biosynthetic routes is impaired due to the high plasticity of metabolic networks, e.g. resulting in activation of compensatory pathways or in an increased exchange of metabolites between cancer cells and the tumor environment. Therefore, such therapies could not be translated into efficient clinical applications so far. Here we show that an enhanced lipogenesis, triggered by a pharmacological activation of the Liver X receptor (LXR), represents a new therapeutic strategy for the treatment of liver carcinoma (HCC). A combination of LXR mediated fatty acid synthesis and concomitant Raf suppression results in oxidative stress, induction of a critical ER stress response and subsequently in apoptosis of different murine and human liver cancer cells. Our mechanistic studies identified Raf as an important regulator of lipid metabolism in liver cancer. We found that Raf-1 directly interacts with Stearoyl-CoA desaturase-1 (Scd1), the central enzyme for the conversion of saturated into mono-unsaturated fatty acids and thereby maintains Scd1 protein stability in HCC cells. Thus, inhibition of Raf by Sorafenib diminished Scd1 protein abundance leading to toxic accumulation of saturated fatty acids and metabolic stress in cancer cells under sustained lipogenesis. Treatment studies in autochthonous liver cancer mouse models and xenograft models of human HCC revealed that a combinatorial therapy, consisting of the LXR agonist T0901317 and Sorafenib is highly potent to suppress liver cancer development and to extend the survival of tumor bearing animals. Such a therapy was efficient against hepatic neoplasia with different metabolic phenotypes and well tolerated by mice, even by animals that already suffer from a fatty liver disease. Taken together, we here propose a pharmacologically induced accumulation of toxic metabolites in cancer cells as a new strategy for efficient metabolic targeting of therapy refractory solid tumors. Citation Format: Ramona Rudalska, Jule Harbig, Marteinn Snaebjoernsson, Lyudmyla Taranets, Florian Heinzmann, Stefan Zwirner, Wei Ciu Hu, Thales Kronenberger, Tae-Won Kang, Antti Poso, Stefan Laufer, Mathias Rosenfeldt, Nisar P. Malek, Bernd Pichler, Nikita Popov, Almut Schulze, Lars Zender, Daniel Dauch. Liver X receptor mediated lipotoxicity represents a treatment option for liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4377.

Abstract 3670: Treatment of epithelial ovarian cancer with folate receptor (α/β) targeted chemotherapy is enhanced by CTLA-4 blockade: Learning from animal models

Abstract Epithelial ovarian cancer (EOC) remains to be the most lethal form of gynecologic malignancies despite aggressive cytoreductive surgery (i.e. debulking) followed by standard chemotherapy. The alpha folate receptor (FRα) has long been recognized as a tumor target for EOC due to its frequently intense overexpression (&amp;gt;80%) in the most common serous histotype. Recently, the beta folate receptor (FRβ) has been discovered on tumor-associated macrophages (TAMs) which are present at various densities in solid tumors but are also found abundant in malignant ascites of EOC patients. The ascites TAMs are strongly immunosuppressive, and high TAM frequency is correlated with poor patient prognosis and treatment resistance. Respectively, tumor- and TAM-associated FRα and FRβ are both functionally active which allows for folate-targeted delivery of small-molecule drug payloads via receptor-mediated endocytosis. Using flow cytometry, we characterized total ascites cells harvested from human IGROV1 xenograft (hFRα) and murine ID8-CL15 syngeneic (mFRα) models at the time of euthanasia. Approximately 70% of IGROV1 and 25% of ID8-Cl15 ascites TAMs (F4/80+ CD11b+) were found to express a functional mFRβ. While ascites fluids from both models caused a dramatic reduction of FRα functionality on tumor cells in-vivo and ex-vivo, FRβ expression and functionality on ascites TAMs were largely unaffected. Interestingly, in the ID8-CL15 model, the ratio of ascites CD8+ T cells to CD4+ T cells decreased as total FR functionality on TAMs increased. Two small-molecule folate-drug conjugates with different mechanisms of action showed activity in extending the survival of tumor-bearing animals. While CTLA-4 blockage alone was completely inactive in the ID8-CL15 model, a combination regimen significantly enhanced the activity of a folate conjugate carrying a potent DNA cross-linking agent. Our data suggest that EOC treatment may require a multi-pronged approach by targeting both FRα-expressing tumor cells and FRβ-expressing TAMs. In addition, immune checkpoint inhibitors may further enhance the FR-targeted therapeutics by modulating the ascites tumor microenvironment. Citation Format: Yingjuan Lu, Theresa P. Johnson, Leroy W. Wheeler, Alex M. Lloyd, Vicky A. Cross, Elaine M. Westrick, Nikki L. Parker, Christopher P. Leamon. Treatment of epithelial ovarian cancer with folate receptor (α/β) targeted chemotherapy is enhanced by CTLA-4 blockade: Learning from animal 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 3670. doi:10.1158/1538-7445.AM2017-3670

Anti-CD47 Antibody As a Targeted Therapeutic Agent for Human Lung Cancer and Cancer Stem Cells.

Accumulating evidence indicates that a small subset of cancer cells, termed the tumor-initiating cells or cancer stem cells (CSCs), construct a reservoir of self-sustaining cancer cells with the characteristic ability to self-renew and maintain the tumor mass. The CSCs play an important role in the tumor initiation, development, relapse, metastasis, and the ineffectiveness of conventional cancer therapies. CD47 is a ligand for signal-regulatory protein-α expressed on phagocytic cells and functions to inhibit phagocytosis. This study was to explore if the expression of CD47 is the mechanism used by lung cancer cells, especially CSCs, to escape phagocytosis in vitro and in vivo. Here, we selected CD133 as the marker for lung CSCs according to previous reports. We analyzed lung cancer and matched adjacent normal (non-tumor) tissue and revealed that CD47 is overexpressed on lung cancer cells, especially on lung CSCs. The mRNA expression levels of CD47 and CD133 correlated with a decreased probability of survival for multiple types of lung cancer. Blocking CD47 function with anti-CD47 antibodies enabled macrophage phagocytosis of lung cancer cells and lung CSCs. Anti-CD47 antibodies inhibited tumor growth in immunodeficient mouse xenotransplantation models established with lung cancer cells or lung CSCs and improved survival in tumor-bearing animals. These data indicate that CD47 is a valid target for cancer therapies, especially for anti-CSC therapies.

Tissue-protective activity of selenomethionine and D-panthetine in B16 melanoma-bearing mice under doxorubicin treatment is not connected with their ROS scavenging potential.

AimTo evaluate molecular mechanisms of tissue-protective effects of antioxidants selenomethionine (SeMet) and D-pantethine (D-Pt) applied in combination with doxorubicin (Dx) in B16 melanoma-bearing-mice.MethodsImpact of the chemotherapy scheme on a survival of tumor-bearing animals, general nephro- and hepatotoxicity, blood cell profile in vivo, and ROS content in B16 melanoma cells in vitro was compared with the action of Dx applied alone. Nephrotoxicity of the drugs was evaluated by measuring creatinine indicator assay, hepatotoxicity was studied by measuring the activity of ALT/AST enzymes, and myelotoxicity was assessed by light microscopic analysis of blood smears. Changes in ROS content in B16 melanoma cells under Dx, SeMet, and D-Pt action in vitro were measured by incubation with fluorescent dyes dihydrodichlorofluoresceindiacetate (DCFDA, H2O2-specific) and dihydroethidium (DHE, O2--specific), and further analysis at FL1 (DCFDA) or FL2 channels (DHE) of FACScan flow cytometer. The impact of aforementioned compounds on functional status of mitochondria was measured by Rhodamine 123 assay and further analysis at FL1 channel of FACScan flow cytometer.ResultsSelenomethionine (1200 µg/kg) and D-pantethine (500 mg/kg) in combination with Dx (10 mg/kg) significantly reduced tumor-induced neutrophilia, lymphocytopenia, and leukocytosis in comparison to Dx treatment alone. Moreover, SeMet and D-Pt decreased several side effects of Dx, namely an elevated creatinine level in blood and monocytosis, thus normalizing health conditions of B16 melanoma-bearing animals.ConclusionsOur results showed that antioxidants selenomethionine and D-pantethine possess significant nephroprotective and myeloprotective activity toward Dx action on murine B16 melanoma in vivo, but fail to boost a survival of B16 melanoma-bearing animals. The observed cytoprotective effects of studied antioxidants are not directly connected with their ROS scavenging.

Abstract B49: Sustained IFNalpha treatment inhibits metastasis in 4T1 mammary carcinoma and correlates with decreasing myeloid-derived suppressor cells

Abstract In order to model pharmacokinetics and pharmacodynamics of pegylated IFNalpha, we utilized hydrodynamic delivery technology using minicircle DNA vectors encoding universal IFNalpha as a rapid and cost-effective method to obtain sustained levels of serum IFNalpha2alpha1. This approach is particularly useful for delivering secreted molecules, such as cytokines, as transfected hepatocytes secrete the transgene product in a paracrine manner providing a serum exposure profile resembling that reported in PEG-intron-treated patients. We found that sustained exposure of IFNalpha2alpha1 induced inhibition of metastasis. Primary 4T1 tumor growth was partially inhibited, while significant decreases in pulmonary metastatic burden were consistently observed. Further evidence that the observed inhibition of metastasis was independent of potential effects on the primary tumor was tested by resecting the primary tumor prior to treatment, which did not alter the outcome. Importantly, sustained IFNalpha2alpha1 treatment correlated with a significant increase in the survival of tumor-bearing animals. IFNalpha effects are plieotropic resulting in potential activation of numerous immune cell types associated with both innate and adaptive immunity. However, the mechanisms associated with IFNalpha-induced inhibition of lung metastasis were largely engendered by innate immune cells since treatment in BALB/c mice deficient for T and B cells did not affect the capacity of IFNalpha to inhibit pulmonary metastasis when compared to wild-type animals. The importance of NK cells has recently been reported as an important innate component for IFNalpha induced reduction of metastatic burden. However, little is known about IFNalpha-regulation of other innate cell types that may contribute to the inhibition of tumor metastasis. Here we report IFNalpha2alpha1 treatment induced marked decreases in 4T1-induced expansion and activation of immature CD11b+ Gr-1+ myeloid cells (aka: MDSC), which have been reported to contribute to metastatic potential. These results correlated with similar decreases in metastatic burden in mice following chlodronate-liposome depletion of CD11b+ Gr-1+ myeloid cells. Citation Format: Christina Kochel, Kalyan Pande, Roanna Ueda, Dan Gorman, Drake Laface. Sustained IFNalpha treatment inhibits metastasis in 4T1 mammary carcinoma and correlates with decreasing myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B49.

Abstract B30: Regulation of the core pre-mRNA splicing machinery by MYC and PRMT5 is essential to sustain lymphomagenesis

Abstract Deregulated expression of the MYC transcription factor occurs in a majority of human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Over-expressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown. Here, we show that during lymphomagenesis in Eµ-myc transgenic mice, MYC directly up-regulates the transcription of the core snRNP assembly genes, including Prmt5, an arginine methyltransferase that methylates Sm proteins. This coordinated regulatory effect is critical for snRNP biogenesis, effective mRNA splicing, cell survival and proliferation. Indeed, Prmt5 haplo-insufficiency significantly delays lymphoma onset in Eµ-myc transgenic mice and the acute deletion of PRMT5 in established lymphomas enhances the survival of tumor-bearing animals. In human lymphomas and leukemias, the expression of core snRNP genes also correlates with that of MYC, and PRMT5 knockdown results in aberrant pre-mRNA splicing and loss of viability. In both human or mouse lymphomas cells, MYC knockdown also reduces the expression of core snRNP components, causing aberrant pre-mRNA splicing. Finally, we identify pre-mRNAs that are particularly sensitive to perturbation of the MYC-PRMT5 axis, resulting in either intron retention or exon skipping. Using antisense oligonucleotides (ASOs), we mimic these splicing defects and demonstrate their individual contribution to the anti-proliferative/apoptotic phenotype observed in Eµ-myc B-cells. Thus, coordinate regulation of the core snRNP assembly machinery and PRMT5 is critical for the maintenance of normal pre-mRNA splicing and cell survival upon oncogenic activation of c-myc, pointing to PRMT5 methyl-transferase activity as a potential therapeutic target in MYC-driven tumors. Citation Format: Cheryl Koh, Marco Bezzi, Diana Low, WeiXia Ang, ShunXie Teo, Florence Gay, Muthafar Al Haddawi, SooYong Tan, Motomi Osato, Arianna Sabo', KengBoon Wee, Bruno Amati, Ernesto Guccione. Regulation of the core pre-mRNA splicing machinery by MYC and PRMT5 is essential to sustain lymphomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B30.

Human neural stem cells expressing carboxyl esterase target and inhibit tumor growth of lung cancer brain metastases

Neural stem cells (NSCs) led to the development of a novel strategy for delivering therapeutic genes to brain tumors. Human NSCs expressing rabbit carboxyl esterase (F3.CE), which activates CPT-11, significantly inhibit the growth of A549 human non-small cell lung adenocarcinoma cells in the presence of CPT-11 in vitro and in vivo. F3.CE cells migrated selectively into the brain metastases located in the opposite hemisphere. The treatment also significantly decreased tumor volume in immune-deficient mice bearing lung cancer when F3.CE cells were transplanted into the contralateral hemisphere. The survival of tumor-bearing animals was significantly prolonged by the treatment with F3.CE and CPT-11. This strategy could be considered as an effective treatment regimen for lung cancer brain metastases.

Abstract LB-36: JAK2/STAT5 inhibition circumvents resistance to PI3K/mTOR blockade: A rationale for co-targeting these pathways in metastatic breast cancer.

Abstract Hyperactive PI3K/mTOR signaling is prevalent in the majority of human malignancies (1) and its inhibition exhibits potent antitumor activity in a wide spectrum of solid cancers. Unfortunately, single-agent targeted cancer therapy is usually short-lived and thwarted by different resistance mechanisms (2). Here, we report the discovery of a JAK2/STAT5-evoked positive feedback loop that causes adaptive resistance to dual PI3K/mTOR inhibition. Mechanistically, PI3K/mTOR inhibition increased IRS1-dependent activation of JAK2/STAT5 and secretion of IL-8 in several cell lines and primary triple-negative breast tumors. Genetic or pharmacological inhibition of JAK2 abrogated this vicious feedback loop. Combined PI3K/mTOR and JAK2 inhibition synergistically reduced cancer cell viability in vitro as well as tumor growth in vivo, and decreased tumor seeding and metastasis due to its impact on the IL-8 receptor CXCR1+ tumor-initiating subpopulation of cells. We further found that combined PI3K/mTOR and JAK2 inhibition increased event-free as a well as overall survival of tumor bearing animals. This study reveals a new link between growth factor signaling, JAK/STAT activation, cytokine secretion and metastasis. Our results provide a rationale for combined targeting of the PI3K/mTOR and JAK2/STAT5 pathways in triple-negative breast cancer, a particularly aggressive and currently incurable disease. Citation Format: Adrian Britschgi, Rita Andraos, Heike Brinkhaus, Inna Klebba, Masato Murakami, Vincent Romanet, Urs Müller, Thomas Radimerski, Mohamed Bentires-Alj. JAK2/STAT5 inhibition circumvents resistance to PI3K/mTOR blockade: A rationale for co-targeting these pathways in metastatic breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-36. doi:10.1158/1538-7445.AM2013-LB-36

The androgen receptor: a biologically relevant vaccine target for the treatment of prostate cancer

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. However, while it has long been the primary molecular target of metastatic prostate cancer therapies, it has not been explored as an immunotherapeutic target. In particular, the AR ligand-binding domain (LBD) is a potentially attractive target, as it has an identical sequence among humans as well as among multiple species, providing a logical candidate for preclinical evaluation. In this report, we evaluated the immune and anti-tumor efficacy of a DNA vaccine targeting the AR LBD (pTVG-AR) in relevant rodent preclinical models. We found immunization of HHDII-DR1 mice, which express human HLA-A2 and HLA-DR1, with pTVG-AR augmented AR LBD HLA-A2-restricted peptide-specific, cytotoxic immune responses in vivo that could lyse human prostate cancer cells. Using an HLA-A2-expressing autochthonous model of prostate cancer, immunization with pTVG-AR augmented HLA-A2-restricted immune responses that could lyse syngeneic prostate tumor cells and led to a decrease in tumor burden and an increase in overall survival of tumor-bearing animals. Finally, immunization decreased prostate tumor growth in Copenhagen rats that was associated with a Th1-type immune response. These data show that the AR is as a prostate cancer immunological target antigen and that a DNA vaccine targeting the AR LBD is an attractive candidate for clinical evaluation.