Human Melanoma Tumor Research Articles

71. Boosting the Immunogenicity of an Oncolytic Vaccinia Virus By Expression of DAI Can Enhance Anti-Tumor Immunity in Humanized Mice

In oncolytic virotherapy the ability of an oncolytic virus to activate immune system against the tumor is nowadays generally understood to be the major mechanism to fully fight the cancer and to achieve long-term anti-tumor effects. Vaccinia viruses (VV) are immunosuppressive viruses encoding for a plethora of genes for neutralization of the host immune responses. To overcome this problem, we developed an oncolytic VV that expresses intracellular pattern recognition receptor DAI to obtain a self-immuno-boosting system to activate the immune responses in the tumor. DAI (DNA-dependent activator of IFN-regulatory factors) is a cytosolic dsDNA sensor and a potent activator of innate immune responses. We showed that DAI is able to recognize vaccinia DNA and to induce IFN-mediated immune responses after VV infection in vitro. We have also shown by wide genome expression profile analyses that infection with DAI-expressing VV increases expression of several genes related to important immunological pathways such as DC migration, immunogenic cell death and chemokine production. Intra-tumoral treatments with DAI-armed VV resulted in significant reduction in the size of B16-OVA tumors in C57BL/6 mice due to increased levels of anti-tumor CD8+ T cells in the tumors. When we re-challenged the mice with the same tumor, the DAI-VV treated mice rejected the growth of the new tumor completely, which also indicates immunity established against the tumor. To even better illustrate the immunogenicity of our virus in vivo we performed studies in PBMC humanized mice – a model closer to human system. Human melanoma tumors (HS294T) were grown in NSG mice and human PBMCs were injected intravenously to create a human-like immune system. Tumors were treated two times with DAI-VV or controls. We showed enhanced control of tumor growth in the DAI-VV treated group of mice. We also observed elevated levels of various human immune cells (e.g. active CD8+ T cells and CD4+ T cells) in the DAI-VV treated mice. In an IFN-gamma ELISpot analysis we could see increase in the amount of activated human T cells against tumor antigens in splenocyte samples of the DAI-VV treated mice.We conclude that expression of DAI by an oncolytic VV is a promising way to self-amplify immunostimulatory and vaccine potency of an oncolytic vaccinia virus to trigger the innate – and eventually the long-lasting adaptive immune responses against cancer. View Large Image | Download PowerPoint Slide

Oncogenic GNAQ and GNA11 Drive Tumorigenesis and Hyper‐Pigmentation in a Zebrafish Model of Human Uveal Melanoma

Uveal melanoma is the most common intraocular cancer, with an estimated 2000 cases diagnosed yearly in the U.S. Tumors arise from melanocytes in the choroid, iris, and ciliary body. Treatment typically includes proton beam therapy, and as such, the availability of tissue samples for research is limited. Approximately 50% of patients diagnosed with the disease develop untreatable liver metastases within 15 years. There is an urgent need to develop an animal model for uveal melanoma, in order to discover new therapeutics and understand disease initiation and progression.
 Approximately 83% of human uveal melanoma tumors have activating mutations in GNAQ or GNA11. Signaling pathways known to cause cell proliferation and survival are active downstream of mutated GNAQ or GNA11, but the exact signals responsible for metastasis remain to be discovered.
 We have developed a zebrafish model for human uveal melanoma by expressing mutated human GNA11Q209L or GNAQQ209L under control of the zebrafish melanocyte‐restricted mitfa promoter. Several lines have been identified that stably express either of the transgenes, Tg(mitfa:GNAQQ209L) or Tg(mitfa:GNA11Q209L). These fish have been crossed to p53M214K fish to facilitate tumor onset through a multi‐hit tumorigenic process. Tg(mitfa:GNAQQ209L);p53M214K and Tg(mitfa:GNA11Q209L);p53M214K zebrafish show hyper‐pigmentation in their skin and eyes. Fish develop tumors as early as 6 months of age. Our current efforts focus on the characterization of the tumors and the pigmentation defects. This model has revealed new mechanisms in pigmentation development and has provided insight into disease pathogenesis and metastasis.

99mTc-DTPA-bis-c(RGDfK) a potential alpha(v)beta3 integrin based homobivalent radioligand for imaging neoangiogenesis in malignant glioma and melanoma

A new99mTc-labeled bivalent DTPA-bis-c(RGDfK) conjugate has been developed and successfully synthesized. Promising results have been obtained for its preclinical evaluation on human glioma and melanoma tumor expressing αvβ3targets.

Abstract 3929: Orthotopic human choroidal melanoma model characterization with bioluminescence and magnetic resonance imaging for therapeutic efficacy evaluation

Abstract BACKGROUND: Intraocular melanomas in the West represents 70% of all primary eye cancers with a mortality rate greater than 50%. Current treatment options include enucleation, plaque radiotherapy, and proton beam radiotherapy. While enucleation is a highly invasive surgical procedure that removes the eye, the side effects of the two different radiotherapies include cataracts, retinopathy, cystoid macular edema, and secondary retinal detachment. Due to the relatively protected nature of the eye within the orbit, most topical treatment applications are precluded and systemic treatments increases the chance and incidence of off-target side effects. Up and coming targeted therapies that may not be constrained by such limitations require a non-invasive, imaging-relevant model in order to evaluate their efficacy. Therefore, the aim of this study was to characterize the imaging of orthotopic luc-enabled OCM-1 human choroidal melanoma tumors using bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) for future use in anti-cancer drug efficacy testing. MATERIALS & METHODS: Human choroidal melanoma cells were modified to express luciferase. 8-12 week old female NIH-Foxn1rnu rats were implanted in the suprachoroidal space with OCM-1-luc tumor spheroids on Day 0. BLI and MRI imaging was performed every 5-7 days starting at Day 7 after implantation of the OCM-1-luc spheroids until animals reached a moribund state to monitor disease progression. For BLI, animals were injected with 150mg/kg luciferin, anesthetized with isoflurane in air and imaged at 10 minutes following luciferin administration. Anatomical MRI was performed using a gradient-echo pulse sequence on a 7T MRI system. After euthanasia, eyes were removed and preserved in formalin for histological analysis. RESULTS & CONCLUSIONS: Animals tolerated the ocular tumor implant well. As a result of these efforts, we have successfully characterized the growth of orthotopic luc-enabled OCM-1 choroidal melanomas using both bioluminescence and magnetic resonance imaging. This provides a simple approach with which to monitor tumor growth and treatment response in a highly efficient way which should allow a high daily animal throughput. These promising results will serve as a solid foundation with which dose routes and levels of anti-cancer drugs can be optimized and their efficacy evaluated with noninvasive imaging. Citation Format: John L. Chunta, Meridith Baugher, Deanne Lister, Erin Trachet, Kevin P. Guley, Chris Bull, Scott Wise, Wilbur R. Leopold, Patrick McConville. Orthotopic human choroidal melanoma model characterization with bioluminescence and magnetic resonance imaging for therapeutic efficacy evaluation. [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 3929. doi:10.1158/1538-7445.AM2014-3929

Abstract 1671: Immunosuppressive myeloid-derived suppressor cells expressing PDL1 are increased in human melanoma tumor tissue

Abstract Despite the ability to elicit active immune responses, most immunotherapies fail to eliminate tumors in human cancer patients. One reason immunotherapy has not met expectations may be due to immunosuppression in the local environment of the tumor. Immunosuppressive myeloid-derived suppressor cells (MDSCs) are increased in the peripheral blood of melanoma patients and the frequency of MDSCs correlates with overall survival and disease progression. Furthermore, cytokines related to the recruitment and differentiation of MDSCs are also increased in melanoma patient serum and changes in these cytokine levels may correlate with disease progression. We find an increased frequency of regulatory T cells, PD-1-expressing T cells, and PD-L1 expressing MDSCs in primary melanoma tumors, suggesting that PD-1/PD-L1 interactions may play a role in immunosuppression in the microenvironment of the tumor. We are currently investigating the mechanisms of immunosuppression used by human MDSCs and whether the suppressive function of MDSCs can be blocked with antibodies targeting PD-L1. In conclusion, we have established MDSCs as an important player in immunosuppression in melanoma patients suggesting that combinatorial treatments that reduce the frequency or activity of MDSCs may improve the effectiveness of future immunotherapies. Citation Format: Kimberly Rae Jordan, Virginia Borges, Martin D. McCarter. Immunosuppressive myeloid-derived suppressor cells expressing PDL1 are increased in human melanoma tumor tissue. [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 1671. doi:10.1158/1538-7445.AM2014-1671

Discovery of tetrahydroisoquinoline-based bivalent heterodimeric IAP antagonists

Bivalent heterodimeric IAP antagonists that incorporate (R)-tetrahydroisoquinoline in the P3′ subunit show high affinity for the BIR2 domain and demonstrated potent IAP inhibitory activities in biochemical and cellular assays. Potent in vivo efficacy was observed in a variety of human tumor xenograft models. The bivalent heterodimeric molecule 3 with a P3–P3′ benzamide linker induced pharmacodynamic markers of apoptosis and was efficacious when administered intravenously at a dose of 1mg/kg to mice harboring A875 human melanoma tumors. Analog 5, with a polyamine group incorporated at the P2′ thiovaline side chain exhibited antiproliferative activity against the P-gp expressing HCT116/VM46 cell line.

A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth

Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC50 = 0.06 nM) and strongly blocks its interaction with SCF (IC50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.

Filamin A expression correlates with proliferation and invasive properties of human metastatic melanoma tumors: implications for survival in patients.

Filamin A (FLNa) cross-links actin filaments into dynamic orthogonal networks and interacts with binding proteins of diverse cellular functions that are implicated in cell growth and motility regulation. Here, we tested the hypothesis that FLNa plays a role in cancer proliferation and metastasis via the regulation of epidermal growth factor receptor (EGFR) function. Ectopic expression and knockdown of FLNa in human melanoma cell lines was performed to investigate changes in cellular proliferation, migration and invasion in vitro and tumor growth in a xenograft model in the mouse. The role of FLNa in EGFR expression and signaling was evaluated by Western blot. Immunohistochemistry was performed on histological sections of human melanoma tumors to determine whether an association existed between FLNa and overall survival. The depletion of FLNa significantly reduced the proliferation, migration and invasion of two melanoma cell lines in vitro and was associated with smaller tumors in a xenograft model in vivo. EGF-induced phosphorylation of EGFR and activation of the Raf-MEK-ERK cascade was negatively affected by the silencing of FLNa both in vitro and in vivo. Cancer patients with low melanoma tumor FLNa expression have improved survival benefit. These data indicate that enhanced tumorigenesis occurs through increase in EGF-induced EGFR activation in FLNa-expressing melanoma cells and that high FLNa levels are predictors of negative outcome for patients with melanoma tumors.

Epigenetics of human melanoma: promises and challenges.

Melanoma is the deadliest form of skin cancer with rising incidence and mortality rates. Although early-stage melanoma is highly curable, advanced-stage melanoma is refractory to treatment. This underscores the importance of prevention and early detection as well as the need to improve treatment and prognostication of human melanoma. Elucidating the underlying mechanisms of the initiation and progression of human melanoma can help identify potential targets of intervention for prevention, diagnosis, therapy, and prognosis of this disease. Aberrant DNA methylation and histone modifications are the best-established epigenetic mechanisms of carcinogenesis. The occurrence of epigenetic changes prior to clinical diagnosis of cancer and their reversibility through pharmacologic/genetic approaches offer a promising avenue for basic and translational research on human melanoma. Candidate gene(s) or genome-wide aberrant DNA methylation and histone modifications have been observed in human melanoma tumor tissues and cell lines, and correlated to cellular and functional characteristics and/or clinicopathological features of this malignancy. The present review summarizes the published researches on aberrant DNA methylation and histone modifications in connection with human melanoma. Representative studies are highlighted to set forth the current state of knowledge, gaps in the knowledgebase, and future directions in these epigenetic fields of research. Examples of epigenetic therapy applied for human melanoma in vitro, and the challenges of its in vivo application for clinical treatment of solid tumors are discussed.

Anti-tumor response in T cells with CD8 independent high affinity TCR remains unaffected with aging (VAC11P.1009)

Abstract Advancement in adoptive cell therapy has led to the use of T cells engineered with tumor antigen reactive high affinity T cell receptors (TCR) in clinical trials. However, T cell engineered with anti-tumor epitope reactive TCR from an aged individual could affect the clinical outcome. In order to study if aging affects the functional efficacy of a CD8 independent high affinity TCR, we compared anti-tumor T cell response from young/old TCR transgenic h3T(human TIL derived Tyrosinase TCR) mice developed in our lab using TIL1383I TCR obtained from TILs of a melanoma patient. In vitro comparison of h3T T cells from old/young mice shows that antigen specific stimulation of the T cells from old mice resulted in an increased expression of CD25 and increased cytokine(IL2,IFNy,TNFa) secretion as compared to T cells from young mice. Remarkably, an overall decreased immune response from old hgp100 reactive T cells from Pmel transgenic mice was observed. Further, adoptively transferred h3T old/young splenocytes in vivo showed comparable control of human melanoma tumor 624-MEL established in a xenograft model. Further, aged T cells engineered with TIL1383I TCR and transferred to treat established melanoma in aged HLA-A2 recipient also showed efficient tumor control. These results demonstrate that aging does not dampen the recognition and activation of CD8+T cells bearing CD8 independent high affinity TCR and thus its broad immunotherapeutic potential in treating patients across all ages.

Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

BackgroundA subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.MethodsshRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA.ResultsWe demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.ConclusionsThese findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

MC13-0074 Correlation between p53 immunohistochemistry and mutational status in human melanoma tumors

MC13-0074 Correlation between p53 immunohistochemistry and mutational status in human melanoma tumors

Abstract A233: Pharmacodynamic Transcriptional markers of carboxyamidotriazole orotate (CTO) exposure in anagen hair.

Abstract Background: Carboxyamidotriazole Orotate (CTO) is the orotic acid salt of carboxyamodotriazole (CAI)which is an inhibitor of calcium -dependent intracellular and extracellular signal transduction pathways, also possessing antiproliferative, antiangiogenic and anti-invasive properties. The activity of CTO alone and combination with temozolomide or 5-fluorouracil was demonstrated in human glioblastoma, melanoma and colon tumor xenografts. In Phase I (NCT01107522), nine patients pretreated with 2 to 8 targeted and non-targeted drugs and having refractory tumors responded to CTO at doses ranging 75mg/m2/day through 427mg/m2/day, and achieved stable disease for different periods (3 to 14 months). Four pretreated tumors were found to have different genomic mutations (PI3KCA, EGFR multiple, BRAFV600 and NRAS) consistent with CTO's suggested mechanism of action to inhibit multiple tyrosine kinases in addition to modulating calcium signaling pathways and calcium-dependent signal transduction pathways, as yet uncharacterized. Method: Using Epistem's ex vivo plucked anagen hair assay platform global gene expression evaluation by microarray analysis was performed to assess transcriptional response to varying doses of CTO (2, 5 and10 μM). Plucked anagen scalp hairs from five healthy donors were exposed to varying doses of CTO over a 24 hr period. Total RNA was isolated form the hair bulbs post culture and used to assess global transcriptional alterations. Results:Significant biologically relevant alteration of the anagen hair bulb transcriptome, ranging from -100 fold to +25 fold differential expression of some mRNAs was observed at CTO levels shown to be clinically relevant. Importantly, transcriptional signatures associated with inhibition of EGFR, MEK, HDAC and HSP90 were strongly suppressed at all doses of CTO. In addition, genes associated with non-voltage dependent calcium signaling were strongly suppressed, together with those associated with RAS and Growth Factor Signature. Modest suppression of transcription signatures of WNT signaling was evident at longer CTO exposure. In contrast, activation of tumor suppressor signatures associated with P53 was observed. Conclusion: These data provide multiple pharmacodynamics markers associated with the suggested mechanism of action of CTO e.g., inhibition of genes of non-voltage dependent calcium signaling and multiple onco-pathways, and also activation of signatures associated with tumor suppressors. These results in the anagen hair assay may provide the molecular MOA of CTO's observed clinical benefit in a broad spectrum of malignant tumors with different genomic types and a tool to design customized combination therapies of CTO with other agents. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A233. Citation Format: Rashida A. Karmali, Gino Miele. Pharmacodynamic Transcriptional markers of carboxyamidotriazole orotate (CTO) exposure in anagen hair. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A233.

Human T Cells Engineered To Express a Programmed Death 1/28 Costimulatory Retargeting Molecule Display Enhanced Antitumor Activity

Adoptive transfer of T cells genetically modified to express cancer-specific receptors can mediate impressive tumor regression in terminally ill patients. However, T cell function and persistence over time could be hampered by the activation of inhibitory costimulatory pathways, such as programmed death 1 (PD1)/programmed death ligand 1, leading to T cell exhaustion and providing tumor cells with an escape mechanism from immunosurveillance. In addition, the lack of positive costimulation at the tumor site can further dampen T cell response. Thus, as T cell genetic engineering has become clinically relevant, we aimed at enhancing T cell antitumor activity by genetically diverting T cell-negative costimulatory signals into positive ones using chimeric costimulatory retargeting molecules and which are composed of the PD1 extracellular domain fused to the signaling domains of positive costimulatory molecules such as CD28 and 4-1BB. After characterizing the optimal PD1 chimera, we designed and optimized a tripartite retroviral vector that enables the simultaneous expression of this chimeric molecule in conjunction with a cancer-specific TCR. Human T cells, transduced to express a PD1/28 chimeric molecule, exhibited enhanced cytokine secretion and upregulation of activation markers upon coculture with tumor cells. These engineered cells also proliferated better compared with control cells. Finally, we tested the function of these cells in two xenograft models of human melanoma tumors and show that PD1/28-engineered human T cells demonstrated superior antitumor function. Overall, we propose that engineering T cells with a costimulatory retargeting molecule can enhance their function, which bears important implications for the improvement of T cell immunotherapy.

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.

Lipid-Coated Cisplatin Nanoparticles Induce Neighboring Effect and Exhibit Enhanced Anticancer Efficacy

Encapsulation of cisplatin (CDDP) into nanoparticles (NPs) with high drug loading and encapsulation efficiency has been difficult due to the poor solubility of CDDP. However, this barrier has been overcome with a reverse microemulsion method appropriating CDDP's poor solubility to our advantage promoting the synthesis of a pure cisplatin nanoparticle with a high drug loading capacity (approximately 80.8 wt %). Actively targeted CDDP NPs exhibited significant accumulation in human A375M melanoma tumor cells in vivo. In addition, CDDP NPs achieved potent antitumor efficacy through the neighboring effect at a dose of 1 mg/kg when injected weekly via iv without inducing nephrotoxicity. The neighboring effect regards an observation made in vivo when the tumor cells that took up CDDP NPs released active drug following apoptosis. Via diffusion, surrounding cells that were previously unaffected showed intake of the released drug and their apoptosis soon followed. This observation was also made in vitro when A375M melanoma tumor cells incubated with CDDP NPs exhibited release of active drug and induced apoptosis on untreated neighboring cells. However, the neighboring effect was unique to rapidly proliferating tumor cells. Liver functional parameters and H&E staining of liver tissue in vivo failed to detect any difference between CDDP NP treated and control groups in terms of tissue health. By simultaneously promoting an increase in cytotoxicity and a lesser degree of side effects over free CDDP, CDDP NPs show great therapeutic potential with lower doses of drug while enhancing anticancer effectiveness.

Selective Inhibition of p300 HAT Blocks Cell Cycle Progression, Induces Cellular Senescence, and Inhibits the DNA Damage Response in Melanoma Cells

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to play critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. Additionally, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells following combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

133 : In vitro eradication of serous ovarian cancer with IFN-α2α and IFN-γ in combination with monocytes

Interferons (IFNs) have been utilized for their antiproliferative and antitumor effects against human cancers, including hairy cell leukemia, chronic myelogenous leukemia, and malignant melanoma. We have previously shown that in combination with human elutriation-purified monocytes, IFN- α 2a and IFN- γ have nearly eradicated certain cancer cell types in vitro and in vivo in a nude mouse model. More specifically, previous in vitro studies have shown the eradication of human osteosarcoma, LOX melanoma, and A549 lung tumor cells, while no such effect seen on the human diploid cell lines WI-38 and MRC-5. Further in vivo analysis of OVCAR-3 and LOX melanoma inoculated intratumorally in female BALB/c nude mice showed that combination therapy significantly reduced tumor growth more profoundly than IFNs or monocytes alone. Such finding may have clinical implications. We have adapted this IFN and monocyte combination treatment model towards human serous ovarian cancer, a subtype accounting for approximately 60–80% of ovarian cancer diagnoses. We further validated IFN-primed monocytes as a novel antitumor treatment against ovarian tumor cells in vitro . Combination treatment has antitumor and antiproliferative effects towards the ovarian cancer cell lines SKOV-3, CaOV3, OVCAR-4, OVCAR-5, and OVCAR-8. These serous tumor cell lines show varying, though prevalent, anti-tumor and anti-proliferative effects following IFN-primed monocyte treatment. SKOV-3, OVCAR-4, and OVCAR-8 cells are moderately sensitive to treatment; however, CaOV3 and OVCAR-5 cells are highly sensitive, with near complete eradication. Results demonstrate that such treatment is a novel candidate for ovarian cancer. Future studies will include in vivo IFN-primed monocyte treatment of intraperitoneal xenografts of serous ovarian cancer cell lines in female BALB/c nude mice, treatment of tumor cells with IFN-primed monocytes in combination with cancer therapeutics, and treatment of ovarian cancer cells with IFN-primed monocytes extracted from patients diagnosed with ovarian cancer.

Two Drugs Are Better than One—Modeling Drug Combinations in Cancer Therapy

Human melanoma tumor samples underlie a mathematical model that predicts responses to combinations of targeted drugs.

Enhanced antitumor activity mediated by human 4‐1BB‐engineered T cells

4-1BB (CD137) is a costimulatory molecule transiently expressed on the T-cell surface after TCR engagement, whereas its ligand 4-1BBL can be found on professional antigen-presenting cells, but more importantly, also on tumor cells. As the role of the 4-1BB/4-1BBL pathway has emerged central to CD8(+) T-cell responses and survival, we sought to test its relevance in the context of genetically modified human T cells. To that end, T cells purified from healthy donors and from vaccinated-melanoma patients were transduced to express high levels of constitutive 4-1BB. 4-1BB-transduced T cells were cocultured with melanoma tumor lines and exhibited enhanced cytokine secretion, upregulation of activation markers as well as increased cytotoxicity in a chick-chorioallantoic membrane model of human melanoma tumors. In addition, these cells expanded and proliferated at a higher rate, expressed heightened levels of the antiapoptotic molecule Bcl(XL) and were also relatively insensitive to immunosuppression mediated by transforming growth factor-β, compared to control cells. We also show that 4-1BBL expression on the target cell is essential to 4-1BB-mediated functional improvement. Overall, we conclude that the modification of human T cells with 4-1BB yields enhanced antitumor function which may have important applications in therapies based on the genetic modification of patient lymphocytes.