Growth Of Human Melanoma Xenografts Research Articles

A Bifunctional Approach of Immunostimulation and uPAR Inhibition Shows Potent Antitumor Activity in Melanoma

Significant advancements of mutation-based targeted therapy and immune checkpoint blockade have been achieved in melanoma. Nevertheless, acquired resistance and nonresponders to therapy require different strategies. An innovative approach is presented here that is based on the combination of innate immune system activation and simultaneous targeting of the oncogene urokinase-type plasminogen activator receptor (uPAR). We generated two triphosphate-conjugated siRNAs targeting uPAR (ppp-uPAR) by invitro transcription. Specific uPAR knockdown and simultaneous activation of the retinoic acid-inducible gene 1 (RIG-I) was shown in different human melanoma cells, fibroblasts, and melanocytes. The compounds induced massive apoptosis in melanoma cells, whereas fibroblasts and melanocytes were less sensitive. The effects were less pronounced when the IFN receptor was blocked. Treatment with ppp-uPAR led to accumulation of p53 and induction of RIG-I-dependent proapoptotic signaling. The apoptotic effects induced by ppp-uPAR were maintained in melanoma cell lines that had acquired double resistance to B-RAF and MEK/extracellular signal-regulated kinase inhibition. Systemic intraperitoneal application of ppp-uPAR in nude mice significantly reduced growth of human melanoma xenografts and elicited a systemic innate immune response with increased serum cytokine levels. Our data suggest that ppp-uPAR represents a therapeutically attractive compound that may help overcome the strong therapy resistance of melanoma.

Effects of a novel Nodal-targeting monoclonal antibody in melanoma.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Abstract 1780: Targeted inactivation of CRL4-CDT2 E3 ubiquitin ligase as a novel therapeutic strategy for malignant melanoma

Abstract The CRL4CDT2 E3 ubiquitin ligase is emerging as a critical regulator cell cycle progression, primarily through the targeted ubiquitylation and degradation of critical cell cycle regulators including CDT1, p21 and SET8 via the 26S proteasome. CDT2, the substrate receptor of the CRL4CDT2 E3 ligase is overexpressed or amplified in several human malignancies, but its role in the development or progression of these cancers remains elusive. We show that CDT2 is overexpressed in primary and metastatic melanoma. Targeted depletion of CDT2 by si-RNA or its pharmacological inhibition by MLN4924, a selective NEDD8-activating enzyme (NAE) inhibitor, suppressed the proliferation of melanoma cancer cells in vitro and inhibited tumor growth of human melanoma xenografts in mice, irrespective of the BRAF mutation status. Inhibition of CRL4CDT2 in melanoma cells with various genetic backgrounds, but not in immortalized human melanocytes, resulted in robust p21- and SET8-dependent rereplication. These results identify the CRL4CDT2 E3 ubiquitin ligase as a promising new therapeutic target for cutaneous melanoma, and demonstrate the efficacy of MLN4924 as a promising anti-melanoma agent. Citation Format: Mouadh Benamar, Fadila Guessous, Patrick Corbett, Craig L. Slingluff, Tarek Abbas. Targeted inactivation of CRL4-CDT2 E3 ubiquitin ligase as a novel therapeutic strategy for malignant 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 1780. doi:10.1158/1538-7445.AM2015-1780

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.

Expression analysis and molecular targeting of cyclin-dependent kinases in advanced melanoma

A major focus of melanoma research continues to be the search for genes/proteins that may be suitable targets for molecular therapy of primary and metastatic melanoma. In line with this effort, the objective of the study presented herein was to determine whether interfering with cell cycle progression and in particular, the expression and function of select cyclin-dependent kinases, would impair the biological features of advanced melanoma. We provide data, which document that unlike nevi and melanoma in situ, primary and metastatic melanomas express high levels of CDK2, CDK1, and CDK5. Furthermore, we present the results of in vitro and preclinical in vivo studies, which demonstrate that treatment with a small-molecule cyclin-dependent kinase inhibitor that selectively blocks the function of CDK2, CDK5, CDK1, and CDK9, leads not only to inhibition of melanoma cell proliferation and apoptosis of melanoma cells, but also impairs the growth of human melanoma xenografts.

Protodynamic Intracellular Acidification by cis-Urocanic Acid Promotes Apoptosis of Melanoma Cells In Vitro and In Vivo

The extracellular tumor microenvironment is acidified, whereas the intracellular pH of tumor and stromal cells is neutral. cis-Urocanic acid (cis-UCA), an endogenous compound of the skin, can acidify the cytosol by transporting protons into the cells. This phenomenon, termed the protodynamic concept, was studied here in human cancer cells. cis-UCA dose-dependently reduced the number of viable human melanoma, cervical carcinoma, and fibrosarcoma cells at weakly acidic extracellular pH. The intracellular pH decreased by up to 0.5 pH units in a concentration-dependent manner with 0.3-30 m cis-UCA at extracellular pH 6.5 but not at pH 7.4. Under the same conditions, 30 mM cis-UCA induced annexin-V binding and activation of caspase-3 in A2058 melanoma cells as signs of apoptotic cell death. Finally, growth of human melanoma xenografts in SCID mice was suppressed by 60% following intratumoral injection of cis-UCA. Accordingly, the percentage of tumor necrosis and active caspase-3-immunopositive cells increased, whereas proliferation activity decreased. These results identify cis-UCA as an anticancer agent inhibiting melanoma growth by immediate intracellular acidification followed by apoptotic cell death in vivo.

CD20-immunotargeting of malignant melanoma.

8565 Background: Melanomas, like other malignancies, contain distinct subpopulations of cells. These subpopulations provide the cellular basis for the complex biology of the disease including phenomena such as self-renewal, differentiation, and tumor initiation, maintenance and progression, and may have major translational impact. In melanoma, several subpopulations - some with stem cell-like characteristics - have been described including one expressing CD20. Methods: multispectral imaging; xenografting onto CB17 SCID and NOD/SCID/γc-/- mice; adjuvant treatment with rituximab in 9 stage IV (M1a-c) melanoma patients, of whom the majority had received multiple prior systemic therapies. Induction treatment at 375 mg/m2 qw for 4 wk followed by maintenance therapy at 375 mg/m2 every 8 wk. Treatment was stopped after 2 years or with development of progressive disease. Results: We show by multispectral imaging that CD20 can be expressed on melanoma cells in human tumor samples. Furthermore, western blot analysis revealed expression of CD20 in lysates from human melanoma xenografts after serial in vivo passaging, consistent with environmental induction. Growth of human melanoma xenografts in two different preclinical in vivo models was significantly reduced by systemic administration of a monoclonal anti-CD20 antibody. Based on these results, we initiated a small phase II pilot trial on adjuvant treatment with an anti-CD20 antibody in stage IV (M1a-c) melanoma patients. Treatment was well tolerated. After 2 years, 6 patients (66%) are still disease-free, the 2 years OS rate was 100%. 2 patients died thereafter (22%). With a mean DFI and OS of 27+ months anti-CD20 treated patients show a > 3-fold longer median DFI and a somewhat higher median OS as compared to data obtained from large prospective clinical trials and retrospective analyses of stage IV melanoma patient cohorts, that have been treated by complete metastasectomy. Conclusions: Though patient numbers included in the present clinical study are too small for definitive conclusions, immunotargeting of CD20 may represent a first example of the potential therapeutic value of targeting melanoma subpopulations. Application of these initial clinical observations in larger trials is under way. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Roche

Phase I study of CNTO 95, a fully human monoclonal antibody (mAb) to αv integrins, in patients with solid tumors

3113 CNTO 95 is a fully human anti-αvintegrin antibody that inhibits the growth of human melanoma xenografts in nude mice and rats by ∼80% and >99%, respectively. The objective of this phase I dose-escalating study was to assess the safety and pharmacokinetics of CNTO 95 in patients with advanced refractory solid tumors. CNTO 95 was infused on days 0, 28, 35 and 42 and standard clinical assessments, DCE-MRI, and FDG-PET were performed. Twenty four patients were enrolled at 0.1, 0.3, 1, 3 and 10 mg/kg. Tumor types included ovarian (6), colorectal (9), angiosarcoma (2), ureteric (1), PNET (1), pseudomyxoma peritonei (1), endometrial (1), metastatic melanoma (1), renal cell carcinoma (1), and carcinosarcoma (1). CNTO95 was associated with one episode of grade III and several episodes of grade II infusion-related fever, which were controllable with acetaminophen. One patient with cutaneous angiosarcoma dosed at 10 mg/kg had a partial response that is ongoing after 2 months on extended treatment (10 mg/kg). A lesion in a patient with ovarian carcinosarcoma and stable disease (10mg/kg) was undetectable on FDG-PET at day 49. Exploratory DCE-MRI analysis indicated a non-dose dependent decrease in area under the gadolinium curve in some patients. Preliminary PK data indicated that drug exposure increased in a greater than dose propor tional manner with dose dependent half-life ranging from 0.19 to 11.81 days over the doses 0.1–10 mg/kg. The initial rapid clearance is probably due to tissue binding at low doses (< 3 mg/kg). At higher doses slower drug clearance could indicate saturation of tissue sites. In conclusion, CNTO 95, a fully human mAb to αv integrins, is well tolerated up to weekly doses of 10 mg/kg. Six patients had extended therapy including one with angiosarcoma who has a partial response. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Centocor Centocor Centocor

BAY 43-9006 in patients with advanced melanoma: The Royal Marsden experience

7506 Background: Approximately 70% of melanomas have an activating mutation in BRAF, leading to elevated RAF kinase activity and cellular proliferation. BAY 43-9006 is a potent RAF kinase inhibitor that retards the growth of human melanoma xenografts. This phase II study aims to determine the clinical benefit and toxicity of BAY 43-9006 in advanced melanoma and to correlate these findings with BRAF status of the patients and their tumours. Methods: Patients with stage IV refractory melanoma were recruited from The Royal Marsden Melanoma Unit and treated with BAY 43-9006 400mg BD orally for 12 weeks after baseline imaging and tumour biopsy. At the 12 week reassessment, patients with objective tumour responses continued BAY 43-9006 until disease progression or unacceptable toxicity. Patients with stable disease were randomised in a double-blind manner between BAY 43-9006 or placebo and followed for on-going response.In the event of disease progression in this cohort, the code was broken and those receiving ...

BAY 43-9006 in patients with advanced melanoma: The Royal Marsden experience

7506 Background: Approximately 70% of melanomas have an activating mutation in BRAF, leading to elevated RAF kinase activity and cellular proliferation. BAY 43-9006 is a potent RAF kinase inhibitor that retards the growth of human melanoma xenografts. This phase II study aims to determine the clinical benefit and toxicity of BAY 43-9006 in advanced melanoma and to correlate these findings with BRAF status of the patients and their tumours. Methods: Patients with stage IV refractory melanoma were recruited from The Royal Marsden Melanoma Unit and treated with BAY 43-9006 400mg BD orally for 12 weeks after baseline imaging and tumour biopsy. At the 12 week reassessment, patients with objective tumour responses continued BAY 43-9006 until disease progression or unacceptable toxicity. Patients with stable disease were randomised in a double-blind manner between BAY 43-9006 or placebo and followed for on-going response.In the event of disease progression in this cohort, the code was broken and those receiving placebo were re-challenged with BAY 43-9006.Tumour responses were evaluated by CT/MRI at 12 weeks and every 6 weeks thereafter. Tumour biopsies were performed at weeks 0,4 and 12 for sequencing BRAF and identification of downstream activity of RAF kinase. Results: Twenty patients were enrolled between October 2002 and April 2003,the median age was 52 (range 18–69) years. At week 12,1 partial response and 3 stable disease were seen. Fifteen patients had progressive disease before or at 12 weeks. Grade 3 skin toxicity was observed in 5 patients and 2 patients developed hypertension requiring intervention. There was no haematological toxicity and all toxicity was reversible. Laboratory analysis of tumour samples to determine BRAF status will be presented. Conclusions: BAY 43-9006 has very modest activity as a single agent. Preliminary data from combination studies are promising and trials are ongoing. The drug is generally well-tolerated and the determination of BRAF status in patients may allow correlation with clinical behaviour of tumours and efficacy of BAY 43-9006 Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bayer Pharmaceutical Corp. Bayer Bayer Bayer Bayer

Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors.

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin (ADAM) activity. We have previously shown that adenovirally expressed tissue inhibitor of metalloproteinases-3 (TIMP-3) induces apoptosis in melanoma cells and inhibits growth of human melanoma xenografts. Here, we have studied the role of death receptors in apoptosis of melanoma cells induced by TIMP-3. Our results show, that the exposure of three metastatic melanoma cell lines (A2058, SK-Mel-5, and WM-266-4) to recombinant TIMP-3, N-terminal MMP inhibitory domain of TIMP-3, as well as to adenovirally expressed TIMP-3 results in stabilization of tumor necrosis factor receptor-1 (TNF-RI), FAS, and TNF-related apoptosis inducing ligand receptor-1 (TRAIL-RI) on melanoma cell surface and sensitizes these cells to apoptosis induced by TNF-alpha, anti-Fas-antibody and TRAIL. Stabilization of death receptors by TIMP-3 results in activation of caspase-8 and caspase-3, and subsequent apoptosis is blocked by specific caspase-8 inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor (Z-DEVD-FMK). Adenovirus-mediated expression of TIMP-3 in human melanoma xenografts in vivo resulted in increased immunostaining for TNF-RI, FAS, and cleaved caspase-3, and in apoptosis of melanoma cells. Taken together, these results show that TIMP-3 promotes apoptosis in melanoma cells through stabilization of three distinct death receptors and activation of their apoptotic signaling cascade through caspase-8.

Retroviral gene transfer of interferon‐inducible protein 10 inhibits growth of human melanoma xenografts

Interferon-inducible protein 10 (IP-10) is an immunomodulatory chemokine recently recognized to have potent antiangiogenic activity in vivo. Due to difficulties in the stability, manufacture and chronic administration of recombinant forms of endogenous antiangiogenic proteins, antiangiogenic gene therapy has emerged as a promising new form of cancer treatment. We retrovirally transduced A375 human melanoma cells with the human IP-10 gene and injected cells subcutaneously into nude mice. IP-10-transduced cells also were mixed with null-transduced cells in varying proportions before injection. In vivo growth of IP-10-transduced melanoma cells was markedly diminished compared to parental or null-transduced cells (p = 0.0002, Kruskal-Wallis test). This growth inhibition was associated with a marked reduction in microvessel density. The degree of growth inhibition of tumors following injection of a mixed population of null- and IP-10-transduced cells was directly associated with the fraction of IP-10-transduced cells present. We conclude that retroviral transduction of human melanoma cells with the IP-10 gene leads to sufficient protein secretion to inhibit angiogenesis and tumor growth. These findings suggest that IP-10 gene therapy might be an effective therapy in patients with cancer.

Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy.

The effect of local and systemic delivery of the angiostatin gene on human melanoma growth was studied in nude mice. Liposome-coated plasmids carrying the cDNA coding for murine and human angiostatin (CMVang and BSHang) were injected weekly, locally or systemically, in mice transplanted with melanoma cells. The treatment reduced melanoma growth by 50% to 90% compared to that occurring in control animals treated with liposome-coated plasmid carrying the lacZ gene or in untreated controls. The growth of both locally injected and controlateral uninjected tumors in mice bearing two melanoma grafts was significantly suppressed after intratumoral treatment. Tumor growth inhibition was also observed in mice treated by intraperitoneal delivery, suggesting that angiostatin gene therapy acts through a systemic effect. Both melanoma growth suppression and delay in the onset of tumor growth were observed in treated mice. PCR performed on tumors and normal tissues showed that the lipofected DNA was present in tissues from treated mice, and angiostatin expression was demonstrated by RT-PCR. Histopathological analysis of melanoma nodules revealed an increase in apoptotic cells and a reduction in vessel density in tumors from treated mice. Our results suggest that systemic, liposome-mediated administration of genes coding for antiangiogenic factors represents a promising strategy for melanoma treatment in humans.

Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice.

Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice, The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths and the median number of long metastases in treated and control mice bearing NG xenografts. c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced celluLar levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of long metastases, and an increase in life span. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-Myc inhibits their growth and is associated with the induction of apoptosis.

Inhibition of the growth of human melanoma xenografts in nude mice by human tumor‐specific cytotoxic T‐cells

Melanoma-specific T-cells (CTLs) are specifically cytotoxic for autologous tumor, when assayed in vitro. To examine their effectiveness in vivo, we tested the ability of these human T-cells to inhibit growth of human melanoma xenografts by using a Winn assay. Nude mice receiving specific CTLs (n = 10) demonstrated a dramatic inhibition of tumor growth. All treated mice were tumor-free at day 50 and nine remained tumor-free at day 65, vs. control mice (n = 10) with average tumor volumes of 321 mm3 and 808 mm3, respectively. To control for the possibility that a non-specific response to the human T-cells could inhibit tumor growth, an additional group received allospecific CTLs. There was no inhibition of tumor growth in this group (n = 8), with the average tumor volume of 2,768 mm3 at day 40 vs. 1,882 mm3 in the control group (n = 10). We conclude that these tumor-specific CTLs can inhibit tumor growth in vivo and may prove useful in the adoptive immunotherapy of melanoma.

Pharmacokinetics and mechanism of action of a doxorubicin-monoclonal antibody 9.2.27 conjugate directed to a human melanoma proteoglycan.

Doxorubicin (DXR) conjugated to a monoclonal antibody (MAb), 9.2.27, which recognizes a human melanoma-associated proteoglycan, effectively suppresses the growth of human melanoma xenografts and prolongs the life span of tumor-bearing athymic nude (nu/nu) mice. We have investigated further the mechanism(s) of this in vivo antitumor activity. Our results indicate that following iv injection, the DXR-MAb 9.2.27 conjugate is cleared from the circulation with typical biphasic kinetics, similar to the clearance process of the unconjugated MAb 9.2.27. In contrast, less than 10% of injected dose per milliliter of blood is found in the circulation at any given time after ip injection. Toxicity studies further indicate that DXR-MAb 9.2.27 conjugate is less toxic in vivo than the freely administered DXR, which is known to cause considerable cardiotoxic effects. Direct autoradiography demonstrates that the DXR-MAb 9.2.27 conjugate binds specifically to the tissue sections derived from a human melanoma xenograft of a nude mouse. A critical evaluation is given of the relevance of these findings and their impact on the design of future strategies for the immunochemotherapy of malignant melanoma.