Melanoma is notoriously resistant to chemotherapy, but variable responses to biotherapies, including the IFNs and IL-2, provide intriguing avenues for further study. Systemic IL-2 treatment has provided significant clinical benefit in a minority of patients with metastatic melanoma, leading to long-term survival in a few cases. We hypothesize that one previously unidentified mechanism of effective IL-2 therapy is through direct upregulation of the tumor suppressor IL-24 in melanoma tumor cells resulting in growth suppression. In this study, five melanoma cell lines were treated with high dose recombinant human IL-2. Three (A375, WM1341, WM793) showed statistically significant increases in IL-24 protein; two (WM35, MeWo) remained negative for IL-24 message and protein. This increase was abolished by preincubating with anti-IL-2 antibody or blocking with antibodies against the IL-2 receptor chains. These IL-2 responsive melanoma cell lines expressed IL-2Rβ and IL-2Rγ mRNA. The IL-2Rβγ complex was functional, as measured by IL-2-induced signal transducers and activators of transcription activation as well as IL-15 signaling through its shared receptor complex. IL-24 upregulation was observed in response to either IL-2 or IL-15. Cell growth was significantly decreased by treatment of IL-24-positive cells with IL-2 or IL-15, whereas no effect was seen in negative cells. Incubating the IL-24 inducible-cells with anti-IL-24 antibody as well as transfecting with IL-24 small interfering RNA effectively reversed the growth suppression seen with IL-2. Thus, we have shown that one mechanism of clinically effective IL-2 therapy may be the direct action of IL-2 on a biologically distinct subset of melanoma cells leading to upregulation of the tumor suppressor IL-24.

BACKGROUND: The role of nonmyeloablative allogeneic stem cell transplantation (NST) in the treatment of chronic lymphocytic leukemia (CLL) is not well established. The authors report on long-term experience with NST in relapsed/refractory CLL and define prognostic factors associated with outcome. METHODS: The authors reviewed the outcome of 86 patients with relapsed/relapsed CLL enrolled in sequential NST protocols. RESULTS: The median patient age was 58 years. Patients were heavily pretreated before transplantation, and 43 required immunomanipulation after NST for persistent or recurrent disease. Immunomanipulation included withdrawal of immunosuppression, rituximab, and step-wise donor lymphocyte infusions. Of 43 patients receiving immunomanipulation, 20 (47%) experienced a complete remission. Patients with human leukocyte antigen (HLA) genotype A1(-)/A2(-)/B44(-) were more likely to experience a complete remission (P = .0009), with rates of 9%, 36%, 50%, and 91%, respectively, for 0, 1, 2, and 3 of these HLA factors. This resulted in significant improvement in progression-free-survival rates of 68.2% at 5 years for patients with all 3 HLA factors. Overall, the estimated 5-year survival rate was 51%. In a multivariate model, a CD4 count of <100/mm(3) and a below normal serum immunoglobulin G level at study entry were associated with a short survival duration (P < .0001). CONCLUSIONS: These results confirm the potential cure of relapsed/refractory CLL with NST and provide the first evidence that immunoglobulin G and CD4 levels are predictive of overall survival after NST in CLL and that human leukocyte antigen alleles predict response to immunomanipulation. Cancer 2011;117:4679-88. (C) 2011 American Cancer Society.

Interleukin-24 (IL-24) is a novel tumor suppressor/cytokine gene expressed in normal human melanocytes but for which expression is nearly undetectable in metastatic melanoma. Overexpression of the IL-24 protein has been shown to inhibit tumor cell proliferation and induce apoptosis in many melanoma cell lines, and is now considered a tumor suppressor. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been widely studied for the treatment of human lung cancer and other solid tumors, but the erlotinib-targeted therapy has not been tested in melanoma. The objective of this study is to investigate the potency of erlotinib in suppressing the growth of human melanoma cells and whether IL-24 could enhance the antitumor activity of erlotinib. In cell viability and apoptosis assays, treatment with erlotinib dependently inhibited the growth of different melanoma cell lines and when combined with adenoviral vector-mediated IL-24 gene therapy, a significant increase in cell growth inhibition and apoptosis induction resulted (P<0.05). Immunoblot assay showed that the combination treatment of erlotinib and IL-24 considerably increased the cleavage of caspase-3 and caspase-9 and the expression of Apaf-1 protein in melanoma cells, inducing activation of the Apaf-1-dependent apoptotic pathways. Moreover, this combination treatment markedly inhibited phosphorylation of the EGFR, phosphatidylinositol-3 kinase, and Akt proteins, inactivating the Akt-dependent cell survival signaling pathway. These results show that a combination of IL-24-mediated molecular therapy and EGFR inhibitors such as erlotinib may be a promising treatment strategy for human melanoma and will serve as a basis for guiding the combination treatment designs in future preclinical and clinical trials.

Abstract Chronic inflammation can facilitate the initiation and progression of malignancies through the production of cytokines and reactive oxidative species. The elevation of interleukin 1 (IL-1) signaling pathway has been observed in melanoma and proposed to drive critical aspects of survival, which is tested in this study. Immunohistochemistry staining for IL-1α and β were performed in 168 biopsy samples from melanoma patients, including 35 nevi, 57 primary and 76 metastatic melanomas. Although only 11.4% of melanoma samples are positive for IL-1β, all the nevus samples are negative. The positive IL-1β percentage and intensity are strongly correlated with advanced disease stage (p < 0.0005 and 0.0002). 93.4% of melanomas and 64% of nevi were positive for IL-1α, with tumors showing significantly higher IL-1α intensity scores than nevi (p < 0.007). Melanoma cell lines A375, WM793, WM35, and SB2 highly express IL-1α, IL-1β, and IL-1 receptor I (IL-1RI), but not MeWo and TXM1 confirmed by RT-PCR and western blot. Therefore, the high expression of IL-1α and/or β was significant in some melanoma tissues and cell lines, which may play a crucial role on the melanoma growth. We further investigated the aberrant IL-1 signaling pathway in two mutant B-Raf melanoma cell lines, A375 and WM793. In both cells, the siRNA knockdown of IL-1α, IL-1β, or MyD88 led to dramatic decreases (about 50%) in several important inflammatory markers, including the Cox-2 expression, the levels of total reactive oxygen and nitrogen species, and the levels of phoshorylated SAPK/JNK and IκB. Moreover, the decrease of IL-1α, IL-1β, or MyD88 levels caused significant increase of p21 and p53 levels. Luciferase assay showed that 100 ng/ml IL-1α and β could increase about 30% of iNOS promoter activity in both cells. Our data suggest that aberrant IL-1 signaling pathway can promote inflammation and oxidative stress in melanoma cells. The treatment of 2.5 μg/ml IL-1RI neutralization antibodies could inhibit 20∼30% of cell growth in A375 and WM793 cells in 72 hours, but did not affect the growth of normal melanocytes as comparing with control goat IgG. Consistently, the siRNA knockdown of IL-1α, IL-1β, or MyD88 inhibited cell growth by 40∼60% in A375, WM793, WM35, and SB2 cells with high IL-1α and β levels. For TXM1 and MeWo cells with lower levels of IL-1α and β, siRNA knockdown of IL-1 system genes did not show any significant inhibitory effects on cell growth. Notably, the inhibition of growth by the decrease of IL-1α, IL-1β, or MyD88 levels in A375 and WM793 cells is due to autophagy, which was confirmed by the increases of acidic vesicular organelles in acridine orange staining and the autophagy marker LC3B levels in western blot. Our studies strongly indicate that the interruption of IL-1 signaling pathway can lead to growth suppression in melanoma cells, which can be a promising therapeutic strategy to sub-group melanoma patients with constitutively high expression of IL-1α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5051. doi:10.1158/1538-7445.AM2011-5051

Abstract Interleukin (IL)-1–mediated inflammation is proposed to contribute to the development and progression of some cancers. IL-1 family member proteins are known to be expressed constitutively in many melanoma tumor cells, and we hypothesize that these support molecular pathways of inflammation and facilitate tumor growth. To investigate the expression of IL-1α and IL-1β in melanoma patients, and their association with disease progression, immunohistochemical staining was carried out on tissues from 170 patients including benign nevi, primary melanomas, and metastatic melanomas. IL-1β levels were low (or zero) in benign nevi and higher in primary and metastatic melanomas (P < 0.0001). IL-1α was expressed in about 73% of nevi and 55% of metastatic melanomas, with levels significantly higher in primary tumors (P < 0.0001); most (98%) primary melanoma samples were positive for IL-1α. In vitro studies with seven human melanoma cell lines showed that five cell lines expressed IL-1α and IL-1β proteins and mRNA. We identified for the first time several important downstream signaling pathways affected by endogenous IL-1, including reactive oxygen and nitrogen species, COX-2, and phosphorylated NF-κB inhibitor (IκB) and stress-activated protein kinase/c-jun-NH2-kinase; all of which were decreased by siRNA to IL-1s. Downregulation of IL-1α, IL-1β, or MyD88 substantially increased p21 and p53 levels. Treatment with IL-1 receptor type I neutralizing antibody or IL-1 pathway–specific siRNAs led to growth arrest in IL-1–positive melanoma cells. Furthermore, blocking the IL-1 pathway increased autophagy in IL-1–positive melanoma cells. These results indicate that the endogenous IL-1 system is functional in most human melanoma and interrupting its signaling inhibits the growth of IL-1–positive melanoma cells. Mol Cancer Res; 9(11); 1537–50. ©2011 AACR.

The role of nonmyeloablative allogeneic stem cell transplantation (NST) in the treatment of chronic lymphocytic leukemia (CLL) is not well established. The authors report on long‐term experience with NST in relapsed/refractory CLL and define prognostic factors associated with outcome.

The interleukin (IL)-24 protein encoded by melanoma differentiation associated-7 (mda-7) gene is a novel IL-10 family cytokine with unique tumor-specific apoptotic and anti-angiogenic properties Additional role(s) for IL-24, including the regulation of skin inflammation, as suggested by recent data, provide a teleologic role for this melanocyte- and monocyte-produced molecule Previous studies by our group led to a Phase I trial for local adenoviral therapy delivery in advanced solid tumor patients These clinical studies employed gene therapy with adenoviral vector mediated delivery of mda-7/IL-24 (Ad-mda-7/IL-24) and clearly demonstrated a bystander apoptotic effect resulting from IL-24 protein production in injected tumor lesion While these studies are useful for "proof of principle" for IL-24, we now realize that we must devise means to use this same product, IL-24, systemically to achieve therapeutic success in patients with melanoma. Therefore, gene therapy with a nanoparticle delivery vehicle is now being pursued We propose that MDA7/IL-24 is a major skin-derived tumor suppressor/cytokine that has profound significance as a biotherapeutic lot melanoma and possibly other cancers

Interleukin (IL)-24 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-24 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-24 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-24 is expressed in keratinocytes during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL-24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor alpha (TGFalpha), TGFbeta, IFNgamma, and IFNbeta, upregulated IL-24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL-24 in both in vitro wound repair and migration assays demonstrated that IL-24 inhibits TGFalpha-induced proliferation and migration of NHEKs. These data support the hypothesis that IL-24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.

Abstract Aberrant expression of proinflammatory IL-1 α and β and the overproduction of nitric oxide (NO) have been shown to promote the progression of some tumors. In previous studies, we had confirmed that inducible nitric oxide synthase (iNOS) was constitutively expressed in most advanced melanomas and highly correlated with poor patient survival. In order to determine whether the imbalance of IL-1 and its natural competitor IL-1 receptor antagonist (IL-1Ra) contributes to the upregulation of iNOS in metastatic melanoma, we initiated an investigation of the expression profile of IL-1 system genes in melanoma and the linkage between IL-1 and iNOS/NO signaling pathways. Immunohistochemistry staining of 38 biopsy samples from metastatic melanoma patients showed that 33 samples highly expressed IL-1α and/or IL-1β. Only 5 samples were negative for both IL-1α and IL-1β expression. RT-PCR confirmed that melanoma cell lines A375, WM793, WM35, and SB2 constitutively expressed high levels of IL-1, IL-1 receptor I (IL-1RI), and iNOS. However, in the same cell lines, there was no detectable IL-1Ra mRNA, which is known to maintain IL-1 balance in normal skin tissues. Our study suggests that the imbalance of IL-1 and IL-Ra is widespread in metastatic melanoma. The engagement of IL-1 and IL-1RI can induce several kinase cascades leading to the activation of SAPK/JNK and NF-κB, and it is well known that transcription of iNOS can be regulated by NF-κB. In our studies, siRNA knockdown of either IL-1α or MyD88 (an important adaptor for IL-1RI signal pathway) in WM375 and A375 led to a dramatic decrease in the phoshorylation of SAPK/JNK and IκB. Furthermore, an 80% decrease of iNOS expression in WM793 was observed after the siRNA knockdown of MyD88. We confirmed that upregulated IL-1 signal pathway can activate NF-κB and the iNOS transcription. Treatment with 200 μM 7-Nitroindazole, a NO synthase inhibitor, not only reduced NO production, but also induced over 50% inhibition of cell growth and a marked increase in IL-1Ra mRNA in WM793 and A375. This indicates that a high level of NO can inhibit the IL-1Ra expression in melanoma cells. Based on our data, we proposed an inflammatory dysfunction model to explain the development of melanoma. It proposes that the constitutive expression of IL-1 can inhibit IL-1Ra expression mediated by the upregulation of iNOS/NO production in melanoma. The constant inhibition of IL-1Ra expression by NO results in a cycle of kinase transduction cascades, which drives NF-κB and its downstream inflammatory genes (including iNOS) constitutively expressed in melanoma. Our studies cast new insight into the mechanism of the pathology of metastatic melanoma. Selective inhibition of iNOS and increasing IL-1Ra levels are promising therapeutic strategies to suppress the aberrant IL-1 inflammation pathway in melanoma. Supported by MDACC SPORE in Melanoma CA093459. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4506.

Abstract Melanoma is one of the most prevalent cancers in the United States, having the fifth highest cancer incidence in males and sixth highest incidence in females. Primary cutaneous melanoma can often be cured by surgery, but for those patients in whom metastasis has occurred, mortality is high in part due to numerous chemoresistance mechanisms. Thus, there is an urgent need to discover and identify novel therapeutic targets for the efficient treatment of this lethal disease. Gene silencing by RNA interference (RNAi) is a powerful tool to identify novel genes involved in specific biological processes. In our study, we used high-throughput druggable siRNA library (Dharmacon) targeting 6028 human genes to screen metastatic melanoma cell lines and found that knockdown of gene expression by siRNA pools targeting 885 genes significantly inhibited melanoma growth (p<0.005). Among them, 67 siRNA pools resulted in >50% inhibition of cell viability in the melanoma A375 cells which we employed for the screening analysis. We validated the top-six cancer-related genes (RRM2, BCAR3, PSMA2, UBB, RRM1, TCP1) as potential melanoma therapeutic target candidates, as knockdown of these 6 genes significantly inhibited cell growth and induced apoptosis in multiple melanoma cell lines. We have hypothesized that antioxidant treatment relieves resistance mechanisms, and showed that among the screened >6000 siRNA pools, treatment with 20 pools significantly increased the growth inhibition in the presence of nontoxic levels of bardoxolone methyl (p<0.005), a novel semi-synthetic triterpenoid (previously known as CDDO-Me) with potent antioxidant activity generously provided by Reata Pharmaceuticals. We also selected 5 additional cancer-related genes (GNPAT, SUMO1, SPINT2, FLI1 and SSX1) for further analysis and validated them as additional candidates for therapeutic targeting in the presence of antioxidant. Our results thus demonstrate the feasibility of the high-throughput siRNA library screen system in discovering new melanoma therapeutic targets and shows that these new candidate genes are promising for the development of novel therapeutic targets for melanoma treatment, many of which are revealed by antioxidant pretreatment. Results from our study provide the basis for significant advances in identifying targets for the treatment of melanoma patients. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the NIH Melanoma SPORE grant P50, CA093459). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2564.

Abstract Melanoma is one of the most prevalent cancers. While early detection and surgical excision can cure melanoma, patients with advanced disseminated disease have a very poor prognosis due to its extremely aggressive growth characteristics and high resistance to chemotherapy and radiation. Improvement of our understanding of the mechanisms that lead to progression of metastatic melanoma, as well as development of novel therapeutic strategies are urgently required for patients. In our study, we applied PCR arrays and bioinformatics analysis to evaluate gene expression of metastatic melanoma cells and normal skin cells. We used the Human Inflammatory Cytokine and Receptors RT2 Profiler™ PCR Array to analyze the differential expression of genes in metastatic melanoma cell lines (A375) and primary normal epidermal melanocytes (NHEM), which are the precursor cell of melanoma. We found the 43 genes were highly expressed in A375 which were upregulated by more than 20 fold in comparison to NHEM. We next selected the top-ten differentially expressed genes (CD70, IL-1β, MGLL, SERPINA3, MDK, IL13RA2, CD74, VEGFA, CXCL1, and IL8), whose expression levels in A375 cells were > 250-fold higher than in the primary normal NHEM, for further validation. The results from our RT-PCR, immunoblot, immunocytochemistry (ICC) and tissue microarray (TMA) analysis showed that CD70, SERPINA3, IL13RA2 and CD74 genes were highly expressed at mRNA and protein levels in various melanoma cell lines and metastatic melanoma tumor tissues. A much lower level of expression of each gene was detected in normal human skin cell lines and normal skin tissues. These results thus demonstrated that the four genes identified by our PCR array are likely of molecular markers and should be pursued as therapeutic targets for melanoma treatment. To further verify their potentials as potential melanoma therapeutic targets, we also evaluated the effects of siRNAs targeting CD70 and CD74 genes and the monoclonal antibodies against CD70 and CD74 proteins for regulation of melanoma growth. Treatment with CD70 or CD74-specific siRNAs and also monoclonal antibodies dramatically inhibited cell growth in various melanoma cell lines, indicating that CD70 and CD74 are also attractive molecular targets in the treatment of human metastatic melanoma. Our findings have important implications in understanding the metastatic process of melanoma and discovering novel therapeutic strategies for melanoma treatment. The molecular markers and therapeutic targets developed in this study will have a direct and immediate translational application. If successful, our study will represent a significant advance in the diagnosis and treatment of melanoma. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Melanoma SPORE grant from the NCI, P50, CA093459). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1175.

Melanoma is the most malignant of skin cancers, highly resistant to chemotherapy and radiotherapy. Temozolomide, a promising new derivative of dacarbazine, is currently being tested for treatment of metastatic melanoma. Resistance to alkylating agents such as temozolomide correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Interleukin-24 (IL-24; mda-7) is a tumor suppressor cytokine that selectively inhibits tumor cell growth by inducing apoptosis and cell cycle arrest in melanoma cell lines and solid tumors. This tumor-selective activity has been observed in multiple preclinical animal models and in clinical trials. In this study, we analyzed the ability of Ad-IL-24 and its protein product, IL-24, to overcome temozolomide resistance in human melanoma cells. We have shown that Ad-IL-24 via exogenous IL-24 protein induces combinatorial synergy of temozolomide-induced cell killing in temozolomide-resistant melanoma cells by inhibition of MGMT. Neutralizing antibodies against IL-24 or its receptors significantly blocked the apoptotic activity of IL-24 + MGMT treatment. We show that accumulation of functional p53 is essential for IL-24-induced down-regulation of MGMT. Using either MGMT small interfering RNA, p53 small interfering RNA, or a p53 dominant-negative mutant to block MGMT protein expression resulted in increased sensitization to temozolomide. However, MGMT blockade in combination with IL-24 + temozolomide resulted in loss of combinatorial synergy, indicating that MGMT expression is required for the reversal of temozolomide resistance in melanoma cells. This study shows that IL-24 can play a significant role in overcoming temozolomide resistance and that the clinical efficacy of temozolomide may be improved by using a biochemotherapy combination with IL-24. [Mol Cancer Ther 2008;7(12):3842–51]

Purpose Our goal was to perform studies on the specificity and antimelanoma mechanism of a novel bis-anthracycline, WP760. WP760 initially identified in the NCI 160 screen as anti-melanoma. Methods The methyl thiazolyl tetrazolium reduction (MTT) assay was used to test tumor cell growth inhibition; confocal microscopy to view WP760 intracellular distribution; flow cytometry for cell-cycle arrest and apoptosis; and Western blotting was employed to identify and compare quantities and kinetics of cell growth related molecule levels. Results WP760 induced G 2 /M-phase cell-cycle arrest and apoptosis in melanoma cell lines and short-term melanoma explants established from clinical specimens in a time and concentration dependent manner at nM concentrations. In contrast, effects on fibroblasts and A549 lung cancer cells required higher concentrations, suggesting that WP760 possesses selectivity for melanoma. Molecular studies indicated that WP760 induced p53 stabilization, checkpoint kinase 2 and p27 Kip1 protein upregulation, and activation of caspase-3. Endogenous nitric oxide (NO) production has been implicated in the chemoresistance of melanoma; WP760 caused inhibition of the inducible nitric oxide synthase (iNOS) protein as well as inhibition of phosphorylation of ERK, known to drive the iNOS pathway. Based on WP760 localization into mitochondria, and caspase-3 inhibitor block the killing of WP760, the intrinsic pathway of apoptosis appears to have been activated. Conclusions Our results indicate that WP760 affects a critical and unique set of growth regulatory effects in melanoma, and is a promising candidate for further preclinical studies.