Glycoprotein NMB Expression Research Articles

GPNMB: a potent inducer of immunosuppression in cancer.

The immune system is comprised of both innate and adaptive immune cells, which, in the context of cancer, collectively function to eliminate tumor cells. However, tumors can actively sculpt the immune landscape to favor the establishment of an immunosuppressive microenvironment, which promotes tumor growth and progression to metastatic disease. Glycoprotein-NMB (GPNMB) is a transmembrane glycoprotein that is overexpressed in a variety of cancers. It can promote primary tumor growth and metastasis, and GPNMB expression correlates with poor prognosis and shorter recurrence-free survival in patients. There is growing evidence supporting an immunosuppressive role for GPNMB in the context of malignancy. This review provides a description of the emerging roles of GPNMB as an inducer of immunosuppression, with a particular focus on its role in mediating cancer progression by restraining pro-inflammatory innate and adaptive immune responses.

Glycoprotein NMB promotes tumor formation and malignant progression of laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma (LSCC), although one of the most common head and neck cancers, has a static or slightly decreased survival rate because of difficulties in early diagnosis, lack of effective molecular targeting therapy, and severe dysfunction after radical surgical treatments. Therefore, a novel therapeutic target is crucial to increase treatment efficacy and survival rates in these patients. Glycoprotein NMB (GPNMB), whose role in LSCC remains elusive, is a type 1 transmembrane protein involved in malignant progression of various cancers, and its high expression is thought to be a poor prognostic factor. In this study, we showed that GPNMB expression levels in LSCC samples are significantly higher than those in normal tissues, and GPNMB expression is observed mostly in growth‐arrested cancer cells. Furthermore, knockdown of GPNMB reduces monolayer cellular proliferation, cellular migration, and tumorigenic growth, while GPNMB protein displays an inverse relationship with Ki‐67 levels. Therefore, we conclude that GPNMB may be an attractive target for future LSCC therapy.

HSP90 inhibitors induce GPNMB cell-surface expression by modulating lysosomal positioning and sensitize breast cancer cells to glembatumumab vedotin.

Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.

GPNMB mitigates Alzheimer’s disease and enhances autophagy via suppressing the mTOR signal

Alzheimer’s disease (AD) is a common neurodegenerative disease which is characterized by amyloid beta (Aβ) accumulation. We found that glycoprotein NMB (GPNMB) was highly expressed in the brain of APP/PS1 mice, a mouse model of AD. However, its role in AD remains unclear. In this study, we aimed to explore the function of GPNMB in AD. The expression of GPNMB in the brain was detected by immunofluorescence and western blot. In addition, the role of GPNMB in AD was explored through gain-of-function. Autophagy, which is beneficial to Aβ clearance, was evaluated by transmission electron microscope and immunofluorescence with beclin-1. Furthermore, 3-MA, an autophagy inhibitor, was employed to evidence whether GPNMB reduced the level of Aβ through autophagy. We found that over-expression of GPNMB improved AD-like behaviors in APP/PS1 mice and reduced Aβ deposition. Further study showed that GPNMB enhanced autophagy, reduced microglial cells and inhibited the activation of the mTOR signal. Additionally, treatment with 3-MA abolished the beneficial effect of GPNMB on Aβ clearance. This study revealed that the high level of GPNMB in AD brain may help Aβ clearance and improve AD-like behaviors through enhancing autophagy via suppressing the mTOR signal. This beneficial role of GPNMB provides us novel strategies for the prevention and treatment of AD.

Adaptive resistance in tumors to anti-PD-1 therapy through re-immunosuppression by upregulation of GPNMB expression

Acquired resistance to the antitumor activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in various types of cancers has increasingly been observed during treatment. To gain insight into the molecular mechanism underlying anti-PD-1 therapy resistance, we developed a mouse MC38 colon adenocarcinoma cell line that was made resistant to anti-PD-1 treatment through repeated in vivo selection. We compared the transcriptomic profiles of anti-PD-1 therapy-resistant and -sensitive tumors using RNA sequencing analysis. The immunosuppressive molecule transmembrane glycoprotein NMB (GPNMB) was significantly upregulated in resistant tumor cells, as determined using quantitative real-time polymerase chain reaction and immunofluorescence analyses. Furthermore, deletion of GPNMB in resistant cells successfully restored sensitivity to anti-PD-1 treatment in vivo. Collectively, our results indicate that tumors may develop resistance to anti-PD-1 therapy by upregulating their expression of the immunosuppressive molecule GPNMB. Furthermore, GPNMB is a potential, targetable biomarker for monitoring adaptive resistance to therapeutic PD-1 blockade, and identification of this immunosuppressive molecule may be a breakthrough for new therapies.

A Phase II Study of Glembatumumab Vedotin for Metastatic Uveal Melanoma.

Glembatumumab vedotin (CDX-011, GV) is a fully human Immunoglobulin G2 monoclonal antibody directed against glycoprotein NMB coupled via a peptide linker to monomethyl auristatin E (MMAE), a potent cytotoxic microtubule inhibitor. This phase II study evaluated the overall response rate and safety of GV, glycoprotein NMB (GPNMB) expression, and survival in patients with metastatic uveal melanoma. Eligible patients with metastatic uveal melanoma who had not previously been treated with chemotherapy received GV 1.9 mg/kg every three weeks. The primary endpoint was the objective response rate (ORR). Secondary endpoints included GPNMB expression, progression-free survival (PFS), overall survival (OS), and toxicity analysis. GPNMB expression was assessed pre- and post-treatment via immunohistochemistry for patients with available tumor tissue. Out of 35 patients who received treatment, two patients had confirmed partial responses (PRs; 6%), and 18 patients had a stable disease (SD; 51%) as the best objective response. 38% of the patients had stable disease >100 days. The grade 3 or 4 toxicities that occurred in two or more patients were neutropenia, rash, hyponatremia, and vomiting. The median progression-free survival was 3.1 months (95% CI: 1.5–5.6), and the median overall survival was 11.9 months (95% CI 9.0–16.9) in the evaluable study population. GV is well-tolerated in metastatic uveal melanoma. The disease control rate was 57% despite a low objective response rate. Exploratory immune correlation studies are underway to provide insight into target saturation, combination strategies, and antigen release.

SAT-141 Glycoprotein NMB (GPNMB) Is Pro-Tumorigenic in TSC2-Null Cancer Cells and Is a Potential Drug Target and Biomarker for Lymphangioleiomyomatosis (LAM)

Lymphangioleiomyomatosis (LAM) is an estrogen-sensitive lung disease found almost exclusively in women that is characterized by hyperproliferation of smooth muscle cells forming small tumors, or LAM lesions throughout the lungs of patients. Growth of these tumors leads to progressive loss of pulmonary function, and sometimes subsequent lung transplantation. LAM tumor cells contain mutations in either the TSC1 or TSC2 genes, leading to activation of the mTORC1 pathway. In fact, mTOR inhibitors such as sirolimus are commonly used to treat LAM; however, these drugs are not always effective and have significant side effects, suggesting the need for new therapeutic targets. Interestingly, another important feature of LAM cells is that they express melanocytic markers that are normally found in melanocytes or melanoma cells. From RNASeq analysis of a mouse model for LAM that we designed, we discovered significant upregulation of the melanocytic marker Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB), a type I transmembrane protein. GPNMB was not only highly expressed in our mouse model (a uterine specific TSC2-null mouse), it was also expressed in TSC2-null cell lines, and human LAM patient lung samples. In our hands, knocking down GPNMB expression by siRNA directed against GPNMB mRNA decreased migration and proliferation in TSC2-null cells. Additionally, we found that GPNMB’s large ectodomain is shed by TSC2-null cells and can be detected in the blood of human patients with LAM. Finally, MMP 2 and 9 can be secreted as a result of ectodomain shedding and its interaction with integrins. Accordingly, we did indeed see a decrease in MMP 2/9 expression in TSC2-null cells with reduced GPNMB expression from treatment with siRNA directed against GPNMB mRNA. Overall, our results demonstrate the potential importance of GPNMB in LAM tumor progression, and suggest that GPNMB may be a possible LAM biomarker and target for its treatment.

Abstract B88: GPNMB expression modulates the tumor immune microenvironment in mouse models of breast cancer

Abstract The role of glycoprotein-NMB (GPNMB) in the immune system is varied. GPNMB expression in macrophages and dendritic cells promotes innate immune responses, whereas GPNMB-expressing myeloid-derived suppressor (MDSC) cells suppress adaptive immune responses (T-cell function). While functional roles for GPNMB expressed within cells of the immune system are emerging, the influence of tumor-derived GPNMB expression on the immune landscape within mammary tumors has not been well characterized. To investigate this further, we have generated two GPNMB-deficient breast cancer cell populations (Lung Metastatic-4T1 [LM-4T1] and E0771 cells) to determine the influence of GPNMB on the tumor immune microenvironment (TME). While loss of GPNMB significantly impaired tumor growth of both these breast cancer models in syngeneic mice, this difference was not apparent with LM-4T1 injected in athymic nude mice. These observations suggest GPNMB may promote tumor growth by modulating the T-cell function. We have used traditional immunohistochemical analyses to broadly characterize the T cells present in the TME of E0771 and LM-4T1 breast tumors, which express or lack GPNMB. We have observed a significant increase in both CD8+ and CD4+ immune cells in GPNMB-deficient LM-4T1 and E0771 tumors compared to parental cells at an experimental endpoint of matched tumor volumes. To determine the temporal response of the immune system to LM-4T1 cells, we have isolated early developing lesions and stained for CD8, CD4, Fox3p, and granzyme B positive cells. We observed a significant increase in CD4+ and a trend for elevated granzyme B+ cells at early timepoints in GPNMB-deficient tumors compared to LM-4T1 parental cells. Taken together, these results suggest GPNMB suppresses an early recruitment of CD4+ cells where, in the absence of GPNMB, this would result in elevated CD8+ and CD4+ cells in these tumors. We are currently characterizing the contribution of CD4+ cells in the progression of these tumor models. Citation Format: Matthew G. Annis, April A.N. Rose, Ryuhjin Ahn, Brian E. Hsu, Josie Ursini-Siegel, Peter M. Siegel. GPNMB expression modulates the tumor immune microenvironment in mouse models of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B88.

Role of the kringle‐like domain in glycoprotein NMB for its tumorigenic potential

Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. Glycoprotein NMB is a type IA transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein‐protein or protein‐carbohydrate interactions, whereas the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi‐immunoreceptor tyrosine‐based activation motif (hemITAM) in its ICD. Furthermore, we showed that the tyrosine residue in hemITAM is involved in induction of the stem‐like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so‐called kringle‐like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of WT GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties, such as subcellular localization, Src‐induced tyrosine phosphorylation at least in overexpression experiments, and homo‐oligomerization. Additionally, GPNMB(ΔKLD) lost its cell migration promoting activity, even though it reduced E‐cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB plays an important role in its tumorigenic potential.

Glycoprotein nmb Is Exposed on the Surface of Dormant Breast Cancer Cells and Induces Stem Cell-like Properties.

Glycoprotein nmb (GPNMB) is a type I transmembrane protein that contributes to the initiation and malignant progression of breast cancer through induction of epithelial-mesenchymal transition (EMT). Although it is known that EMT is associated with not only cancer invasion but also acquisition of cancer stem cell (CSC) properties, the function of GPNMB in this acquisition of CSC properties has yet to be elucidated. To address this issue, we utilized a three-dimensional (3D) sphere culture method to examine the correlation between GPNMB and CSC properties in breast cancer cells. Three-dimensional sphere cultures induced higher expression of CSC genes and EMT-inducing transcription factor (EMT-TF) genes than the 2D monolayer cultures. Three-dimensional culture also induced cell surface expression of GPNMB on limited numbers of cells in the spheres, whereas the 2D cultures did not. Therefore, we isolated cell surface-GPNMBhigh and -GPNMBlow cells from the spheres. Cell surface-GPNMBhigh cells expressed high levels of CSC genes and EMT-TF genes, had significantly higher sphere-forming frequencies than the cell surface-GPNMBlow cells, and showed no detectable levels of proliferation marker genes. Similar results were obtained from transplanted breast tumors. Furthermore, wild-type GPNMB, but not mutant GPNMB (YF), which lacks tumorigenic activity, induced CSC-like properties in breast epithelial cells. These findings suggest that GPNMB is exposed on the surface of dormant breast cancer cells and its activity contributes to the acquisition of stem cell-like properties.Significance: These findings suggest that cell surface expression of GPNMB could serve as a marker and promising therapeutic target of breast cancer cells with stem cell-like properties. Cancer Res; 78(22); 6424-35. ©2018 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Increases in transmembrane glycoprotein NMB (GPNMB), phospho‐ERK1/2, and matrix metallopeptidase (MMP)‐9 follow decline in arylsulfatase B in cystic fibrosis

In previous reports, decline in the enzyme arylsulfatase B (ARSB; N‐acetylgalactosamine‐ 4‐sulfatase) was identified in cystic fibrosis (CF) cells in culture and in circulating leukocytes from CF patients. Recently, the transmembrane glycoprotein NMB (GPNMB) was shown to be increased in a CF gene array and in Arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfatase)‐null mice. These findings are consistent with previous reports that ARSB is reduced in cystic fibrosis (CF). A mechanism by which decline in ARSB, and the resultant increase in chondroitin 4‐sulfate, increased GPNMB expression has been reported. Subsequently, experiments were designed to assess the potential role of GPNMB in CF and the mechanisms by which GPNMB might contribute to CF pathophysiology. In serum and circulating leukocytes from CF and control patients, GPNMB levels were determined by ELISA. GPNMB was also measured in cells and spent media of CFTR‐corrected, CFTR‐uncorrected, and normal human bronchial epithelial cells (BEC) following ARSB siRNA or control siRNA. The GPNMB which co‐immunoprecipitated with β‐1 integrin was determined by ELISA. Matrix metalloproteinase (MMP)‐9 was measured, and the correlations between GPNMB and MMP‐9 in serum and spent media were determined. Phospho‐ERK1/2 in the circulating leukocytes and in the cultured cells was measured. The impact of excess exogenous RGD peptide and GPNMB siRNA on phospho‐ERK1/2 content was determined, and the impact of the RGD peptide, GPNMB siRNA, and ERK phosphorylation inhibitor on MMP‐9 mRNA expression was determined. GPNMB was markedly increased in cells and serum of CF patients compared to controls (p<0.0001, unpaired t‐test, two‐tailed). In uncorrected CF cells and in BEC following ARSB silencing, the GPNMB was significantly increased (p<0.0001; p<0.001). Silencing GPNMB or treatment with excess RGD peptide significantly inhibited the ARSB‐silencing induced increase in phospho‐ERK1/2 in the BEC. Silencing GPNMB, treatment with excess RGD peptide, or treatment with ERK phosphorylation inhibitor blocked the ARSB silencing‐induced increases in MMP‐9 expression in the normal BEC. Findings suggest that decline in ARSB activity caused by decline in CFTR function leads to increased GPNMB, phospho‐ERK1/2, and MMP‐9 expression, which may contribute to disruption of cell signaling and to organ dysfunction in CF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The transcription factor MAFK induces EMT and malignant progression of triple-negative breast cancer cells through its target GPNMB.

Triple-negative breast cancer (TNBC) is particularly aggressive and difficult to treat. For example, the transforming growth factor-β (TGF-β) pathway is implicated in TNBC progression and metastasis, but its opposing role in tumor suppression in healthy tissues and early-stage lesions makes it a challenging target. Therefore, additional molecular characterization of TNBC may lead to improved patient prognosis by informing the development and optimum use of targeted therapies. We found that musculoaponeurotic fibrosarcoma (MAF) oncogene family protein K (MAFK), a member of the small MAF family of transcription factors that are induced by the TGF-β pathway, was abundant in human TNBC and aggressive mouse mammary tumor cell lines. MAFK promoted tumorigenic growth and metastasis by 4T1 cells when implanted subcutaneously in mice. Overexpression of MAFK in mouse breast epithelial NMuMG cells induced epithelial-mesenchymal transition (EMT) phenotypes and promoted tumor formation and invasion in mice. MAFK induced the expression of the gene encoding the transmembrane glycoprotein nmb (GPNMB). Similar to MAFK, GPNMB overexpression in NMuMG cells induced EMT, tumor formation, and invasion, in mice, whereas knockdown of MAFK in tumor cells before implantation suppressed tumor growth and progression. MAFK and GPNMB expression correlated with poor prognosis in TNBC patients. These findings suggest that MAFK and its target gene GPNMB play important roles in the malignant progression of TNBC cells, offering potentially new therapeutic targets for TNBC patients.

Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF.

Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events.

The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells.

BackgroundDendritic cells (DC) are the most potent antigen-presenting cells (APC) with the unique ability to activate naïve T cells and to initiate and maintain primary immune responses. Immunosuppressive and anti-inflammatory stimuli on DC such as the cytokine IL-10 suppress the activity of the transcription factor NF-κB what results in downregulation of costimulatory molecules, MHC and cytokine production. Glycoprotein NMB (GPNMB) is a transmembrane protein, which acts as a coinhibitory molecule strongly inhibiting T cell responses if present on APC. Interestingly, its expression on human monocyte-derived dendritic cells (moDC) is dramatically upregulated upon treatment with IL-10 but also by the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib, nilotinib or dasatinib used for the treatment of chronic myeloid leukemia (CML). However, the molecular mechanisms responsible for GPNMB overexpression are yet unknown.ResultsThe immunosuppressive cytokine IL-10 and the BCR-ABL TKI imatinib or nilotinib, that were examined here, concordantly inhibit the PI3K/Akt signaling pathway, thereby activating the downstream serine/threonine protein kinase GSK3ß, and subsequently the microphthalmia-associated transcription factor (MITF) that is phosphorylated and translocated into the nucleus. Treatment of moDC with a small molecule inhibitor of MITF activity reduced the expression of GPNMB at the level of mRNA and protein, indicating that GPNMB expression is in fact facilitated by MITF activation. In line with these findings, PI3K/Akt inhibition was found to result in GPNMB overexpression accompanied by reduced stimulatory capacity of moDC in mixed lymphocyte reactions (MLR) with allogeneic T cells that could be restored by addition of the GPNMB T cell ligand syndecan-4 (SD-4).ConclusionsIn summary, imatinib, nilotinib or IL-10 congruently inhibit the PI3K/Akt signaling pathway thereby activating MITF in moDC, resulting in a tolerogenic phenotype. These findings extend current knowledge on the molecular mechanisms balancing activating and inhibitory signals in human DC and may facilitate the targeted manipulation of T cell responses in the context of DC-based immunotherapeutic interventions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-015-0099-5) contains supplementary material, which is available to authorized users.

Abstract 3984: Elucidating expression patterns of GPNMB and potential anti-tumor effects of the antibody-drug conjugate, glembatumumab vedotin (CDX-011) in human osteosarcoma primary samples and cell lines

Abstract Despite numerous clinical trials to improve outcomes for osteosarcoma treatment, there has been no significant improvement in cure rates over the last three decades. Glycoprotein NMB (GPNMB), also known as osteoactivin, is a transmembrane glycoprotein that is expressed in various normal cell types including osteoclasts and osteoblasts, and plays a role in development and differentiation, respectively. Studies have also shown that GPNMB is highly expressed in cancers including breast cancers and melanomas. CDX-011 is an antibody-drug conjugate that targets the GPNMB protein. A human monoclonal antibody glembatumumab is linked to monomethyl auristatin E, an antimitotic agent. Recent studies have shown the drug is effective in treating patients with metastatic triple- negative GPNMB-expressing breast cancer, and melanoma. The pediatric preclinical testing program (PPTP) has recently shown the drug is efficacious in xenograft models of osteosarcoma. In this study, we explore the feasibility of GPNMB as a target for antibody-mediated therapy in osteosarcoma and assess the anti-tumor effects of CDX-011. Since CDX-011 activity appears to correlate with GPNMB protein expression, a cohort of osteosarcoma tissue sections from patients at the time of initial biopsy, definitive surgery, and recurrence was stained immunohistochemically to determine the presence or absence of GPNMB protein. The majority of samples show positive staining suggesting further that osteosarcoma might be a candidate for CDX-011 treatment. Flow cytometry was performed on human osteosarcoma xenografts and cell lines to determine the surface expression level of GPNMB prior to treatment with CDX-011. Of the 9 cell lines studied, 5 lines showed high surface expression of GPNMB protein. Cytotoxicity assays were performed on the same 9 cell lines representing both high and low GPNMB surface expression patterns to determine the in vitro effectiveness of CDX-011 in osteosarcoma cell lines and its correlation with GPNMB expression. IC50 on those cell lines ranged from 3.5- 56 µM. Three out of 5 cell lines, which showed high GPNMB surface expression pattern, demonstrated lower IC50 (3- 14 µM) compared to the others. These findings suggest that CDX-011 may be a potential targeted treatment in osteosarcoma patients with elevated GPNMB expression patterns. In addition, further studies are ongoing, including quantitative PCR for GPNMB gene expression, and ELISA to further validate expression of GPNMB protein to better understand the mechanism of CDX-011 in osteosarcoma. Citation Format: Sajida Piperdi, Vicky Kuo, Amy Park, Michael Roth, Richard Gorlick. Elucidating expression patterns of GPNMB and potential anti-tumor effects of the antibody-drug conjugate, glembatumumab vedotin (CDX-011) in human osteosarcoma primary samples and cell lines. [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 3984. doi:10.1158/1538-7445.AM2014-3984

GPNMB expression in uveal melanoma: a potential for targeted therapy

Uveal melanoma is an aggressive disease without effective adjuvant therapy for metastases. Despite genomic differences between cutaneous and uveal melanomas, therapies based on shared biological factors could be effective against both tumor types. High expression of glycoprotein-NMB (GPNMB) in cutaneous melanomas led to the development of CDX-011 (glembatumumab vedotin), a fully human monoclonal antibody against the extracellular domain of GPNMB conjugated to the cytotoxic microtubule toxin monomethylauristatin E. Ongoing phase II trials suggest that CDX-011 has activity against advanced cutaneous melanomas. To determine the potential role of CDX-011 in uveal melanomas, we studied their GPNMB expression. Paraffin-embedded tissues from 22 uveal melanomas treated by enucleation from 2004-2007 at one institution were evaluated immunohistochemically for expression of GPNMB using biotinylated CDX-011 (unconjugated) antibody. Melanoma cells were evaluated for percentage and intensity of expression. Spectral imaging was used in one case with high melanin content. Clinical data were reviewed. Twelve women and 10 men with a median age of 58.7 years (range: 28-83 years) were included. Eighteen of 21 tumors evaluated immunohistochemically (85.7%) expressed GPNMB in 10-90% of tumor cells with variable intensity (5 tumors, 1+; 11, 2+; and 2, 3+). Eleven of 18 tumors (61.1%) expressed GPNMB in >or=50% of cells. Spectral imaging showed diffuse CDX-011 (unconjugated) reactivity in the remaining case. Uveal melanoma, like cutaneous melanoma, commonly expresses GPNMB. Ongoing clinical trials of CDX-011 should be extended to patients with metastatic uveal melanoma to determine potential efficacy in this subset of patients with melanoma.

A Phase (Ph) I/II Study of CR011-VcMMAE, an Antibody-Drug Conjugate, in Patients (Pts) with Locally Advanced or Metastatic Breast Cancer (MBC).

Abstract Background: Glycoprotein NMB (GPNMB), also known as osteoactivin, is expressed in 25-40% of breast cancers and is associated with an increased risk of recurrence. This is the first study of CR011-vcMMAE, a fully-human monoclonal anti-GPNMB antibody conjugated to the tubulin inhibitor monomethylauristatin E (MMAE), in pts with breast cancer.Methods: Eligible pts with MBC had ³ 2 prior chemotherapy regimens, including a taxane, an anthracycline, and capecitabine; and ECOG PS ≤ 2. Doses were escalated to 1.88 mg/kg IV q3w (the maximum tolerated dose [MTD] in a previous study of patients with melanoma) using a standard 3+3 design followed by a Ph II expansion at the MTD. Pts received CR011-vcMMAE until disease progression (PD) or intolerable toxicity. The primary endpoint of the Ph II study was 12-week progression free rate, defined as the proportion of patients who were alive and free of progressive disease 12 weeks from the first dose of CR011-vcMMAE. A Simon two-stage minimax design was used (p0=0.1; p1=0.3, α=β=0.1) with 16 patients in the first stage and a total of 25 pts. Secondary endpoints included objective response rate and duration of response. IHC with a goat polyclonal antibody to GPNMB was performed on pt biopsy specimens.Results: In Ph I, 14 pts were treated with CR011-vcMMAE at 1.0 mg/kg (n=3), 1.34 mg/kg (n=5), and 1.88 mg/kg (n=6). In the first 2 pts at 1.34 mg/kg, dose limiting toxicity of worsening peripheral sensory neuropathy was observed. Pts with baseline neuropathy worse than grade 1 were subsequently excluded. There were 3 partial responses, one confirmed, and 1.88 mg/kg q3w was selected for Ph II. In Ph II, 14 pts with MBC (median age 57 years, range 34 - 76) had a median of 5 prior regimens; median follow up was 6 weeks; 12 pts are ongoing (median duration 6 weeks, range 1 - 14 weeks). Two pts were progression-free at 12 weeks, therefore the study has met the criteria for advancement to the second stage. The most common AEs were rash (61%), fatigue (50%) and alopecia (50%). The most common grade 3/4 AEs were neutropenia (17%) and neuropathy (11%). IHC staining of 5 patient biopsies revealed 2 with positive GPNMB expression, including one of the patients with PR.Conclusions: CR011-vcMMAE is active and well-tolerated in heavily pretreated pts with advanced breast cancer. The Phase II study has met the criteria for advancement into the second stage, and enrollment is ongoing. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6096.

CR011, a fully human monoclonal antibody-auristatin E conjugate, for the treatment of melanoma.

Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to identify melanoma-associated cell surface molecules and to develop as well as preclinically test immunotherapeutic reagents designed to exploit such targets. By transcript profiling, we identified glycoprotein NMB (GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody, designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. These preclinical results support the continued evaluation of CR011-vcMMAE for the treatment of melanoma.