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Purpose: Although the murine orthologue of glycoprotein nonmetastatic B (GPNMB), Osteoactivin, promotes breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer.Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB levels in several published gene expression data sets and two independent tissue microarrays derived from human breast tumors. GPNMB-expressing human breast cancer cell lines were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent.Results: GPNMB expression correlates with shorter recurrence times and reduced overall survival of breast cancer patients. Epithelial-specific GPNMB staining is an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells and sensitizes them to killing by CDX-011 (glembatumumab vedotin), a GPNMB-targeted antibody-drug conjugate.Conclusions: GPNMB expression is associated with the basal/triple-negative subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic triple-negative breast cancers, a patient population that currently lacks targeted-therapy options. Clin Cancer Res; 16(7); 2147–56. ©2010 AACR.

Supplementary Fig. S1. Heatmap of mRNA expression for individual melanosomal differentiation antigens and a MITF transcriptional signature using a published dataset of melanoma samples (2). Supplementary Fig. S2. GPNMB expression is induced in multiple BRAF-mutant cells in response to MAPK pathway inhibition. Supplementary Fig. S3. Gene expression of MITF and GPNMB in melanoma cells following MAPK pathway inhibition. Supplementary Fig. S4. Dabrafenib and Trametinib stimulate MITF nuclear localization. Supplementary Fig. S5. Correlation of MITF and GPNMB mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway 7 inhibitors. RT-qPCR data for MITF and GPNMB was expressed as the ratio relative to pretreatment levels. Supplementary Fig. S6. Correlation between the expression of individual melanosomal genes with MITF mRNA expression in melanoma samples from patients that were on-treatment or that progressed on therapy with MAPK pathway inhibitors. RT-qPCR data for each gene and MITF was normalized to GAPDH and expressed as the ratio relative to pre-treatment levels. Supplementary Fig. S7. MAPK pathway inhibition results in elevated levels of soluble GPNMB released from melanoma cells. Table S1: Clinical characteristics of patients biopsied for evaluation of MITF and GPNMB mRNA expression Table S2: Correlation coefficients between individual gene expression and MITF or a MITF Signature Table S3: Complete Responses Following Trametinib Treatment in Cohorts Injected with A375 or WM-2664 Melanoma Cells. Table S4: List of Primers for RT-qPCR Analyses

Supplementary legends and methods

Contains supplementary methods, references and figure legends.

Purpose: To determine if BRAF and/or MEK inhibitor–induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB.Experimental Design: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth.Results: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor–treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone.Conclusions: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088–98. ©2016 AACR.

Supplementary Data from Glycoprotein Nonmetastatic B Is an Independent Prognostic Indicator of Recurrence and a Novel Therapeutic Target in Breast Cancer

FIGURE S1: LY3009120 is an effective inhibitor of BRAF class II mutant melanoma cells. FIGURE S2: Bioinformatic characterization of PDX models. FIGURE S3: Inhibitory effects of BRAF and MEK inhibitors in melanoma cells. FIGURE S4: Representative images from cell growth clonogenic assays. FIGURE S5: Encorafenib and binimetinib are an effective combination for class II BRAF mutant melanoma. FIGURE S6: Dual MAPK inhibition slows progression of class II BRAF mutant melanoma brain metastases.

Purpose:Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi.Experimental Design:Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi.Results:BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi.Conclusions:Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma.See related commentary by Johnson and Dahlman, p. 6107.

PURPOSE Non-V600 mutations comprise approximately 35% of all BRAF mutations in cancer. Many of these mutations have been identified as oncogenic drivers and can be classified into three classes according to molecular characteristics. Consensus treatment strategies for class 2 and 3 BRAF mutations have not yet been established. METHODS We performed a systematic review and meta-analysis with published reports of individual patients with cancer harboring class 2 or 3 BRAF mutations from 2010 to 2021, to assess treatment outcomes with US Food and Drug Administration–approved mitogen-activated protein kinase (MAPK) pathway targeted therapy (MAPK TT) according to BRAF class, cancer type, and MAPK TT type. Coprimary outcomes were response rate and progression-free survival. RESULTS A total of 18,167 studies were screened, identifying 80 studies with 238 patients who met inclusion criteria. This included 167 patients with class 2 and 71 patients with class 3 BRAF mutations. Overall, 77 patients achieved a treatment response. In both univariate and multivariable analyses, response rate and progression-free survival were higher among patients with class 2 compared with class 3 mutations, findings that remain when analyses are restricted to patients with melanoma or lung primary cancers. MEK ± BRAF inhibitors demonstrated greater clinical activity in class 2 compared with class 3 BRAF-mutant tumors than BRAF or EGFR inhibitors. CONCLUSION This meta-analysis suggests that MAPK TTs have clinical activity in some class 2 and 3 BRAF-mutant cancers. BRAF class may dictate responsiveness to current and emerging treatment strategies, particularly in melanoma and lung cancers. Together, this analysis provides clinical validation of predictions made on the basis of a mutation classification system established in the preclinical literature. Further evaluation with prospective clinical trials is needed for this population.

Although combination BRAF/MEK inhibition has produced significant survival benefits for BRAF p.V600 mutant melanomas, targeted therapies approved for BRAF non-p.V600 mutant melanomas remain limited. Through the analysis of 772 cutaneous melanoma exomes, we reveal that BRAF non-p.V600 mutations co-occurs more frequently with NF1 loss, but not with oncogenic NRAS mutations, than expected by chance. We present cell signaling data, which demonstrate that BRAF non-p.V600 mutants can signal as monomers and dimers within an NF1 loss context. Concordantly, BRAF inhibitors that inhibit both monomeric and dimeric BRAF synergize with MEK inhibition to significantly reduce cell viability in vitro and tumor growth in vivo in BRAF non-p.V600 mutant melanomas with co-occurring NF1 loss-of-function mutations. Our data suggest that patients harboring BRAF non-p.V600 mutant melanomas may benefit from current FDA-approved BRAF/MEK inhibitor combination therapy currently reserved for BRAF p.V600 mutant patients.

Purpose: Dual MAPK pathway inhibition (dMAPKi) with BRAF and MEK inhibitors improves survival in BRAF V600E/K mutant melanoma, but the efficacy of dMAPKi in non-V600 BRAF mutant tumors is poorly understood. We sought to characterize the responsiveness of class II (enhanced kinase activity, dimerization dependent) BRAF mutant melanoma to dMAPKi. Experimental Design: Tumors from patients with BRAF wild-type (WT), V600E (class I), and L597S (class II) metastatic melanoma were used to generate patient-derived xenografts (PDX). We assembled a panel of melanoma cell lines with class IIa (activation segment) or IIb (p-loop) mutations and compared these with WT or V600E/K BRAF mutant cells. Cell lines and PDXs were treated with BRAFi (vemurafenib, dabrafenib, encorafenib, and LY3009120), MEKi (cobimetinib, trametinib, and binimetinib), or the combination. We identified 2 patients with BRAF L597S metastatic melanoma who were treated with dMAPKi. Results: BRAFi impaired MAPK signaling and cell growth in class I and II BRAF mutant cells. dMAPKi was more effective than either single MAPKi at inhibiting cell growth in all class II BRAF mutant cells tested. dMAPKi caused tumor regression in two melanoma PDXs with class II BRAF mutations and prolonged survival of mice with class II BRAF mutant melanoma brain metastases. Two patients with BRAF L597S mutant melanoma clinically responded to dMAPKi. Conclusions: Class II BRAF mutant melanoma is growth inhibited by dMAPKi. Responses to dMAPKi have been observed in 2 patients with class II BRAF mutant melanoma. These data provide rationale for clinical investigation of dMAPKi in patients with class II BRAF mutant metastatic melanoma. See related commentary by Johnson and Dahlman, p. 6107.

BackgroundMetastatic uveal melanoma (mUM) is a rare disease for which no systemic therapy has demonstrated overall survival (OS) benefit. There are no robust data on prognostic factors for OS in patients with mUM treated with immune checkpoint inhibitors (ICI).Retrospective and non-randomized prospective studies have reported response rates of 0-37% for anti-PD1/L1 +/-anti-CTLA4 ICI in mUM, indicating a potential benefit only in a subset of patients. This study evaluates the characteristics associated with ICI benefit in patients with mUM.MethodsWe performed a single-center retrospective cohort study of patients with mUM who received anti-PD1/L1 +/-anti-CTLA4 ICI between 2014–2019. Clinical and genomic characteristics were collected from chart review. Treatment response and clinical progression were determined by physician assessment. Multivariable Cox regression models and Kaplan-Meier log-rank tests were used to assess differences in clinical progression-free survival (cPFS) and OS between groups and to identify clinical variables associated with ICI outcomes.ResultsWe identified 71 mUM patients who received 75 lines of ICI therapy. Of these, 54 received anti-PD1/L1 alone, and 21 received anti-PD1/L1 + anti-CTLA4. Patient characteristics were: 53% female, 48% were 65 or older, 72% received one or fewer lines of prior therapy. Within our cohort, 53% of patients had developed stage IV disease < 2 years after their initial diagnosis. Bone metastases were present in 12% of patients. For the entire cohort, the median cPFS was 2.7 months and median OS was 10.0 months. In multivariable analyses for both cPFS and OS, the following variables were associated with good prognosis: ≥ 2yrs from initial diagnosis to stage IV (n=25), LDH Metastatic Uveal Melanoma Prognostic risk Score (MUMPS). Patients were divided into 3 MUMPS risk groups based on the number of the above-mentioned prognostic variables: Poor risk (0-1), Intermediate risk (2) and Good risk (3). Good risk patients experienced longer cPFS (6.0 months) and OS (34.5 months) than patients with intermediate (2.3 months cPFS, 9.4 months OS) and poor risk disease (1.8 months cPFS, 3.9 months OS); PConclusionWe developed a MUMPS risk score, based on retrospective data, that is comprised of 3 readily available clinical variables (time to stage IV diagnosis, presence of bone metastases, and LDH). This MUMPS risk score has potential prognostic value. Further validation in independent datasets is warranted to determine the role of this MUMPS risk score in selecting ICI treatment management for mUM.

BackgroundPatients with cancer who are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more likely to develop severe illness and die compared with those without cancer. The impact of immune checkpoint inhibition (ICI) on the severity of COVID-19 illness is unknown. The aim of this study was to investigate whether ICI confers an additional risk for severe COVID-19 in patients with cancer.MethodsWe analyzed data from 110 patients with laboratory-confirmed SARS-CoV-2 while on treatment with ICI without chemotherapy in 19 hospitals in North America, Europe and Australia. The primary objective was to describe the clinical course and to identify factors associated with hospital and intensive care (ICU) admission and mortality.FindingsThirty-five (32%) patients were admitted to hospital and 18 (16%) died. All patients who died had advanced cancer, and only four were admitted to ICU. COVID-19 was the primary cause of death in 8 (7%) patients. Factors independently associated with an increased risk for hospital admission were ECOG ≥2 (OR 39.25, 95% CI 4.17 to 369.2, p=0.0013), treatment with combination ICI (OR 5.68, 95% CI 1.58 to 20.36, p=0.0273) and presence of COVID-19 symptoms (OR 5.30, 95% CI 1.57 to 17.89, p=0.0073). Seventy-six (73%) patients interrupted ICI due to SARS-CoV-2 infection, 43 (57%) of whom had resumed at data cut-off.InterpretationCOVID-19–related mortality in the ICI-treated population does not appear to be higher than previously published mortality rates for patients with cancer. Inpatient mortality of patients with cancer treated with ICI was high in comparison with previously reported rates for hospitalized patients with cancer and was due to COVID-19 in almost half of the cases. We identified factors associated with adverse outcomes in ICI-treated patients with COVID-19.

PurposeAnti-programmed cell death protein 1 (PD1)±anti-cytotoxic T-lymphocyte associated protein 4 (CTLA4) immune checkpoint inhibitors (ICIs) are standard therapeutic options for metastatic melanoma. We assessed whether biologic subtype according to primary tumor type or genomic subtype can function as predictive biomarkers for anti-PD1±anti-CTLA4 ICI in patients with advanced melanoma.MethodsWe performed a single-center retrospective cohort analysis of patients who received anti-PD1±anti-CTLA4 ICI for advanced melanoma between 2012 and 2019. Primary tumor type, BRAF and NRAS mutation status, and other covariates were abstracted from chart review. Log-rank tests and multivariable Cox regression models were used to assess differences in clinical progression-free (cPFS) and overall survival (OS).ResultsWe identified 230 patients who received 249 lines of anti-PD1±anti-CTLA4 ICI for unresectable or metastatic disease. Of these patients, 74% were cutaneous, 11% mucosal, 8% unknown primary and 7% acral. BRAF and NRAS mutations were identified in 35% and 28% of patients, respectively. In multivariable analyses of the entire cohort, acral or mucosal primary tumor type, >3 metastatic sites, elevated LDH were predictive of shorter cPFS and OS. Combination ICI therapy was associated with longer cPFS (HR 0.57, 95% CI 0.38 to 0.86, p=0.007) and OS (HR 0.42, 95% CI 0.28 to 0.65, pNRAS (HR 0.24, 95% CI 0.10 to 0.62, p=0.003, n=69) and BRAF V600E/K (HR 0.47, 95% CI 0.24 to 0.90, p=0.024, n=86) mutant melanoma but not BRAF/NRAS wild-type (n=94) melanoma.ConclusionsIn our cohort, primary melanoma tumor type and genomic subtype were independent predictive markers of cPFS and OS for patients with metastatic melanoma receiving anti-PD1 ICI. Primary tumor type and genomic subtype—including NRAS—should be further evaluated in prospective clinical trials to determine their value as predictive markers. Biologic subtypes may facilitate clinical decision-making when recommending combination ICI treatment (anti-PD1±anti-CTLA4) versus anti-PD1 alone for patients with metastatic melanoma.

BACKGROUND: People with cancer are at risk for initial, late, and long-term effects of cancer and its treatments. Cancer rehabilitation (CR) focuses on prevention/treatment of these sequelae and optimization of physical, social, and vocational functioning. Our center has a multidisciplinary impairment-driven outpatient CR program, but referrals of patients with GI cancer were low. AIMS: We aimed (for 2019, relative to 2018) (1) to increase CR referrals of patients with GI cancer by 50% and (2) to increase the proportion of referrals coming from oncologists. Balancing measures included inappropriate referrals and cancellations. METHODS: A rapid cycle improvement approach was used to optimize GI referrals to the CR program. Barriers to CR referral were identified through a literature review and informal interviews of GI clinicians. Barriers included (a) knowledge of CR program existence, (b) awareness of the referral process, (c) time, and (d) lack of CR program exposure. The team used Plan-Do-Study-Act (PDSA) cycles every 2 months from January to December 2019 to address barriers. A p-chart was used to analyze the results. RESULTS: PDSA cycles included CR program advertisement, a presentation to GI staff, nurse-led patient identification, patient-facing posters, and clinician thank-you emails. The p-chart showed a 100% relative increase in referral numbers and an improvement in the percentage of patients referred by oncologists from 51% to 75%. There was no significant change in inappropriate referrals or cancellations. CONCLUSION: Through PDSA cycles, we improved the total number of patients with GI cancer and percentage referred by an oncologist to a CR program. Future work will assess sustainability.

The RAS-RAF-MEK-ERK signaling cascade is among the most frequently mutated pathways in human cancer. Approximately 50% of melanoma patients possess a druggable hotspot V600E/K mutation in the BRAF protein kinase. FDA-approved combination therapies of BRAF and MEK inhibitors are available that provide survival benefits to patients with a BRAF V600 mutation. Non-V600 BRAF mutants are found in many cancers, and are more prevalent than V600 mutations in certain tumor types. For example, between 50-80% of BRAF mutations in non-small cell lung cancer and 22-30% in colorectal cancer encode for non-V600 mutants. As next generation sequencing becomes increasingly used in clinical practice, oncologists are frequently identifying non-V600 BRAF mutations in their patient's tumors, but are uncertain of viable therapeutic options that could be employed for optimal treatment. From recent studies, a new classification system is emerging for BRAF mutations based on biochemical and signaling mechanisms associated with these mutants. Class I BRAF mutations affect amino acid V600 and signal as RAS-independent active monomers, class II mutations function as RAS-independent activated dimers, and class III mutations are kinase impaired but increase signaling through the MAPK pathway due to enhanced RAS binding and subsequent CRAF activation. These distinct classes of BRAF mutations predict response to targeted therapies and have important implications for future drug development. Herein, we discuss pre-clinical and clinical findings that may lead to improved treatments for all classes of BRAF mutant cancers.

GPNMB has emerged as an immunomodulator and an important positive mediator of tumor progression and metastasis in numerous solid cancers. Tumor intrinsic GPNMB-mediated effects on cellular signaling, coupled with the ability of GPNMB to influence the primary tumor and metastatic microenvironments in a non-cell autonomous fashion, combine to augment malignant cancer phenotypes. In addition, GPNMB is often overexpressed in a variety of cancers, making it an attractive therapeutic target. In this regard, glembatumumab vedotin, an antibody-drug conjugate (ADC) that targets GPNMB, is currently in clinical trials as a single agent in multiple cancers. In this review, we will describe the physiological functions of GPNMB in normal tissues and summarize the processes through which GPNMB augments tumor growth and metastasis. We will review the pre-clinical and clinical development of glembatumumab vedotin, evaluate on-going clinical trials, explore emerging opportunities for this agent in new disease indications and discuss exciting possibilities for this ADC in the context of combination therapies.

Background: TNBC is an aggressive subtype that constitutes ~15% of all BC. Currently there are no targeted therapies available for patients with. As such, there is much interest in developing targeted therapies for this disease. Recently we identified GPNMB as a transmembrane protein that promotes breast tumor growth and metastasis. CDX-011 is an antibody drug conjugate that targets GPNMB, and has recently shown promising clinical activity in patients with GPNMB+TNBC. In subset analyses of the EMERGE trial, patients with high GPNMB expressing TNBC had a median OS of 10 vs. 5.5 months for CDX011 versus chemotherapy, respectively. Response rates to CDX011 correlated with degree of GPNMB expression. These findings support the hypothesis that TNBC with high GPNMB will respond better to CDX011. As such, we sought to identify therapies with intrinsic activity against TNBC that would also induce GPNMB expression, in order to synergize with CDX-011. We have recently shown that MAPK pathway inhibition induces GPNMB expression in melanoma. Therefore we sought to determine whether Mek inhibition (Mek-i) induced GPNMB in TNBC and the targeted therapies could synergize with CDX-011. Results: We interrogated the TCGA breast datasets to determine whether the MAPK pathway is more frequently altered in TNBC. Indeed, this pathway is altered in 94% of Basal compared to 60% LumA, 83% LumB, 73% Her2 subtypes. Furthermore, we show that a Mek-activation transcriptional signature is significantly higher in basal compared to Her2 or Luminal subtypes. Indeed, we show that Fra, a downstream target of activated Erk, is most highly expressed in BC cells of the basal subtype. We used immunoblot and FACS analysis to assess GPNMB expression in response to Mek-i; trametinib and cobimetinib markedly induced GPNMB protein expression in 12 of the 14 TNBC cell lines tested. Moreover, we show that in the TCGA dataset, low expression of the Mek-activity signature correlates with higher GPNMB, specifically within the basal subtype, thus providing clinical corroboration for our in vitro observations. We go on to show that Mek-i mediated GPNMB up-regulation is regulated by TFE3. We find that EGFR is upregulated in response to Mek inhibition in several TNBC cells. GPNMB heterodimerizes with EGFR in immunoprecipitation experiments. Using siRNA to knockdown GPNMB or ectopic GPNMB overexpression, we found that GPNMB is both necessary and sufficient for enhanced EGFR activation in response to Mek-i in TNBC. Finally, we used CRISPR-CAS9 to delete genomic GPNMB from murine lung-metastatic TNBC cell lines. 533LM2 grow in syngeneic Balb/c mice. Here we show that GPNMB is required for tumor growth and metastasis in vivo. Mek-i slows tumor growth; but the combination of GPNMB deletion with Mek-i led to tumor regression, and significantly impaired tumor growth and metastasis relative to all other groups. Conclusions: Together our data show that the MAPK pathway is hyperactivated in TNBC; inhibiting this pathway impairs tumor growth, but enhances GPNMB, which facilitates mammary tumor growth and metastasis in the setting of Mek-i. These data provide rationale for combined targeting of GPNMB and the MAPK pathway in TNBC. Citation Format: Rose A, Annis M, Perkins D, Siegel P. GPNMB activates EGFR to overcome Mek-inhibition: Implications for the development of rational targeted therapy combinations in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-03.

Purpose: To determine if BRAF and/or MEK inhibitor–induced GPNMB expression renders melanomas sensitive to CDX-011, an antibody-drug conjugate targeting GPNMB. Experimental Design: The Cancer Genome Atlas melanoma dataset was interrogated for a panel of MITF-regulated melanosomal differentiation antigens, including GPNMB. BRAF-mutant melanoma cell lines treated with BRAF or MEK inhibitors were assessed for GPNMB expression by RT-qPCR, immunoblot, and FACS analyses. Transient siRNA-mediated knockdown approaches were used to determine if MITF is requirement for treatment-induced GPNMB upregulation. GPNMB expression was analyzed in serial biopsies and serum samples from patients with melanoma taken before, during, and after disease progression on MAPK inhibitor treatment. Subcutaneous injections were performed to test the efficacy of MAPK inhibitors alone, CDX-011 alone, or their combination in suppressing melanoma growth. Results: A MITF-dependent melanosomal differentiation signature is associated with poor prognosis in patients with this disease. MITF is increased following BRAF and MEK inhibitor treatment and induces the expression of melanosomal differentiation genes, including GPNMB. GPNMB is expressed at the cell surface in MAPK inhibitor–treated melanoma cells and is also elevated in on-treatment versus pretreatment biopsies from melanoma patients receiving MAPK pathway inhibitors. Combining BRAF and/or MEK inhibitors with CDX-011, an antibody-drug conjugate targeting GPNMB, is effective in causing melanoma regression in preclinical animal models and delays the recurrent melanoma growth observed with MEK or BRAF/MEK inhibitor treatment alone. Conclusions: The combination of MAPK pathway inhibitors with an antibody-drug conjugate targeting GPNMB is an effective therapeutic option for patients with melanoma. Clin Cancer Res; 22(24); 6088–98. ©2016 AACR.

BRAF is a constituent of the mitogen-activated protein kinase (MAPK) signaling pathway, which serves to activate downstream MEK, and is one of the most commonly mutated oncogenes in human tumors. Indeed, BRAF V600 mutations are present in approximately 40% of metastatic melanoma tumors. Encorafenib (LGX-818, Braftovi) and binimetinib (MEK-162, Mektovi) are small-molecule inhibitors of BRAF and MEK, respectively. BRAF and MEK inhibitors have been shown to improve overall and progression-free survival among patients with metastatic melanoma. Of these inhibitors, encorafenib and binimetinib are the newest combination, which received approval by the Food and Drug Administration (FDA) for the treatment of BRAF V600E/K-mutated melanoma in June 2018. This review will focus on the preclinical pharmacology, pharmacokinetics and clinical utility of encorafenib and binimetinib in BRAF V600-mutated melanoma.

Background: ICI are widely used in the treatment of various cancer types. It has been hypothesized that ICI could confer an increased risk of severe acute lung injury or other complications associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We analyzed data from 113 patients with laboratory-confirmed COVID-19 while on treatment with ICI without chemotherapy in 19 hospitals in North America, Europe, and Australia. Data collected included details on symptoms, comorbidities, medications, treatments and investigations for COVID-19, and outcomes (hospital admission, ICU admission, and mortality). Results: The median age was 63 years (range 27–86); 40 (35%) patients were female. Most common malignancies were melanoma (n=64, 57%), non-small cell lung cancer (n=19, 17%), and renal cell carcinoma (n=11, 10%); 30 (27%) patients were treated for early (neoadjuvant/adjuvant) and 83 (73%) for advanced cancer. Most patients received anti-PD-1 (n=85, 75%), combination anti-PD-1 and anti-CTLA-4 (n=15, 13%), or anti-PD-L1 (n=8, 7%) ICI. Comorbidities included cardiovascular disease (n=31, 27%), diabetes (n=17, 15%), and pulmonary disease (n=14, 12%). Symptoms were present in 68 (60%) patients; 46 (68%) had fever, 40 (59%) cough, and 23 (34%) dyspnea. Overall, ICI was interrupted in 58 (51%) patients. At data cutoff, 33 (29%) patients were admitted to hospital, 6 (5%) to ICU, and 9 (8%) patients died. COVID-19 was the primary cause of death in 7 patients, 3 of whom were admitted to ICU. Cancer types in patients who died were melanoma (2), non-small cell lung cancer (2), renal cell carcinoma (2), and others (3); all (9) patients had advanced cancer. Administered treatments were oxygen therapy (8), mechanical ventilation (2), vasopression (2), antibiotics (7), antiviral drugs (4), glucocorticoids (2), and anti-IL-6 (2). Of all hospitalized patients, 20 (61%) had been discharged and 4 (12%) were still in hospital at data cutoff. Conclusion: The mortality rate of COVID-19 in patients on ICI is higher than rates reported for the general population without comorbidities but may not be higher than rates reported for the cancer population. Despite these preliminary findings, COVID-19 patients on ICI may not have symptoms and a proportion may continue ICI. Correlative analyses are ongoing and will be presented. Citation Format: Aljosja Rogiers, Carlo Tondini, Joe M. Grimes, Megan H. Trager, Sharon Nahm, Leyre Zubiri, Neha Papneja, Arielle Elkrief, Jessica Borgers, April Rose, Johanna Mangana, Michael Erdmann, Ines Pires da Silva, Christian Posch, Axel Hauschild, Lisa Zimmer, Paola Queirolo, Caroline Robert, Karijn Suijkerbuijk, Paolo A. Ascierto, Paul Lorigan, Richard Carvajal, Osama E Rahma, Mario Mandala, Georgina V. Long. Clinical characteristics and outcomes of coronavirus 2019 disease (COVID-19) in cancer patients treated with immune checkpoint inhibitors (ICI) [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S02-01.

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.

Purpose Glycoprotein NMB (gpNMB), a novel transmembrane protein overexpressed in 40% to 60% of breast cancers, promotes metastases in animal models and is a prognostic marker of a poor outcome in patients. The antibody-drug conjugate glembatumumab vedotin consists of a fully human anti-gpNMB monoclonal antibody, conjugated via a cleavable linker to monomethyl auristatin E. Glembatumumab vedotin is generally well tolerated, with observed objective responses in advanced melanoma. This is, to our knowledge, the first study of glembatumumab vedotin in breast cancer. Patients and Methods Eligible patients had advanced/metastatic breast cancer with at least two prior chemotherapy regimens, including taxane, anthracycline, and capecitabine. A standard 3+3 dose escalation was followed by a phase II expansion. Immunohistochemistry for gpNMB was performed retrospectively for patients with available tumor tissue. Results Forty-two patients were enrolled. Dose-limiting toxicity (DLT) consisted of worsening neuropathy at 1.34 mg/kg. After excluding patients with baseline neuropathy more than grade 1, no DLT occurred through 1.88 mg/kg (the phase II dose). The phase II primary activity end point was met (12-week progression-free survival [PFS12] = 9 of 27 patients; 33%). Sixteen of 19 (84%) patients tested had gpNMB-positive tumors. At the phase II dose, median PFS was 9.1 weeks for all patients, 17.9 weeks for patients with triple-negative breast cancer (TNBC), and 18.0 weeks for patients with gpNMB-positive tumors. Two patients had confirmed partial responses; both had gpNMB-positive tumors and one had TNBC. Conclusion Glembatumumab vedotin has an acceptable safety profile. Preliminary evidence of activity in treatment-resistant metastatic breast cancer requires confirmation, such as the phase II randomized trial (EMERGE) that also examines the relationship between activity and gpNMB distribution/intensity.

Background: Anti-PD1 immunotherapy, when used alone or in combination (combo) with anti-CTLA4, prolongs overall (OS) and progression-free survival (PFS) of patients (pts) with advanced melanoma, compared to single-agent (SA) anti-CTLA4. Combo immunotherapy is more toxic than SA anti-PD1, therefore it is of interest to identify patients most likely benefit from SA anti-PD1 to spare these pts the toxicity of combo immunotherapy. We asked whether genomic driver mutations (mts) in BRAF, NRAS or KIT could predict anti-PD1 responses in melanoma pts. Methods: We preformed a single-center retrospective analysis of 292 melanoma pts who received SA or combo anti-PD1 immunotherapy at Princess Margaret Hospital between 2012-17. BRAF/NRAS/KIT mts and other covariates were abstracted from chart review. Multivariable Cox models were used to assess differences in OS/PFS. Results: We identified 209 and 83 melanoma patients who received SA-anti-PD1 or combo immunotherapy, respectively. Pt characteristics were: mean age 59 yrs; 56% male; 77% cutaneous/unknown primary 23% uveal or mucosal; 17% brain metastases; 34% stage M0/M1a/M1b, 66% stage M1c/M1d, 51% BRAF/NRAS/KIT mt. Among pts receiving SA anti-PD1, presence of a BRAF, NRAS, or KIT mt was an independent predictor of poor PFS (HR 1.84; 95% CI 1.21-2.81) and OS (HR 1.93; 95% CI 1.17-3.22) in multivariate models. Other negative predictors of poor survival were: elevated LDH, multiple previous lines of therapy, 3 or more sites of metastasis, and uveal or mucosal histology. Combo immunotherapy was independently associated with longer OS compared to SA anti-PD1 in BRAF/NRAS/KIT mt pts (HR 0.43; 95% CI 0.20-0.90), but not in BRAF/NRAS/KIT wild-type pts (HR 1.34; 95% CI 0.46-3.90). Conclusions: In this retrospective study, the presence of a BRAF NRAS or KIT mt was an independent predictor of poor PFS/OS in melanoma pts treated with SA anti-PD1. Combo immunotherapy prolonged survival vs SA anti-PD1 in BRAF/NRAS/KIT mt melanoma, but not in BRAF/NRAS/KIT wild-type melanoma. These data suggest that BRAF/NRAS/KIT wild-type melanoma pts may derive less added benefit from combo immunotherapy (vs single agent anti-PD1) compared to pts with BRAF/NRAS/KIT mutations. Validation analyses are on-going. Citation Format: April A.N. Rose, Susan M. Armstrong, David Hogg, Marcus Butler, Anthony M. Joshua, Danny Ghazarian, Suzanne Kamel-Reid, Ayman Al Habeeb, Mary Anne Chappell, Kendra Ross, Eitan Amir, Phillippe L. Bedard, Lillian Siu, Anna Spreafico. The relationship between BRAF/NRAS/KIT genomic driver mutations and anti-PD1 immunotherapy responses in patients with advanced melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3141.

We read with great interest the recent article by Dagogo-Jack and colleagues, wherein they interrogated the clinical outcomes of patients with lung cancer whose tumors expressed different classes of BRAF mutations ([1][1]). Clinicians are increasingly requesting next-generation sequencing analyses

Vitamin D regulates expression of genes important in development and progression of breast cancer. The association of vitamin D with breast cancer outcomes among breast cancer patients is controversial. We conducted a systematic review and meta-analysis of this association in early stage breast cancer outcome. We searched MEDLINE (1982-May 1, 2013), the American Society of Clinical Oncology (2009-2012), and the San Antonio Breast Cancer Symposium (2010-2012) for abstracts, using the following keywords: "breast cancer" and "prognosis" or "survival", and "vitamin D" or" calcitriol" to identify studies reporting the associations of blood vitamin D levels (drawn close to diagnosis) with breast cancer outcomes. Meta-analyses were performed using an inverse-variance weighted fixed-effects model with Stata Version 12. Eight studies including 5,691 patients were identified. Vitamin D deficiency was variably categorized across studies; a median of 36.8 % of patients were classified as deficient. Low vitamin D levels were associated with a pooled hazard ratio of 2.13 (95 % CI 1.64-2.78) and 1.76 (95 % CIs 1.35-2.30) for recurrence (six studies) and death (four studies), respectively, with no evidence of significant heterogeneity across studies. There was potential evidence of a publication bias in studies examining associations with death (but not in those examining associations with recurrence). These findings support an association of low levels of vitamin D with increased risk of recurrence and death in early stage breast cancer patients. Given the observational nature of the included studies, it cannot be concluded that this association is causal. Further research is warranted to investigate the potential beneficial effects of vitamin D in breast cancer.

12093Background: dMAPKi with BRAFi and MEKi improves survival in BRAF V600E/K mt MM, but the effects of these inhibitors on nonV600 BRAF mt MM is poorly understood. We sought to characterize nonV60...

Purpose: Although the murine orthologue of glycoprotein nonmetastatic B (GPNMB), Osteoactivin, promotes breast cancer metastasis in an in vivo mouse model, its importance in human breast cancer is unknown. We have examined the significance of GPNMB expression as a prognostic indicator of recurrence and assessed its potential as a novel therapeutic target in breast cancer. Experimental Design: The clinical significance of GPNMB expression in breast cancer was addressed by analyzing GPNMB levels in several published gene expression data sets and two independent tissue microarrays derived from human breast tumors. GPNMB-expressing human breast cancer cell lines were further used to validate a toxin-conjugated anti-GPNMB antibody as a novel therapeutic agent. Results: GPNMB expression correlates with shorter recurrence times and reduced overall survival of breast cancer patients. Epithelial-specific GPNMB staining is an independent prognostic indicator for breast cancer recurrence. GPNMB is highly expressed in basal and triple-negative breast cancers and is associated with increased risk of recurrence within this subtype. GPNMB expression confers a more migratory and invasive phenotype on breast cancer cells and sensitizes them to killing by CDX-011 (glembatumumab vedotin), a GPNMB-targeted antibody-drug conjugate. Conclusions: GPNMB expression is associated with the basal/triple-negative subtype and is a prognostic marker of poor outcome in patients with breast cancer. CDX-011 (glembatumumab vedotin) is a promising new targeted therapy for patients with metastatic triple-negative breast cancers, a patient population that currently lacks targeted-therapy options. Clin Cancer Res; 16(7); 2147–56. ©2010 AACR.

Background: We identified a patient who presented with brain metastatic melanoma. Her tumor expressed a rare BRAF mutation (mt) (L597S). L597S is a non-V600 BRAF mt with intermediate BRAF kinase activity. Little is known about whether melanomas bearing these non-V600 mt are amenable to treatment with the same targeted therapies currently used for BRAF V600 mutant melanoma. Therefore, we employed a translational approach to characterize non-V600 mts in melanoma and to investigate their responsiveness to targeted therapies. Methods: We performed systemic review to quantify the incidence of non-V600 mts. Tumor fragments from patients BRAF WT, V600E and L597S melanoma brain metastases were used to generate 3 patient-derived xenograft and cell lines. We obtained several other cell lines bearing these mts, and mts within the p-loop of BRAF. Cells were treated with clinical inhibitors of BRAF (BRAFi; vemurafenib (V), dabrafenib (D), encorafenib (E)), and MEK (MEKi; cobimetinib (C), trametinib (T), binimetinib (B)). BRAFi, MEKi, or the combination thereof were tested in the following assays: immunoblots to analyze cell signalling, long-term in vitro growth assays, and subcutaneous and intracranial in vivo tumor growth experiments. Results: MEKi inhibited Erk in all cell lines whereas BRAFi induced paradoxical Erk activation in WT cells, but impaired Erk phosphorylation in V600 and L597 mutant cells. In long-term growth assays, all MEKi were capable of inhibiting growth in all 12 cell lines tested to varying degrees. BRAFi inhibited the growth of V600 and L597S mt cells but not BRAF WT or p-loop mutant cells. The addition of BRAFi consistently inhibited the growth of MEKi-treated L597S mt cells. E was more effective than either D or V at impairing the growth of MEKi-treated non-V600 cells. Remarkably, V potentiated the growth of MEKi-treated cells with BRAF p-loop mts, whereas E further inhibited their growth. In the L597S subcutaneous PDX model, all vehicle-treated tumors grew progressively, whereas T led to overall stability of tumor growth (25% ORR) and D + T caused 100% of tumors to shrink significantly. Moreover, D + T improved the survival of mice with intracranial L597S metastases, compared to vehicle or T treated mice. Conclusions: Non-V600 BRAF mts comprise 15% of all BRAF mts in melanoma. Like V600 mutant melanoma, L597S BRAF mutant melanoma are synergistically growth inhibited by the combination of BRAFi +MEKi in vitro and in vivo. MEKi-treated cells with BRAF p-loop mutations are further inhibited by E, whereas V potentiates growth, suggesting unique mechanisms of BRAFi between inhibitors. Taken together, our data provide a rationale for investigating the efficacy of dual MAPK inhibition with E or D + MEKi in patients with non-V600 mutant melanoma. Citation Format: April A.N. Rose, Matthew Dankner, Shivshankari Rajkumar, Ian R. Watson, Kevin Petrecca, Catalin Mihalcioiu, Peter M. Siegel. Non-V600 BRAF mutations in melanoma: actionable targets for rational drug combinations [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B056.

The loss of expression of the transcription factor GATA3 in breast tumors has been linked to aggressive tumor development and poor patient survival. In the present work, we address potential roles for GATA3 in breast tumor lung metastasis and progression. Using an aggressive breast cancer cell line, which metastasizes specifically to the lung, we show that GATA3 expression results in reduced tumor outgrowth in the mammary fat pad and lower lung metastatic burden in nude mice. Specifically, GATA3 expression inhibits breast cancer cell expansion inside the lung parenchyma. This phenotype correlates with the ability of GATA3 to negatively regulate the expression of several genes that promote breast cancer lung metastasis (ID1/-3, KRTHB1, LY6E and RARRES3). Conversely, the expression of genes encoding known inhibitors of lung metastasis (DLC1 (deleted in liver cancer 1) and PAEP (progestagen-associated endometrial protein)) is upregulated by GATA3. These data correlate with microarray data from human breast cancer patients, showing a strong correlation between high GATA3 expression and absence of metastases specifically to the lungs. We conclude that GATA3 inhibits primary breast tumor outgrowth and reduces lung metastatic burden by regulating key genes involved in metastatic breast tumor progression.

Introduction Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells. Methods Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs. Results We show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis. Conclusions Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0558-3) contains supplementary material, which is available to authorized users.

Glycoprotein nmb (GPNMB) promotes breast tumor growth and metastasis and its expression in tumor epithelium correlates with poor prognosis in breast cancer patients. Despite its biological and clinical significance, little is known regarding the molecular mechanisms engaged by GPNMB. Herein, we show that GPNMB engages distinct functional domains and mechanisms to promote primary tumor growth and metastasis. We demonstrate that neuropilin-1 (NRP-1) expression is increased in breast cancer cells that overexpress GPNMB. Interestingly, the GPNMB-driven increase in NRP-1 expression potentiated vascular endothelial growth factor signaling in breast cancer cells and was required for the growth, but not metastasis, of these cells in vivo. Interrogation of RNAseq data sets revealed a positive correlation between GPNMB and NRP-1 levels in human breast tumors. Furthermore, we ascribe pro-growth and pro-metastatic functions of GPNMB to its ability to bind α5β1 integrin and increase downstream signaling in breast cancer cells. We show that GPNMB enhances breast cancer cell adhesion to fibronectin, increases α5β1 expression and associates with this receptor through its RGD motif. GPNMB recruitment into integrin complexes activates Src and Fak signaling pathways in an RGD-dependent manner. Importantly, both the RGD motif and cytoplasmic tail of GPNMB are required to promote primary mammary tumor growth; however, only mutation of the RGD motif impaired the formation of lung metastases. Together, these findings identify novel and distinct molecular mediators of GPNMB-induced breast cancer growth and metastasis.

Background: The duration of survival benefit from MAPKi in B-raf mutant melanoma (MEL) is limited by the near universal development of resistance (RES). Recently, MiTF, a transcription factor and master regulator of melanocyte differentiation - was identified as a hub of MAPKi RES. Given its dual role as both a tumor promoter and inducer of MEL senescence, MiTF is a problematic therapeutic target. Pigmentation is a MiTF-dependent feature of MELs correlates with poor survival. We have interrogated the MAPKi/MiTF-dependent regulation of a panel of MITF-regulated melanosomal differentiation antigens (MDAs) for their potential as therapeutic targets to overcome MAPKi RES. Methods: The TCGA MEL gene expression data set was interrogated. B-raf mutant MEL cells were exposed to dabrafenib (D), or trametinib (T). qRT-PCR, Immunoblot or FACS were used to assess MiTF and MDA expression. Serial biopsy tissue and serum samples from MEL patients taken before treatment, during treatment, and at progression with MAPKi therapy were obtained, and analyzed by IHC and qRT-PCR, RNAseq, and ELISA. Transient siRNA-mediated knockdown was used to assess the requirement for MiTF, in MAPKi-mediated induction of MDAs, including GPNMB. To assess the interaction between MAPKi and CDX011 (C), a GPNMB- targeted antibody drug conjugate, mice bearing A375 and WM2664 xenografts were treated with T or DT + C in combination. Results: MiTF is strongly correlated with MDAs, including GPNMB in gene-expression datasets derived from human MEL tumors. A MiTF driven MDA-gene-signature correlates with poor survival among 291 melanoma patients. MAPKi treatment increased MiTF and MDAs in MEL cells, and induced pigmentation in vivo. This phenotype was reversible as MEL acquired RES to MAPKi. Knockdown of MiTF leads to abrogation of MAPKi-mediated GPNMB induction. GPNMB promotes MEL invasion in vitro. MiTF and MDA were elevated in tumors and serum from patients receiving MAPKi treatment and returned to baseline in tumors that acquired RES. The addition of C to either T or DT led to, decreased pigmentation, prolonged tumor regression and inhibited the development RES to T or DT treatment in vivo. Tumors that acquired RES to DT displayed increased c-Met expression and MAPK activation; tumors treated with DT+C failed to develop this phenotype. Conclusions: MAPKi induce MDA in a MiTF-dependent manner in MEL. This process leads to melanocytic differentiation and increased pigmentation, which is subsequently reversed as a tumor acquires RES to MAPKi. This demonstrates the plasticity of MEL in response to MAPKi. The pigmented phenotype is less proliferative but more invasive which is in part mediated by GPNMB. This transient phenotype may allow tumors to acquire characteristics (ie. c-Met upregulation and MAPK re-activation) which contribute to MAPKi RES. Targeting a pro-invasive MDA, GPNMB with CDX-011 subverts this plasticity and impairs the development of MAPKi RES. Citation Format: April A.N. Rose, Matthew G. Annis, Dennie T. Frederick, Zhifeng Dong, Lawrence Kwong, Tibor Keller, Thomas Hawthorne, Ian R. Watson, Keith T. Flaherty, Peter M. Siegel. Targeting the melanosome: overcoming MAPK-inhibitor resistance in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 296.

Background: Triple negative breast cancer (TNBC) is an aggressive subtype that constitutes ∼15% of all BC. Currently there are no targeted therapies available for patients with TNBC and these patients have a poor prognosis. As such, there is much interest in developing targeted therapies for this disease. Recently we identified GPNMB as a transmembrane protein that promotes breast tumor growth and metastasis. CDX-011 is an antibody drug conjugate that targets GPNMB, and has recently shown promising clinical activity in patients with GPNMB+TNBC. In subset analyses of the EMERGE trial, patients with high GPNMB expressing TNBC had a median OS of 10 vs. 5.5 months for CDX011 versus chemotherapy, respectively. Response rates to CDX011 correlated with degree of GPNMB expression. These findings support the hypothesis that TNBC with high GPNMB will respond better to CDX011. As such, we sought to identify therapies with intrinsic activity against TNBC that would also induce GPNMB expression, in order to synergize with CDX-011. Recently there has been much interest in targeting the MAPK pathway in TNBC. We have recently shown that MAPK pathway inhibition induces GPNMB expression in melanoma. Therefore we sought to determine whether mek inhibition induced GPNMB in TNBC and the targeted therapies could synergize with CDX-011. Results: We interrogated the TCGA breast dataset to determine whether the MAPK pathway is more frequently altered in TNBC. Indeed, we find that the MAPK pathway is altered in 93% of basal BC compared to 56%, 82%, and 81% of Lum A, LumB, and Her2 subtypes, respectively. We used immunoblot and FACS analysis to assess GPNMB expression in response to MAPK-inhibition. We found that mek inhibitors (trametinib, cobimetinib) markedly induced GPNMB protein expression in several TNBC cell lines. RTK upregulation has been proposed as an adaptive resistance mechanism to mek inhibition in TNBC. Indeed, we find that EGFR is upregulated in response to Mek inhibition in MDA-MB-468 and Hs578T cells. Using shRNA to knockdown GPNMB expression in MDA-MB-468 cells or ectopic GPNMB overexpression in Hs578t cells, we found that GPNMB is both necessary and sufficient for enhanced EGFR activation in response to Mek inhibition in TNBC. Interestingly, we also find that Hs578T cells overexpressing GPNMB show less growth inhibition in response to Mek inhibitors compared to control cells in vitro. These in vitro data are corroborated by our analyses of 1097 breast tumors from the TCGA dataset. GPNMB alterations were found in 7% of all BC and correlates significantly with increased EGFR, Mek and Erk activation. Finally, we are investigating the efficacy of combining trametinib with CDX011 to treat TNBC using in vivo mouse models. Preliminary data from this experiment suggest that MDA-MB-468 tumors treated with both drugs are more growth restricted than tumors treated with either drug alone. Final data will be presented at the meeting. Conclusions: Mek inhibition induces GPNMB expression in TNBC. GPNMB promotes EGFR activation and protects from mek-inhibitor induced growth inhibition. The combination of a mek inhibitor with CDX011 shows promise in pre-clinical models and warrants further investigation in clinical trials. Citation Format: Rose AA, Annis MG, Maric G, Siegel PM. Targeting glycoprotein non-metastatic B (GPNMB) to overcome EGFR-mediated resistance to Mek inhibition in triple negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-03.

International audience; Galectins are members of a family of beta-galactosides-binding proteins that have recently emerged as novel modulators in different aspects of cancer. The expression of galectins in tumors and/or the tissue surrounding them has been well documented. Since galectin-7 expression has been associated with epithelial tissues and varies significantly in various types of cancer, we have investigated for the first time its role in breast cancer. Using two preclinical mouse models, high levels of galectin-7 expression in breast cancer cells drastically increased their ability to metastasize to lungs and bones. Significant increases in the number of pulmonary metastases and osteolytic lesions were induced by overexpression of galectin-7 compared with control cells. In human tissues, galectin-7 was specifically found in myoepithelial cells of normal human breast tissue, but not in luminal cells. Its expression was severely altered in breast carcinoma, many samples showing greater than 70% of galectin-7 positive cells. High expression levels of galectin-7 were restricted to high-grade breast carcinomas, including HER2 overexpressing and basal-like groups. In HER2 overexpressing cases, galectin-7 expression was associated with lymph node axillary metastasis. Taken together, our results indicate that galectin-7 may represent a potential target for both specific detection and therapeutic inhibition of metastatic breast cancer.

In the previous issue of Breast Cancer Research, Metz and colleagues characterize hematopoietic growth factor inducible neurokinin-1 type (HGFIN; osteoactivin, Gpnmb, Dc-HIL) as a gene expressed in normal mammary epithelial cells that encodes a protein with tumor suppressor functions in breast cancer [1]. In contrast, we have recently identified osteo-activin as a novel mediator promoting breast cancer metastasis [2] and thus feel it is premature to classify osteoactivin as a tumor suppressor in breast cancer. In their article, Metz et al. claim that osteoactivin expression is "detectable in non-tumorigenic cells, but low to undetectable in malignant cells". This conclusion was based on RNA expression in three breast cell lines and three primary breast tissues. In our study, we analyzed microarray data from a recently published data set [3] that examined gene expression profiles of three non-malignant and 51 malignant human breast cell lines. We confirmed these microarray data by performing quantitative real-time polymerase chain reaction (PCR) of osteoactivin expression in nine of these cell lines [2]. Our analysis revealed that 48 of the 51 breast cancer cell lines examined possessed higher osteoactivin expression than normal breast MCF-12A cells. In contrast to the data from Metz et al., the T47D and HCC70 breast cancer cell lines possessed 2.4 and 48.5 fold higher osteoactivin expression, respectively, relative to MCF-12A cells. The authors also performed in situ hybridization for osteo-activin mRNA in paraffin embedded breast biopsies. They conclude that osteoactivin is found at higher levels in benign tissues than in malignant tissues, and that no correlation exists between osteoactivin expression and estrogen receptor (ER) status. It must be noted that their conclusions are in contrast to several published large scale analyses of primary human breast tumors that are available through the online cancer microarray database, Oncomine [4]. These studies indicate that osteoactivin expression is significantly higher in both breast cancer versus normal breast specimens and in ER negative versus ER positive breast tumors [2]. Metz et al. state: "Osteoactivin is expressed in breast cancer cells and has been associated with bone invasion, and aggressive form of the tumor". They repeatedly refer to Rich et al. to corroborate this remark, however, this particular reference does not provide any evidence to support this statement [5]. Rich et al. were, in fact, investigating a role for osteoactivin in promoting the invasion of a defined glioma model. This study did not employ breast cancer cells in any experiments, nor was bone metastasis investigated. In our study, we report, for the first time, that ectopic osteoactivin expression in breast cancer cells is sufficient to induce a significant increase in the incidence of osteolytic bone metastases in vivo. Finally, Metz et al. refer to a study that links osteoactivin expression to the low metastatic potential of melanoma cell lines to support the notion that osteoactivin functions as a tumor suppressor [6]. However, the authors have overlooked more recent studies that identify osteoactivin as being highly expressed in metastatic melanoma-derived cell lines and clinical specimens. These studies report the development of a cytotoxin-conjugated, anti-osteoactivin antibody (CR011-vcMMAE) that is currently in Phase I/II clinical trials as a treatment for patients with Stage III and Stage IV melanoma [7,8]. In summary, osteoactivin is emerging as an important, yet controversial effector of tumor progression in several cancer types, including breast. As such, the pursuit of rigorous, functional studies – aimed at addressing the mechanisms of osteoactivin-mediated effects on tumor progression and metastasis in vivo – will be critical to assess its feasibility as a target for therapeutic intervention in breast cancer.

The skeleton is a preferred site of metastasis in patients with disseminated breast cancer. We have used 4T1 mouse mammary carcinoma cells, which metastasize to bone from the mammary fat pads of immunocompetent mice, to identify novel genes involved in this process. In vivo selection of parental cells resulted in the isolation of independent, aggressively bone metastatic breast cancer populations with reduced metastasis to the lung. Gene expression profiling identified osteoactivin as a candidate that is highly and selectively expressed in aggressively bone metastatic breast cancer cells. These cells displayed enhanced migratory and invasive characteristics in vitro, the latter requiring sustained osteoactivin expression. Osteoactivin depletion in these cells, by small interfering RNA, also lead to a loss of matrix metalloproteinase-3 expression, whereas forced osteoactivin expression in parental 4T1 cells was sufficient to elevate matrix metalloproteinase-3 levels, suggesting that this matrix metalloproteinase may be an important mediator of osteoactivin function. Overexpression of osteoactivin in an independent, weakly bone metastatic breast cancer cell model significantly enhanced the formation of osteolytic bone metastases in vivo. Finally, high levels of osteoactivin expression in primary human breast cancers correlate with estrogen receptor–negative status and increasing tumor grade. Thus, we have identified osteoactivin as a protein that is expressed in aggressive human breast cancers and is capable of promoting breast cancer metastasis to bone. (Mol Cancer Res 2007;5(10):1001–14)

Background: Recent advances with B-raf and Mek targeted therapies have transformed the therapeutic landscape for metastatic melanoma and improved survival times for patients. Despite this progress, resistance to targeted therapies remains a universal problem that limits treatment efficacy. Combination therapy strategies represent a viable option to overcome the problem of therapeutic resistance. Glycoprotein Non-Metastatic B (GPNMB) is a cell surface protein that is highly expressed in a wide variety of cancer types, and regulates: tumor growth, cancer cell migration, invasion and metastasis. Glembatumumab vedotin (CDX-011) is a GPNMB-targeted antibody-drug conjugate that is in clinical trials for the treatment of melanoma and breast cancer. Interestingly, a wide spectrum of kinase inhibitors (KI) has been reported to induce GPNMB expression in a variety of cell types. Here we investigate the mechanism(s) responsible for elevated GPNMB levels in melanoma cells treated with clinically relevant KI, determine whether KI-mediated induction of GPNMB expression synergizes with CDX-011 in the treatment of melanoma. Methods: B-raf mutant (A375, WM-2664) and N-ras mutant (SkMel2) melanoma cells were exposed to inhibitors of B-raf (vemurafenib, dabrafenib), or Mek (trametinib, selumetinib). Immunoblot analysis or fluorescence-activated cell sorting was used to assess GPNMB expression. B-raf mutant cells were passaged in the presence of vemurafenib over the course of several weeks to months to generate vemurafenib-resistant cells. Transient siRNA knockdown studies were used to assess the requirement for the transcription factor, MiTF, in KI-mediated induction of GPNMB. To assess the interaction between individual KI and CDX011, dose response curves for the KI and CDX-011 alone or in combination in melanoma cells in vitro were generated. Melanoma cells were either 1) pretreated or 2) pre and co-treated with KI for 48 hours prior to treatment with CDX011 for 96 hours. Cell viability was assessed by XTT assay. Results: B-raf inhibition led to an induction of total and cell surface GPNMB protein in B-raf mutant, but not in B-raf WT melanoma cells. Mek inhibition induced GPNMB expression in all three melanoma cell lines. In addition to acute inhibitor treatment, Vemurafenib-resistant cells also expressed higher levels of GPNMB when compared to parental cells (inhibitor sensitive). Knock-down of the MiTF transcription factor abrogated the vemurafenib-mediated induction of GPNMB. Pretreatment of melanoma cells with Mek inhibitors led to a reduction in cell viability, compared to untreated cells, that was further exacerbated by exposure to CDX-011, in a dose-dependent manner. Interestingly, sub-optimal doses with B-raf inhibitors did not significantly affect cell viability of WM-2664 cells when used in isolation, but did enhance the sensitivity of melanoma cells to CDX-011. Conclusions: Induction of GPNMB in response to B-raf/Mek inhibition is a MiTF-dependent process that occurs in both B-raf mutant and WT cells. In this context, induction of GPNMB expression sensitizes cells to CDX-011 mediated cell killing. As such, the combination of B-raf/Mek inihibitors with CDX-011 represents an intriguing new combination therapy strategy for the treatment of melanoma, and warrants further validation in in vivo mouse models. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C232. Citation Format: April A.N. Rose, Peter M. Siegel. B-raf/Mek/Erk Pathway inhibition induces GPNMB expression and sensitizes melanoma cells to the antibody-drug conjugate glembatumumab vedotin. [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 C232.

Molecularly targeted therapies are rapidly growing with respect to their clinical development and impact on cancer treatment due to their highly selective anti-tumor action. However, many aggressive cancers such as triple-negative breast cancer (TNBC) currently lack well-defined therapeutic targets against which such agents can be developed. The identification of tumor-associated antigens and the generation of antibody drug-conjugates represent an emerging area of intense interest and growth in the field of cancer therapeutics. Glycoprotein non-metastatic b (GPNMB) has recently been identified as a gene that is over-expressed in numerous cancers, including TNBC, and often correlates with the metastatic phenotype. In breast cancer, GPNMB expression in the tumor epithelium is associated with a reduction in disease-free and overall survival. Based on these findings, glembatumumab vedotin (CDX-011), an antibody-drug conjugate that selectively targets GPNMB, is currently being investigated in clinical trials for patients with metastatic breast cancer and unresectable melanoma. This review discusses the physiological and potential pathological roles of GPNMB in normal and cancer tissues, respectively, and details the clinical advances and challenges in targeting GPNMB-expressing malignancies.

Background: Glycoprotein non-metastatic melanoma protein B (GPNMB)/Osteoactivin (OA) is a transmembrane protein expressed in approximately 40–75% of breast cancers. GPNMB/OA promotes the migration, invasion and metastasis of breast cancer cells; it is commonly expressed in basal/triple-negative breast tumors and is associated with shorter recurrence-free and overall survival times in patients with breast cancer. Thus, GPNMB/OA represents an attractive target for therapeutic intervention in breast cancer; however, little is known about the functions of GPNMB/OA within the primary tumor microenvironment. Methodology/Principal Findings: We have employed mouse and human breast cancer cells to investigate the effects of GPNMB/OA on tumor growth and angiogenesis. GPNMB/OA-expressing tumors display elevated endothelial recruitment and reduced apoptosis when compared to vector control-derived tumors. Primary human breast cancers characterized by high vascular density also display elevated levels of GPNMB/OA when compared to those with low vascular density. Using immunoblot and ELISA assays, we demonstrate the GPNMB/OA ectodomain is shed from the surface of breast cancer cells. Transient siRNA-mediated knockdown studies of known sheddases identified ADAM10 as the protease responsible for GPNMB/OA processing. Finally, we demonstrate that the shed extracellular domain (ECD) of GPNMB/OA can promote endothelial migration in vitro. Conclusions/Significance: GPNMB/OA expression promotes tumor growth, which is associated with enhanced endothelial recruitment. We identify ADAM10 as a sheddase capable of releasing the GPNMB/OA ectodomain from the surface of breast cancer cells, which induces endothelial cell migration. Thus, ectodomain shedding may serve as a novel mechanism by which GPNMB/OA promotes angiogenesis in breast cancer.

Background: Breast cancer is the second most frequent type of cancer affecting women. We are increasingly aware that changes in mRNA splicing are associated with various characteristics of cancer. The most deadly aspect of cancer is metastasis, the process by which cancer spreads from the primary tumor to distant organs. However, little is known specifically about the involvement of alternative splicing in the formation of macroscopic metastases. Our study investigates transcript isoform changes that characterize tumors of different abilities to form growing metastases. Methods and Findings: To identify alternative splicing events (ASEs) that are associated with the fully metastatic phenotype in breast cancer, we used Affymetrix Exon Microarrays to profile mRNA isoform variations genome-wide in weakly metastatic (168FARN and 4T07) and highly metastatic (4T1) mammary carcinomas. Statistical analysis identified significant expression changes in 7606 out of 155,994 (4%) exons and in 1725 out of 189,460 (1%) intronic regions, which affect 2623 out of 16,654 (16%) genes. These changes correspond to putative alternative isoforms—several of which are novel—that are differentially expressed between tumors of varying metastatic phenotypes. Gene pathway analysis showed that 1224 of genes expressing alternative isoforms were involved in cell growth, cell interactions, cell proliferation, cell migration and cell death and have been previously linked to cancers and genetic disorders. We chose ten predicted splice variants for RT-PCR validation, eight of which were successfully confirmed (MED24, MFI2, SRRT, CD44, CLK1 and HNRNPH1). These include three novel intron retentions in CD44, a gene in which isoform variations have been previously associated with the metastasis of several cancers. Conclusion: Our findings reveal that various genes are differently spliced and/or expressed in association with the metastatic phenotype of tumor cells. Identification of metastasis-specific isoforms may contribute to the development of improved breast cancer stage identification and targeted therapies.

1067 Background: Glycoprotein NMB (GPNMB), also known as osteoactivin, has been shown to regulate metastasis of breast cancer in vivo. CR011-vcMMAE, a fully-human monoclonal anti-GPNMB antibody conjugated to the tubulin inhibitor monomethylauristatin E, is safe and active in pts with advanced melanoma. This is the first study of CR011-vcMMAE in breast cancer. Methods: Eligible pts with MBC had ≥ 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 melanoma) using a standard 3+3 design. Immunohistochemistry (IHC) with goat polyclonal antibody to GPNMB was performed on pt biopsy specimens and on tissue microarrays containing normal breast, DCIS, breast tumor and lymph node metastases. Results: 10 pts with MBC (median age 57, range 36 - 69) had a median of 7 prior regimens and were treated with CR011-vcMMAE for a median of 2 cycles (range 1–4). 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. Pts were treated at 1.0 mg/kg (n = 3), 1.34 mg/kg (n = 5), and 1.88 mg/kg (n = 2); enrollment at 1.88 mg/kg is continuing. Other adverse events (AEs) were grade 1/2 anorexia and pain in 4 pts; diarrhea, rash, fatigue, and neuropathy in 3 pts; and grade 3 rash in 1 pt. Evidence of antitumor activity has been observed. A response of 37% tumor shrinkage was seen in a pt after only 2 cycles and is ongoing. A second pt had a 51% reduction after 2 cycles, but had PD after 12 weeks. Breast tumor samples were more likely to stain positive for GPNMB than normal breast tissues. Conclusions: CR011-vcMMAE 1.34 mg/kg IV q3w is well-tolerated in heavily pretreated pts with MBC. The 1.88 mg/kg q3w dose is being assessed and phase II expansion is planned at the MTD. IHC of pt tumor specimens is being evaluated. [Table: see text]