Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells

Recurrence is the major cause of melanoma death due to cell dissemination from primary/metastatic tumor (CTCs). During asymptomatic periods, these cells reside in bone marrow (bone-marrow resident tumor [BMRT cells]) and remain quiescent. We hypothesized that disseminated tumor cells survive in bone marrow (BM) during these periods and evolve to metastatic potency during disease progression. First, we isolated CTC-enriched, Lin-neg population from clinically advanced melanoma patients and performed biomarker expression and mutational profiling to confirm presence of putative CTCs. Lin-neg CTC population contained unique transcriptomics signature with elevated melanoma markers expression (BAGE, MAGEA1, B4GALNT1, S100A3). Further, downstream IPA analysis demonstrated that an upregulation of transcripts for genes involved in cell survival and pro-development functions with concomitant decrease in cell proliferative and inflammation properties. Second, we implanted Lin-neg population in murine xenograft models and isolated HLA+/ Melan-A+ cells population from blood and BM at endpoint. Expression of human (HLA-ABC) and melanoma markers (Melan-A, S100, NG2, CD146) on BMRTCs and CTCs was confirmed by employing: (a) immunofluorescence staining, (b) single-cell DEPArrayTM and CellSearchTM capture, (c) genomic profiling, and (d) organ-site colonization of CTCs with concomitant to BMRTCs in BM. Third, we discovered distinct transcriptomic signature of BMRTCs vs CTCs by differential gene expression profiling. Subsequent pathway analysis showed top five altered canonical pathways in BMRTCs: protein ubiquitination pathway, EIF2, actin cytoskeleton, systemic lupus erythematosus and hypoxia signaling. Of note, a strikingly elevated expression of PTEN in BMRTCs was detected. Fourth, because PTEN binds to USP7, a key component of protein ubiquitination pathway, we evaluated USP7/PTEN axis in CTC-driven BMRTCs modulated metastatic competency. The use of two clinically approved USP7 inhibitors to study their effects on the proliferative capacity of BMRTCs led to a significant reduction of CTCs at metastatic sites. This study provides critical insights to identify biomarkers of melanoma recurrence during the asymptomatic periods of the disease, fostering application of USP7 inhibitors for innovative melanoma therapies for patients with metastasis undetectable disease and/or yet to develop metastasis. Citation Format: Monika Vishnoi, Debasish Boral, Haowen N. Liu, Marc L. Sprouse, Wei Yin, Michael A. Davies, Isabella C. Glitza Oliva, Dario Marchetti. Targeting USP7/PTEN axis regulates the metastatic competency of bone marrow-resident melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3601.