Abstract 6714: ALCAM promotes neuroblastoma proliferation, migration, and immune evasion

Abstract

BACKGROUND: Neuroblastoma is a cancer arising from the developing sympathetic nervous system. Less than half of children diagnosed with high-risk neuroblastoma survive five years. Two transcriptional subpopulations have been identified: aggressive, chemotherapy-sensitive adrenergic (ADRN) and indolent, chemotherapy-resistant mesenchymal (MES). ALCAM is a cell adhesion molecule that is overexpressed in neuroblastoma. ALCAM is a cancer stem cell marker in colorectal and small cell lung cancer and promotes bone metastasis in prostate cancer. ALCAM also interacts with the CD6 receptor on T cells, which facilitates adhesion in the immunological synapse and inhibits T cell activation. METHODS: We characterized ALCAM expression in 24 neuroblastoma cell lines using immunoblotting. We investigated ALCAM expression in ADRN and MES neuroblastoma using RNA-sequencing data from two patient datasets. Next, we developed doxycycline (dox)-inducible ALCAM depletion cell lines using CRISPR inhibition (CRISPRi) in three high-ALCAM cell lines with relevant oncogenic aberrations. Using the Incucyte® SX5 Live-Cell Analysis System, we interrogated the effects of ALCAM depletion on proliferation and scratch wound migration. To determine the mechanism of ALCAM overexpression, we leveraged ChIP-sequencing data for MYC and MYCN in 9 cell lines. We probed the effects of the MYC(N)-MAX disrupter MYCi975 on ALCAM expression using immunoblotting. To investigate the effects of the ALCAM-CD6 interaction on T cells, we stimulated CD4+ and CD8+ T cells with CD3/28 antibodies in the presence or absence of recombinant ALCAM and measured activation by ELISA for IFN-γ, and TNF-α and flow cytometry for CD69 expression. RESULTS: ALCAM is highly expressed in most neuroblastoma primary tumors and cell lines. ALCAM mRNA expression is higher in the ADRN subpopulation (p ≤ 0.0045), although both ADRN and MES tumors and cell lines express high levels of ALCAM. Dox-inducible CRISPRi mediated robust ALCAM depletion in all three cell lines, and ALCAM knockdown significantly delayed proliferation (p ≤ 0.0025) and impaired scratch wound migration. ChIP-sequencing showed robust MYC(N) peaks at the ALCAM promoter, and treatment with IC50 concentrations (3.5 - 9.0 μM) of MYCi975 depleted MYC(N) and ALCAM. Activation of CD4+ and CD8+ T cells in the presence of recombinant ALCAM mediated dose-dependent reduction in expression of T cell activation marker CD69 after 3 days and inhibited IFN-γ and TNF-α release. CONCLUSIONS: ALCAM is overexpressed in neuroblastoma and its expression is driven, at least in part, by MYC(N). ALCAM is a mediator of cellular proliferation and migration and may contribute to immune evasion by inhibiting T cell activation. Ongoing in vivo studies will characterize the effects of ALCAM depletion on tumor growth and metastasis. We propose ALCAM is a tractable immunotherapy target through multi-specific antibodies or the ALCAM-CD6 interaction. Citation Format: Jarrett Lindsay, Minu Samanta, Nathan Kendsersky, Jonathan Gaither, Molly Christie, Kyabeth Torres-Rodriguez, Catherine Wingrove, John M. Maris. ALCAM promotes neuroblastoma proliferation, migration, and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6714.

Developing targeted therapies for neuroblastoma by dissecting the effects of metabolic reprogramming on tumor microenvironments and progression

“…MPS-I NB cells exhibited a distinctive characteristic of self-communication which markedly accelerated their malignant progression (Figure 13 N). In the previous study, ALCAM signaling pathway was found to promote proliferation, migration, and immune evasion of NB cells [66]. L1CAM, overexpressed in various tumors including NB in the present study, was reported to markedly facilitate cell adhesion, proliferation, and migration [67][68][69]; thus, therapies of CAR T cells targeting L1CAM have been investigated in the stage of clinical test NB [69].…”