Abstract 2525: Identification of differentially expressed myeloma-specific pathways in the absence of VLA-4
Authors: Hathi Deep K. , Chanswangphuwana Chantiya , Rettig Michael P. , Achilefu Samuel , DiPersio John F. , Shokeen Monica 
Published: August 2018

Multiple myeloma (MM) is a cancer of terminally differentiated plasma B-cells in the hematopoietic bone marrow (BM). Despite advances in MM therapy and stem cell transplantation, MM accounts for approximately 20% of all mortalities resulting from hematologic malignancies. One of the major proteins involved in modulating myeloma cell survival and adhesion within the BM is the very late antigen-4 (VLA-4; also known as α4β1 or CD49d/CD29). VLA-4 is a bidirectional integrin overexpressed in myeloma cells that is implicated in key MM tumorigenesis pathways, including cell proliferation, survival, and adhesion-mediated drug resistance.

Previous studies have demonstrated that VLA-4 is inconsistently overexpressed in myeloma cell lines and clinical studies. This heterogeneous expression of VLA-4 may affect disease progression, localization of myeloma lesions, and efficacy of myeloma therapies. Here, we generated VLA-4 knockout (KO) versions of the murine myeloma cell line 5TGM1-GFP to study changes in disease kinetics and identify clinically relevant proteins and pathways that are influenced by VLA-4 expression in myeloma cells.

VLA-4 KO 5TGM1-GFP cell lines were generated using CRISPR/Cas9 to induce a single base pair deletion in the ITGA4 (α4) gene. Lack of α4 surface expression and VLA-4 functionality was verified in KO cell lines in vitro with fluorescence activated cell sorting (FACS) of anti-CD49d and recombinant soluble vascular cell adhesion protein-1 staining, respectively. Implantation of the KO clones in the immunocompetent C57Bl/KaLwRij model via intravenous dissemination of myeloma cells resulted in minimal, but heterogeneous, uptake in the BM and spleen. FACS confirmed the absence of VLA-4 expression in the excised MM tumors, suggesting the differences in disease kinetics and tumor localization between the KO cell lines were induced by changes in MM pathways affected by the knockdown of VLA-4 function.

To identify proteins and pathways affected by knockdown of α4, we performed RNA sequencing on over 100,000 genes in 5TGM1-GFP wild-type (WT) and KO cell lines. Hierarchical clustering on statistically significant differentially expressed genes (false discovery rate-corrected p&lt;0.001) identified 591 overlapping genes, and 975 non-overlapping genes. Comparison with clinical gene expression data from the MIT Broad Institute MM database identified endoglin, myc, and estrogen receptor-1 as prominent genes with high mutation rates in patients. Finally, myeloma-specific differentially expressed genes were analyzed using graph-based network analysis and gene ontology to identify key proteins and pathways. Our results demonstrate the impact of VLA-4 on BM localization and oncologic pathways that may be exploited in the development of novel therapeutic strategies.

Citation Format: Deep K. Hathi, Chantiya Chanswangphuwana, Michael P. Rettig, Samuel Achilefu, John F. DiPersio, Monica Shokeen. Identification of differentially expressed myeloma-specific pathways in the absence of VLA-4 [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 2525.