Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival

Abstract

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α4β1) is a key player in cell–cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α4) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4+ 5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models.

First-in-Humans Evaluation of Safety and Dosimetry of64Cu-LLP2A for PET Imaging

“…were examined by flow cytometry using LLP2A-Cy5 (19). Using a 14-color flow cytometry panel, we identified eighteen different hematopoietic cell populations within these seven samples (Supplemental Figure 8).…”

“…Purified cells were > 97% for CD38 + CD138 + plasma cells. LLP2A-Cy5 synthesis and flow cytometry were performed as previously described (19) IGS mice and was considered NOEL (no observable effect level) (Supplemental Figure 4).…”

“…Encouragingly, there is a well-characterized preclinical PET probe, 64 Cu-LLP2A, specific for the activated conformation of VLA4 (20,21). We have previously demonstrated the utility of 64 Cu-LLP2A-PET in diverse human and mouse models of MM (19,(22)(23)(24).…”

Molecular Impact of the Tumor Microenvironment on Multiple Myeloma Dissemination and Extramedullary Disease

“…The VCAM-1 ligand, very-late antigen 4 (VLA-4), is frequently overexpressed on malignant plasma cells in the BM and plays an important role in plasma cell homing and cell adhesion (30,31). In this regard, Hathi et al showed that CRISPR/Cas9 induced depletion of VLA-4 in murine 5TGM1 myeloma cells resulted in increased extramedullary spread to soft tissue sites, liver and spleen and to reduced intramedullary tumor burden compared to mice injected with 5TGM1-wildtype cells where no manifestation of EMD occurred (32). In line with these findings, heparanase-induced shedding of CD138 (Syndecan-1), expressed on normal and malignant plasma cells, promotes myeloma cell migration and invasion by coupling VLA-4 to the vascular endothelial cell growth factor receptor-2 (VEGFR2) (33).…”

Effects of Non-Opioid Analgesics on the Cell Membrane of Skin and Gastrointestinal Cancers

“…One of these drugs is NONOnaproxen, which at a concentration of 0.1 mM, decreases surface activation of β1 integrin on human melanoma M624 cells after one hour of incubation [41]. The combination of β1 and α4 subunits(very late antigen-4 VLA-4, integrin α4β1) is a receptor for vascular cellular adhesion molecule-1 (VCAM-1) [42]. Downregulation of integrin activity leads to diminished melanoma cell adhesion to endothelial VCAM-1.…”