Fig 2: Ectopic expression of SLAMF7 variant X secreted sSLAMF7 and enhanced multiple myeloma cell growth. (A) Structure-based prediction of SLAMF7 variant 1-variant X interactions using the AlphaFold2 program. (B) We used the lentiviral vector CSII-CMV-MCS-IRES-VENUS (provided by Dr. Hiroyuki Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) containing the coding regions of SLAMF7 variant 1 and SLAMF7 variant X cDNA for gain-of-function experiments. Whole cell lysates and supernatants were prepared from RPMI8226 cells transduced with mock, SLAMF7 variant 1, or SLAMF7 variant X after 24 hours (hr) of culture. Immunoblotting was carried out according to a standard method using the following antibodies: anti-SLAMF7 (#98611), anti-BSA (#23053) (Cell Signaling Technology, Beverly, MA, USA); and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoblot shows the expression levels of SLAMF7 protein, with GAPDH as the loading control of cell lysate, and BSA as the loading control of supernatant (left panel). The sSLAMF7 levels in the culture supernatants were measured using ELISA (right panel). Bars indicate the mean of 3 independent experiments. P value was determined by one-way ANOVA with Tukey’s multiple comparison test. (C) SLAMF7-negative (SLAMF7–) RPMI8226 and KMM.1 cells and SLAMF7-positive (SLAMF7+) KMS28-BM and MM.1S cells were cultured with the supernatant derived from 1x105 RPMI8226 cells transduced with SLAMF7 variant 1 or SLAMF7 variant X after 24 hr of culture. Cell proliferation was assessed after 72 hr using the MTT reduction assay and is shown relative to the variant 1 group. Bars indicate the means of 3 independent experiments. P value was calculated using a paired Student t test.

Fig 3: Soluble SLAMF7 is generated from alternative splicing in multiple myeloma cells. (A) The full length SLAMF7 or GAPDH (internal control) was amplified from the cDNA of CD138+ plasma cells derived from multiple myeloma (MM) patients, normal plasma cells (NPC) from healthy volunteers, and bone marrow mononuclear cells (BM-MNC). PCR was conducted for 30 cycles, and the products were analyzed using agarose gel electrophoresis and ethidium bromide staining. TA-cloning and complete sequencing of the PCR products identified the amplicons as variant 1 (1008 bp), variant 6 (891 bp), variant 8 (726 bp), variant 4 (687 bp), variant 2 (498 bp), and an unknown variant X (771 bp). (B) The schematics of the SLAMF7 variants including exon numbers encoding the IgV-domain (V), IgC2-domain (C2), and membrane anchor portion (MA) of SLAMF7.

Fig 4: (A): Intron retention due to PRMT5 inhibition was evaluated by re-analysis of RNA-seq data (as described earlier) using IRFinder (n = 2 for each condition and per cell line). Volcano plot shows IRFinder output in JJN3 cells, identifying different gene targets affected by intron retention. Only the plot for JJN3 is shown here, other plots are shown in the supplementary data. (B): Venn diagram presentation of common genes affected by intron retention (n = 2 × 2 per HMCL/treatment condition). A set of 45 common affected genes were identified. Samples were analysed per cell line type to account for cell type specific effects. (C): Reactome enrichment analysis was subsequently performed using the 45 common genes. Selected pathways that were deregulated are shown. (D): Western blot analysis of identified targets MMSET/WHSC1 HELLS and SLAMF7 in JJN3, OPM2, XG7 and AMO1 cells. For MMSET, * depicts the wild type MMSET isoform where § depicts the aberrant MMSET protein affected by t (4; 14), which is present in OPM2 cells. Tubulin and actin were used as loading control. One experiment representative of 3 experiments performed is shown (n = 3).

Fig 5: MM cell lines were co-cultured with adhesion to UBE6T-7 cells under normoxic (20% O2) (Normoxia + UBE) or hypoxic (5% O2) (Hypoxia + UBE) conditions and treated with various concentrations of T-5224 for 72 h. The mRNA expression level of SLAMF7 was determined by qPCR and is shown relative to the untreated control values set at 1.0. Data were quantified using the 2−∆∆Ct method with GAPDH as a reference. The means ± S.D. (bars) of three independent experiments. p-values were determined by one-way ANOVA with the Student−Newman−Keuls multiple comparison test (upper panels). Whole cell lysates were simultaneously prepared and subjected to immunoblotting for SLAMF7 and GAPDH proteins (lower panels).