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: Sequence of SLAMF7 variant X cDNA and the expression in multiple myeloma cells. (A) Sequence of SLAMF7 variant X cDNA and its translation product. The C-terminus unique to the variant X protein is underlined. The variant X-specific forward and reverse primers are shown in italics. The primer pair was used for semi-quantitative and real-time quantitative RT-PCR (Q-PCR) analyses. (B) Total cellular RNA was isolated from 1-10x104 cells using an RNeasy Kit (Qiagen, Valencia, CA, USA), reverse-transcribed into complementary DNA using ReverTra Ace and oligo(dT) primers (Toyobo, Tokyo, Japan), and subjected to semi-quantitative RT-PCR or real-time quantitative RTPCR (Q-PCR) using the specific primers described in Online Supplementary Table S1, and TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). SLAMF7 variant 1, variant X and GAPDH (internal control) mRNA expression in multiple myeloma (MM) cells (patients 1-5) (upper panel), CD34+/CD38– normal hematopoietic stem cells (CD34+/CD38-), CD34+/CD38+ normal hematopoietic progenitor cells (CD34+/ CD38+), CD34– normal bone marrow cells (CD34–), peripheral blood mononuclear cells (PB-MNC), HEK293 cells (HEK293), stromal cell lines (UBE6T-7 and stroma-natural killer), fibroblasts (BJ), and KMS28-BM were analyzed using semi-quantitative RT-PCR. The PCR products were analyzed using agarose gel electrophoresis and ethidium bromide staining. The results of suboptimal amplification cycles (35 cycles) are shown. KMS28-BM was used as a positive control. (C) Expression levels of SLAMF7 variant X in 14 MM patients were determined by Q-PCR, normalized to that of GAPDH, and quantified using the 2-∆∆Ct method with the values of BM-MNC set at 1.0. Serum levels of soluble SLAMF7 (sSLAMF7) were also measured using a SLAMF7 ELISA Kit (Aviva Systems Biology, San Diego, CA, USA). The correlation coefficient (R) between SLAMF7 variant X expression and sSLAMF7 was evaluated by calculating Pearson’s correlation coefficient.

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).