Association of ADAM family members with proliferation signaling and disease progression in multiple myeloma

Multiple myeloma (MM) is a hematological malignancy whose curability is greatly challenged by recurrent patient relapses and therapy resistance. We have previously proposed the high expression of ADAM8, ADAM9 and ADAM15 (A Disintegrin And Metalloproteinase 8/9/15) as adverse prognostic markers in MM. This study focused on the so far scarcely researched role of ADAM8/9/15 in MM using two patient cohorts and seven human MM cell lines (HMCL). High ADAM8/9/15 expression was associated with high-risk cytogenetic abnormalities and extramedullary disease. Furthermore, ADAM8/15 expression increased with MM progression and in relapsed/refractory MM compared to untreated patient samples. RNA sequencing and gene set enrichment analysis comparing ADAM8/9/15high/low patient samples revealed an upregulation of proliferation markers and proliferation-associated gene sets in ADAM8/9/15high patient samples. High ADAM8/9/15 expression correlated with high Ki67 and high ADAM8/15 expression with high MYC protein expression in immunohistochemical stainings of patient tissue. Conversely, siRNA-mediated knockdown of ADAM8/9/15 in HMCL downregulated proliferation-related gene sets. Western blotting revealed that ADAM8 knockdown regulated IGF1R/AKT signaling and ADAM9 knockdown decreased mTOR activation. Lastly, high ADAM8/9/15 expression levels were verified as prognostic markers independent of Ki67/MYC expression and/or high-risk abnormalities. Overall, these findings suggest that ADAM8/9/15 play a role in MM progression and proliferation signaling.


Secondary antibodies
Anti-rabbit 1:2 000 in 5 % milk-TBS-T CST 7074 Anti-mouse 1:2 000 in 5 % milk-TBS-T CST 7076 Table S1: siRNA sequences and antibodies.siRNAs targeting the maximum amount of transcripts were chosen.BLAST results of targets are summarized in supplementary Table S2.
All siRNAs are predesigned Silencer Select siRNAs purchased from Ambion by Life technologies (Thermo Fisher Scientific, Waltham, MA, USA).

Figure S1 :
Figure S1: High expression of ADAMs is associated with shorter survival in the validation cohort.(A-C) Correlation of high GE (> mean of all samples) of ADAM8 (A), ADAM9 (B) and ADAM15 (C) with OS in the validation cohort.(D-F) Correlation of high GE (> mean of all samples) of ADAM8 (D), ADAM9 (E) and ADAM15 (F) with PFS in the validation cohort.(G)Since the association between high ADAM9 GE and PFS was not significant with GE > mean defining GE as high, the analysis was also performed considering a higher threshold (> 10 TPM) as high ADAM9.Survival correlations were performed using the Kaplan Meier method and log rank test.n=51 patients.(A, D) ADAM8 GE > mean: n=15 patients; ADAM8 GE < mean: n= 36 patients.(B, E) ADAM9 GE > mean: n=16 patients; ADAM9 GE < mean: n= 35 patients.ADAM15 GE > mean: n=13 patients; ADAM15 GE < mean: n= 38 patients.(G) ADAM9 GE > 10 TPM: n=5 patients; ADAM9 GE < 10 TPM: n=46 patients.

Figure S2 :
Figure S2: PCA (A,C,E) and volcano plots (B,D,F) for RNA-Seq data concerning ADAM8 comparisons.PCA was performed on vst transformed data.(A,B): Comparison of ADAM8 high vs.ADAM8 low patient samples (top/lowest 10%) from the MMRF cohort.Positive log2fold change signifies upregulation in the ADAM8 high samples.(C,D): Comparison of ADAM8 high vs.ADAM8 low patient samples (top/lowest 25%) from the validation cohort.Positive log2fold change signifies upregulation in the ADAM8 high samples.(E,F): Comparison of HMCL transfected with scr-siRNA (scr) or siRNA targeting ADAM8 (si).Negative log2 fold change signifies downregulation in the ADAM8 siRNA samples.ADAM8 high vs.ADAM8 low

Figure S4 :
Figure S4: PCA (A,C,E) and volcano plots (B,D,F) for RNA-Seq data concerning ADAM15 comparisons.PCA was performed on vst transformed data.(A,B): Comparison of ADAM15 high vs.ADAM15 low patient samples (top/lowest 10%) from the MMRF cohort.Positive log2fold change signifies upregulation in the ADAM15 high samples.(C,D): Comparison of ADAM15 high vs.ADAM15 low patient samples (top/lowest 25%) from the validation cohort.Positive log2fold change signifies upregulation in the ADAM15 high samples.(E,F): Comparison of HMCL transfected with scr-siRNA (scr) or siRNA targeting ADAM15 (si).Negative log2 fold change signifies downregulation in the ADAM15 siRNA samples.

Figure S11 :Figure S12 :
Figure S11: ADAM8, ADAM9 and ADAM15 expression in HMCL.20 µg protein were used and representative WB of three independent rounds of experiments is shown.Antibody specificity was verified by siRNA knockdowns in HMCL.Bands that were downregulated by the siRNA knockdown are marked with a red <.Ab: antibody.

Table S5 :
Cox proportional hazards model considering high ADAM8, ADAM9 and ADAM15 GE and the cytogenetic abnormalities associated with a high expression of the respective ADAM gene in the MMRF cohort.PFS: Progression-free survival.OS: Overall survival.HR: Hazard ratio.CI: Confidence interval.(A) Information about ADAM8 GE, 1q amplification, p53 abnormality and t(4;14) was available for 358 patients.76 of these patients had an ADAM8 GE > mean; 1q amplification was detected in 125; p53 abnormality in 64 and t(4;14) in 55 patients.(B) Information about ADAM9 GE, 1q amplification, t(4;14) and t(14;16) was available for N=494 patients.206 of these patients had an ADAM9 GE > mean.The 1q amplification was detected in 188; t(4;14) in 77 and t(14;16) in 46 patients.(C) Information about ADAM15 GE and 1q amplification status was available for 547 patients.ADAM15 GE was > mean in 176 of these patients and the 1q amplification was present in 212 patients.
Distribution of samples with and without the translocation t(11;14) in the (A) ADAM9 low/high and (B) ADAM15 low/high groups used for DESeq and GSEA in the MMRF and validation cohort (top/lowest 10% or 25% of samples).Analysis includes only cases where both gene expression and translocation status was available.Group sizes are summarized above the bars.Statistical test was Fisher's exact.
(A, B) Cox proportional hazards model for progression-free (A) and overall survival (B) including ADAM9 and Ki67 expression status as variables.ADAM9 high : ADAM9 GE > mean of all samples.Ki67 high : ≥30% Ki67 + CD138 + cells.(C,D)Since high ADAM9 GE was only significantly associated with shorter PFS in the univariate analysis at higher thresholds defining ADAM9 GE as high (> 10 TPM, FigureS1), Cox proportional hazards model for progression-free (A) and overall survival (B) was also performed including ADAM9 and Ki67 expression status as variables with ADAM9 high : ADAM9 GE > 10 TPM.Ki67 high : ≥30% Ki67 + CD138 + cells.n=29 patients from the validation cohort.HR: Hazard ratio.CI: Confidence interval.
) (A, B) Cox proportional hazards model for progression-free (A) and overall survival (B) including ADAM8 and Ki67 expression status as variables.n=29patientsfrom the validation cohort.ADAM8 high : ADAM8 GE > mean of all samples.Ki67 high : ≥ 30% Ki67 + CD138 + cells.(C)Coxproportional hazards model for overall survival including ADAM8 and MYC expression status as variables.n=44patients.MYC high : ≥ 40% MYC + CD138 + cells.Analysis was not performed for PFS because high MYC expression has only been shown to correlate with OS in univariate analyses (see (33)).HR: Hazard ratio.CI: Confidence interval.(A, B) Cox proportional hazards model for progression-free (A) and overall survival (B) including ADAM15 and Ki67 expression status as variables.n=29 patients from the validation cohort.ADAM15 high : ADAM15 GE > mean of all samples.Ki67 high : ≥ 30% Ki67 + CD138 + cells.(C) Cox proportional hazards model for overall survival including ADAM15 and MYC expression status as variables.Analysis was not performed for PFS because high MYC expression has only been shown to correlate with OS in univariate analyses (see (33)).n=44 patients.MYC high : ≥ 40% MYC + CD138 + cells.HR: Hazard ratio.CI: Confidence interval.