SK-MM-2

Impact Of ASO-Mediated Targeting of V Exon 3'ss in Myeloma Cells SK-MM-2

Systemic AL amyloidosis is caused by organ deposition of amyloid fibrils derived from monoclonal immunoglobulin light chains produced by a deregulated plasma-cell clone. Lambert et al. explored whether splice-switching antisense oligonucleotides (ASOs) directed against the variable (V) exon of the clone-specific pre-mRNA can force expression of a V-domain-less, truncated light chain that triggers ER stress-mediated apoptosis.

3' splice sites (3'ss) are more conserved than 5'ss, so ASOs targeting 3'ss excise exons more efficiently. In SK-MM-2 myeloma cells expressing IGKV3-15IGKJ4 κ light chain, ASO-Vκ3-15-3'ss (Fig. 1A) triggered near-complete V-exon skipping: ΔV-κLC mRNA rose to 67.7 ± 7.1 % (24 h) and 79.5 ± 9.0 % (48 h) of total Igκ transcripts (Fig. 1B). A ~12 kDa truncated ΔV-κLC protein appeared while full-length κ chains declined (Fig. 1C). ΔV-κLC accumulation coincided with increased cell death (Fig. 1D). RNA-seq (|log2FC| ≥ 0.75, adj p < 0.05) revealed 1 100 up- and 524 down-regulated genes enriched for apoptosis, autophagy, ubiquitination and ER-stress pathways, indicating that ER-stress-driven apoptosis underlies the massive lethality of ASO-Vκ3-15-3'ss (Fig. 1E).

OTUD1 is a Regulator of Ig Production and Proliferation in Myeloma Cells

Serum Ig quantity fails to stratify MM prognosis; intracellular Ig load does. Integrating CRISPR screening with transcriptomic/proteomic data, Vdovin et al. identified OTUD1 as a deubiquitinase essential for Ig synthesis, proteasome-inhibitor (PI) sensitivity and tumor burden.

To probe OTUD1's role in Ig production, they generated MM lines (RPMI8226, MM.1 S, HEK 293, JJN-3, SK-MM-2, and U2OS) with dox-inducible OTUD1 overexpression (OTUD1 oe) or two shRNA knock-downs (sh OTUD1_1/2) (Fig. 2a-c); controls lacked dox or carried non-targeting shRNA. OTUD1 oe raised intracellular Ig light chain (iIgL) (Fig. 2d), whereas sh OTUD1 reduced it (Fig. 2e), without altering IgL mRNA or secretion. Knock-down of other DUBs had no effect, and catalytically dead OTUD1-C320R failed to change iIgL, confirming that OTUD1 enzymatic activity specifically sustains high IgL levels. Consistent with public data, OTUD1 curbed myeloma aggressiveness: OTUD1 oe slowed proliferation, whereas sh OTUD1 accelerated it (Fig. 2f, g). In vivo, the growth difference was magnified (Fig. 2h-k), and intracellular iIgL in xenograft tumors tracked OTUD1 levels (Fig. 2l, m). OTUD1 oe imposed a partial S-phase arrest without compromising viability, recapitulating patient observations and validating the model for dissecting OTUD1 function in myeloma.