SKP2/CKS1B as a rational drug target in 1q21 amplified multiple myeloma.

Abstract Chromosome 1q21 amplification is closely associated with disease progression and adverse prognosis in multiple myeloma (MM), rising from approximately 20% incidence in MGUS to 70–80% in relapsed or refractory MM. The CKS1B gene, located in the 1q21 region, acts as an important prognostic biomarker; patients with the highest CKS1B mRNA levels in the COMPASS study exhibited the poorest survival. As a core partner of SKP2 in the SCF E3 ubiquitin ligase complex, CKS1B promotes degradation of the cell cycle inhibitor p27 and contributes to MM cell proliferation. Using CRISPR-Cas9 gene editing of 17 genes located in 1q21 individually knocked out in two MM cell lines with genomic 1q21 amplification, CKS1B was identified as key vulnerability. To validate and extend these findings, CKS1B, as well as SKP2, were knocked out in 19 MM cell lines of varying 1q21 copy number. Strong proliferation inhibition was observed in 13 of 19 cell lines, characterized by increased G2/M cell cycle arrest, and induction of senescence. While no clear association was found between 1q21 copy number and response, 13q loss was present in 4 of 6 robustly inhibited lines but only one of three lines with limited response. To explore the mechanism of action, systematic analysis of 22 published SKP2/CKS1B substrates in the MM1R cell line after SKP2/CKS1B knockout demonstrated increased protein levels of p27, Cyclin E, ORC1, and FOXO3a. The levels of p27 were further explored across all 19 MM cell lines, resulting to be upregulated in a large majority (17/19) including two insensitive lines. Strikingly, knocking out p27 in SKP2- or CKS1B-dependent cell lines rescued the proliferation defect, confirming the central role of p27 in mediating the effect. To further investigate the SKP2/CKS1B dependency in MM, we analyzed Cks1b, Skp2, and p27 expression along with whole-exome sequencing in high-risk P53-BIcγ1-derived mouse MM cell lines (Larrayoz et al., Nat Med 2023). Based on molecular profiling, the MM5080 cell line exhibited amplification of the chromosome 3q region syntenic to the human 1q21, leading to high expression of Cks1b. MM5080 cells were then used to generate stable, doxycycline-inducible shSkp2 clones, which progressively led to a significant proliferation reduction and cell cycle arrest ex vivo, together with upregulation of p27 levels. Notably, these effects were reversed upon doxycycline withdrawal. Finally, in vivo Skp2 inhibition in MM5080 cells engrafted into in Rag2−/−γc−/− mice delayed disease onset and extended mouse survival (median OS: 26 vs 22 days, p<0.001). These results validate SKP2 and CKS1B as critical dependencies in MM, support the therapeutic potential of targeting this pathway—especially in high-risk, 1q21-amplified or 13q-deleted disease—and provide a strong rationale for further preclinical development and patient stratification with SKP2/CKS1B inhibitors.