PYGO2 IS OVEREXPRESSED IN MULTIPLE MYELOMA PATIENTS WITH 1Q21 AMPLIFICATION DRIVING PLASMA CELL PROLIFERATION AND CELL CYCLE PROGRESSION

Chromosome 1q21 copy number alterations (1q21+), including gain (three copies) and amplification (four or more copies), are among the most frequent secondary genetic events in multiple myeloma (MM). These alterations are associated with poor prognosis, increased disease aggressiveness, and resistance to proteasome inhibitors (PIs). Importantly, 1q21+ is already detectable in approximately 30-40% of newly diagnosed MM (NDMM) patients, reaching up to 70% in relapsed/refractory MM (RRMM), suggesting its prevalence increases with disease progression. Despite its clinical relevance, the exact genes that drive the aggressiveness of 1q21+ remain unknown. In this study, we identify PYGOPUS2 (PYGO2), a transcriptional co-activator of the Wnt/β-catenin signaling pathway located on chromosome 1q21, as a key effector of MM progression in patients harboring 1q21+ alterations. Through transcriptomic analysis of CD138+ plasma cells (PCs) derived from MM bone marrow (BM) aspirates and validation in public datasets (GSE227907 and CoMMpass), we demonstrated that PYGO2 expression is significantly upregulated in 1q21+ patients and positively correlated with the degree of chromosomal amplification. Immunohistochemical analysis further confirmed elevated nuclear PYGO2 protein levels in 1q21+ MM bone biopsy samples. High PYGO2 expression is associated with adverse cytogenetic features, including del(13q) and t(4;14), and correlates with significantly shorter progression-free survival (PFS) and overall survival (OS). In fact, stratification of patients by PYGO2 expression quartiles revealed that those in the highest quartile (Q1) exhibit the worst clinical outcomes, supporting the prognostic significance of PYGO2 in MM. Functional studies in human myeloma cell lines (HMCLs) demonstrated that PYGO2-knock-down (KD) in 1q21+ models (JJN3 and OPM2) leads to impaired cell proliferation, increased cell death, and cell cycle arrest. Specifically, KD of PYGO2 resulted in a reduction of Cyclin D1 (CCND1) mRNA levels and downregulation of H3K4me3, a histone mark associated with active transcription and located on the promoter of CCND1. These findings suggest that PYGO2 regulates CCND1 expression through chromatin remodeling mechanisms. Cell cycle analysis reveals that PYGO2-KD induces G0/G1 phase arrest in OPM2 cells and increases the Sub-G0 apoptotic fraction in JJN3 cells, highlighting cell line-specific effects on cell cycle dynamics. Conversely, overexpression (OE) of PYGO2 in OCI-MY5 cells, which are wild-type for 1q21, promoted cell proliferation, accelerated S-phase entry, and reduced sensitivity to Carfilzomib. These results confirm the oncogenic role of PYGO2 and its contribution to PIs resistance in MM. To further elucidate the transcriptional landscape regulated by PYGO2, we performed RNA-sequencing on JJN3 cells following PYGO2-KD. Differential expression analysis identified 452 genes significantly modulated by PYGO2, with enrichment of pathways related to Hedgehog signaling, epithelial–mesenchymal transition (EMT), TGF-β signaling, and interferon-γ response in cells with higher expression of PYGO2. Gene ontology (GO) analysis revealed biological processes involved in cell adhesion, integrin-mediated signaling, and matrix interaction, suggesting that PYGO2 may also influence tumor–microenvironment interactions and immune modulation. Collectively, our findings establish PYGO2 as a copy number-driven oncogene in MM, functionally implicated in cell proliferation, cell cycle regulation, and therapeutic resistance. Its overexpression in 1q21+ patients provides a mechanistic link between chromosomal amplification and aggressive disease behaviour. Given its role, PYGO2 emerges as a promising candidate for targeted therapeutic intervention. This study not only advances our understanding of the biological consequences of 1q21+ in MM, but also highlights PYGO2 as a potential biomarker for risk stratification and a novel target for precision medicine approaches. Future investigations should explore the druggability of PYGO2 and its downstream effectors, with the goal of improving outcomes in high-risk 1q21+ MM patients resistant to PIs therapies.