Objective <p>TP53 mutations are infrequently found in multiple myeloma (MM), a malignancy characterized by the clonal proliferation of plasma cells that secrete monoclonal immunoglobulin. This study explored the mechanism of caspase-activated DNase (CAD) and B-cell lymphoma 2 (BCL-2) in regulating proliferation and drug resistance of TP53-mutant MM cells.</p> Methods <p>TP53 wild-type (H929) and TP53-mutant (U266 and RPMI-8226) MM cell lines were cultured under standard conditions. TP53-mutant cells were transfected with lentiviral shRNA plasmids targeting CAD and BCL-2 (Lv-sh-CAD and Lv-sh-BCL-2). Cell viability, proliferation, apoptosis, and cell cycle, as well as the levels of CAD, BCL-2, cell cycle-related proteins (P21, Cyclin D1, Cyclin-dependent kinase 1 [CDK1]) and drug resistance-associated proteins (adenosine triphosphate-binding cassette subfamily B member 1 [ABCB1], ABCG2) were assessed by CCK-8, colony formation, flow cytometry, qRT-PCR and western blot assays. A xenograft tumor model was established and treated with bortezomib for in vivo validation.</p> Results <p>Compared to H929 cells, U266 and RPMI-8226 cells exhibited elevated CAD and BCL-2 levels as well as reduced drug resistance. Knockdown of CAD or BCL-2 in TP53-mutant MM cells decreased cell proliferation, and the expression of ABCB1, ABCG2, Cyclin D1 and CDK1, while increasing apoptosis, G2/M arrest level and P21 expression. These changes also reduced resistance to bortezomib and doxorubicin. Co-knockdown of CAD and BCL-2 further promoted G2/M arrest, thereby inhibiting proliferation and drug resistance, and facilitating apoptosis. In vivo, co-knockdown of CAD and BCL-2 suppressed the growth of TP53-mutant MM cells and enhanced their sensitivity to bortezomib.</p> Conclusions <p>CAD and BCL-2 were highly expressed in TP53-mutant MM cells, and their co-knockdown boosted G2/M cell cycle arrest, thereby preventing cell proliferation, enhancing apoptosis, and reducing drug resistance.</p>