Zadro, A.; Arribas, A. J.; Robusto, M.; Vultaggio, S.; Andronache, A.; Faga, G.; Kupcova, K.; Cannas, E.; Spriano, F.; Sartori, G.; Cascione, L.; Tarantelli, C.; Napoli, S.; Rinaldi, A.; Rossi, D.; Zucca, E.; Moretti, M.; Havranek, O.; Stathis, A.; Varasi, M.; Mercurio, C.; Bertoni, F.

B-cell receptor (BCR) signaling is a key therapeutic target in B-cell lymphomas, and Bruton tyrosine kinase inhibitors (BTKi) have demonstrated clinical efficacy in marginal zone lymphoma (MZL). However, the rate of complete remissions is relatively low, and resistance remains a significant clinical challenge, underscoring the need for novel combination strategies. To identify compounds that enhance the activity of BTKi and overcome resistance, we conducted a high-throughput screen of 1,695 compounds using a previously developed MZL model of acquired resistance to BTK and PI3K inhibitors derived from the Karpas1718 cell line. Thirty-three compounds showed single-agent anti-proliferative activity in the nanomolar range, both in the Karpas1718-resistant and parental cells. Based on their clinical potential in combination with BTKi, seven compounds were selected for further validation. The polo-like kinase 1 (PLK1) inhibitor rigosertib emerged as a top candidate, showing strong activity in both parental and BTKi-resistant cell lines. Combination treatment of rigosertib and BTKi zanubrutinib resulted in broad transcriptomic changes, characterized by the downregulation of pathways involved in B-cell activation, proliferation, and BCR signaling. Mechanistically, the combination reduced phosphorylation of key BCR pathway components and inhibited NF-{kappa}B nuclear translocation. These findings suggest that PLK1 inhibition can overcome BTKi resistance by further inhibiting BCR signaling through the canonical NF-{kappa}B pathway. Therefore, the dual pharmacological inhibition of BTK and PLK1 is a promising therapeutic approach for patients with MZL and warrants further preclinical and clinical investigations.