Large-scale drug screening identifies clinically actionable combinations to overcome BTK/PI3K inhibitor resistance in marginal zone lymphoma

Background. BTK inhibitors (i), and, more recently, BTK degraders have become clinically relevant drugs for patients with B-cell malignancies, including marginal zone lymphoma (MZL). PI3Ki are also active drugs, although the first- and second-generation molecules are now less used due to toxicity. Here, we present data from a large pharmacological screen involving over 3,500 compounds in 2 MZL models with secondary resistance to BTK/PI3Ki, developed through prolonged exposure to idelalisib (Arribas 2022) or ibrutinib (Arribas 2025). Methods. Parental VL51 and resistant derivatives were exposed to DMSO or a library of 3,527 compounds (Mariotto 2023) as single agents (5µM) or in combination with either PI3Ki idelalisib (1µM) or BTKi ibrutinib (500nM) for 72 hours. Spatial and intra- and inter-plate effects were corrected. Robust Z-values were then DMSO-normalized. Highly active compounds across parental and resistant lines, as well as those with improved responses in either idelalisib-resistant or ibrutinib-resistant lines, were selected for further validation by combination with idelalisib or ibrutinib (MTT assay, 72 hours). Synergy of combinations was evaluated according to the Chou-Talalay combination index (CI) and to the MuSyC, HSA and ZIP algorithms. Results. We identified 127 highly active compounds as single agents in both parental and the 2 resistant (Z<0.5), 10 compounds with higher activity in both resistant lines (Z<0.3), and 27 and 54 with increased response in BTKi and PI3Ki resistant (Z<0.3), compared to the parental counterpart, respectively. The identified molecules targeted apoptosis, cell cycle, PI3K/AKT/mTOR, chromatin/epigenetics, GPCR, DNA damage and repair, and immune signaling. Among these compounds, we selected those more advanced in the clinical setting for further testing in combination with BTKi and PI3Ki. The AChE/cytochrome-P450-i acetylshikonin was synergistic when combined with idelalisib or ibrutinib in six cell lines. The addition of the PAK4/NAMPTi KPT-0247 or the antibiotic diclazuril restored sensitivity to BTKi and improved the potency of BTKi in resistant cells, but not in the parental cells. Adding the alisertib (AURKAi), rigosertib (PLKi), fimepinostat (PI3K/HDACi), lanatoside-C (Na+K+ATPase), astragalin (apoptosis), astragaloside-I (WNT) and oridonin (AKT) was of benefit (additivity or synergism) in both parental and resistant cells. Conclusions. This large-scale screen identifies multiple actionable vulnerabilities in BTKi- and PI3Ki-resistant MZL. Several clinically advanced agents-particularly PAK4/NAMPT, AURKA, WNT, and Na⁺/K⁺-ATPase inhibitors-enhanced or restored the activity of BTKi/PI3Ki. These findings highlight new therapeutic strategies for relapsed/refractory MZL and support clinical evaluation of targeted combinations to overcome acquired resistance.