Anti-MM activity of dual Asn/Gln depletion plus Kar. MM cell lines (A) and primary CD138⁺ cells (NDMM and RRMM) (B) were treated with ASNase, Kar, or their combination. Cell viability was measured and presented as a percentage of control cells (untreated). Synergism was calculated by CI analysis, with heat maps depicting the CI values at increasing doses of Erw-ASNase and Kar. CIs were generated with CalcuSyn software for each drug combination. CI < 1, CI = 1, and CI > 1 denote synergism, additive effect, and antagonism, respectively. Data are mean ± SD (n = 3). (C) Viability of U266 NanoLuc⁺ cells treated with Erw-ASNase, Kar, or their combination for 48 hours, alone and in the presence of MM patient–derived BMSCs (gray), measured by luciferase-based luminescence assay. Data are mean ± SD in all graphs (n = 3). (D) Healthy donor PBMCs were exposed to increased doses of each drug, alone or in combination, and viability was measured using an MTS assay. (E) LP1 cells were treated with Erw-ASNase (0.5 U/mL), Kar (3 nM), or both. Apoptotic cell death was assessed by flow cytometry using annexin V and propidium iodide double staining after 48 hours. (F) Immunoblots for PARP1, caspase-3, caspase-9, and GAPDH on the indicated MM cell lines at 24 hours posttreatment with Erw-ASNase (0.5 U/mL), Kar (5 nM), or both. (G) Western blot showing that amino acid depletion recapitulates apoptotic features triggered by Erw-ASNase treatment on Kar-exposed U266 cells. Cleavage of PARP1, caspase-3, caspase-9, and GAPDH (loading control) was detected. One representative western blot of 3 is shown. **P < .01, ***P < .0001, unpaired Student t test. ns, not significant.