The MLN4924/belinostat regimen is active against primary AML/MDS cells and diminishes the primitive CD34⁺/CD38⁻/CD123⁺ population enriched for LICs, while displaying minimal toxicity toward normal CD34⁺ cells. (A) A representative primary sample from a patient with AML carrying the indicated cancer hotspot mutations (top panels) and normal CB CD34⁺ cells (bottom panels) was exposed to 300 nM PXD-101 ± 200 nM MLN4924 for 24 hours, after which the percentage of Annexin V⁺ cells was determined by FCM. (B) Parallel experiments were carried out with 47 primary samples (AML = 42; MDS = 5, indicated by squares, treatment with 500 nM PXD-101 ± 500 nM MLN4924). Cell viability was analyzed by 7-aminoactinomycin D (7AAD) staining and FCM. Lines indicate means and SD. (C) After exposure to 300 nM PXD-101 ± 200 nM MLN4924 for 24 hours, the percentage of the primitive CD34–phycoerythrin-positive (PE⁺)/CD38–peridinin chlorophyll protein-negative (PerCP⁻)/CD123–allophycocyanin-positive (APC⁺) population, which is enriched for LICs, was determined by multicolor FCM in BM mononuclear cells from a primary AML sample carrying the indicated cancer hotspot mutations. (D) Parallel experiments were carried out with 25 primary AML samples. Viability of CD34⁺/CD38⁻/CD123⁺ cells was analyzed by 7AAD staining and FCM. (E) Alternatively, western blot analysis was performed to monitor the selected markers for inhibition of neddylation, disruption of the DDR, and induction of DNA damage in 2 representative primary samples that responded to the MLN4924/belinostat regimen. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI, propidium iodide.