Targeted sequencing of IDH1 and IDH2 genes was performed using bone marrow specimens from matched diagnosis and relapsed AML patients with IDH baseline mutation before or after IDHmut inhibitor treatment. gDNA was extracted from the bone marrow clot sections using the QuickExtract FFPE DNA extraction kit (Epicenter Biotechnologies) following the manufacturer's instructions. The exon sequences of IDH1 and IDH2 genes were PCR amplified. The DNA fragments for each exon were purified with QIAquick spin columns (Qiagen), mixed at equal molarity, and fragmented using Covaris acoustic shearing following the manufacturer's instructions. Sonicated DNA was processed for library generation using NEBNext Ultra II DNA Kits (New England Biolabs) following the manufacturer's protocol. Libraries with different index sequences were quantified, pooled, and sequenced on an Illumina Nextseq500 system using the 75bp high output sequencing kit.

Acquired resistance to allosteric inhibition of cancer-associated IDH mutations has been described through mutations to restore the production of oncometabolite R-2HG, yet the complete repertoire of IDH second-site mutations and underlying mechanisms remain unknown. Here we establish isogenic leukemia cell lines containing common IDH mutations by CRISPR-mediated base editing. Through mutational scanning of IDH single-amino acid variants, we identify secondary mutations responsible for resistance to IDH inhibition through disabling NADPH-dependent uncompetitive inhibition. Acquired mutations at NADPH binding sites act in cis or trans to prevent the formation of stable IDH enzyme-inhibitor complexes, restore mutant enzyme activity, and drive therapy resistance. Hence, our findings uncover a new class of pathogenic mutations and mechanisms for acquired resistance to a targeted cancer therapy.