2-HG–restoring mutations emerging at relapse or disease progression. (A) Second-site IDH1 mutations emerging during ivosidenib treatment (n = 20). Each circle represents a single patient with detection of the mutation. Direct denotes that the mutation makes contact with the ivosidenib or cofactor (NADPH) binding pocket by 3-dimensional modeling. Indirect denotes that it induces structural changes in vicinity of ivosidenib or cofactor binding pockets (hypothesized). Novel mutations (not previously published) are mostly the indirect type. (B) Crystal structure of IDH1-R132H in the inactive, open conformation. The cocrystal structure of IDH1-R132H with inhibitor 20a (PBD accession no. 5L57)⁴⁰  was obtained at 2.7 A^o resolution and used to develop the model for the ivosidenib analog AGI-14686 (supplemental Methods). The homodimeric protein structure is shown with the inhibitor (AGI-14686) in yellow, occupying the middle of the tetra-helical domain. The protein monomers are shown in green and cyan, the mutated 132H residue in magenta and cofactor NADPH in orange. (C) The mIDH1-AGI-14686 binding model for the IDH1-R132H-S280F mutation places the di-F cyclopentane of AGI-14686 near Ser280 (left). Mutation of Ser280 to Phe (right) will create a steric interference with ivosidenib (or its analogs), and thus will no longer bind to mIDH1. The mIDH1 protein is depicted in cartoon (green and blue for each monomer), the ivosidenib analog AGI-14686 in yellow sticks, and residue 280 in spheres (Ser in the left panel, Phe in the right panel). R132H is shown in magenta. (D) Biochemical 50% inhibitory concentration (IC₅₀) for second-site IDH1 mutations. We expressed various combinations of IDH1 mutants with second-site mutations and examined biochemically whether they were still inhibited by ivosidenib.