Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance

Abstract

Mutations in IDH genes occur frequently in acute myeloid leukemia (AML) and other human cancers to generate the oncometabolite R-2HG. Allosteric inhibition of mutant IDH suppresses R-2HG production in a subset of AML patients; however, acquired resistance emerges as a new challenge and the underlying mechanisms remain incompletely understood. Here we establish isogenic leukemia cells containing common IDH oncogenic mutations by CRISPR base editing. By mutational scanning of IDH single-amino acid variants in base-edited cells, we describe a repertoire of IDH second-site mutations responsible for therapy resistance through disabling uncompetitive enzyme inhibition. Recurrent mutations at NADPH binding sites within IDH heterodimers act in cis or trans to prevent the formation of stable enzyme-inhibitor complexes, restore R-2HG production in the presence of inhibitors, and drive therapy resistance in IDH-mutant AML cells and patients. We therefore uncover a new class of pathogenic mutations and mechanisms for acquired resistance to targeted cancer therapies.

Forward Genetic Screens Identify Mechanisms of Resistance to Small-Molecule Lactate Dehydrogenase Inhibitors

“…Targeted mutagenesis methods such as CRISPR-Cas9 base editing screens or saturation mutagenesis screens could expand the number of known LDHi compound-resistant alleles. 57,60,61 Additionally, our results raise the possibility that LDHB upregulation might be a more common resistance mechanism than compound-resistant mutations. Beyond conferring resistance to diverse LDH inhibitors, upregulation of wildtype LDHB does not involve a trade-off between blocking a drug interaction and preserving enzymatic function.…”

“…Given that NCGC00420737 is a competitive inhibitor, this is not surprising as active-site residues that are required for enzyme function also coordinate compound binding. By contrast, greater mutational diversity can be seen with allosteric inhibitors or other compounds that do not bind enzyme-active sites. , Another possibility is that the mutational spectrum generated by MSH2 loss and MMR deficiency, which favors C > T and T > C substitutions (), , does not fully capture the mutation space that could drive resistance. Targeted mutagenesis methods such as CRISPR-Cas9 base editing screens or saturation mutagenesis screens could expand the number of known LDHi compound-resistant alleles. ,, …”

Forward genetic screens identify mechanisms of resistance to small molecule lactate dehydrogenase inhibitors

“…Given that NCGC00420737 is a competitive inhibitor, this is not surprising as active site residues that are required for enzyme function also coordinate compound binding. By contrast, greater mutational diversity can be seen with allosteric inhibitors or other compounds that do not bind enzyme active sites (38, 56). Another possibility is that the mutational spectrum generated by MSH2 loss and MMR deficiency, which favors C>T and T>C substitutions (https://cancer.sanger.ac.uk/signatures/sbs/) (57, 58), does not fully capture the mutation space that could drive resistance.…”

Viewing AML through a New Lens: Technological Advances in the Study of Epigenetic Regulation

“…Many of these mutations are hotspot (DNMT3A-R882) or gain-of-function mutations (IDH1-R132 and IDH2-R140). Therefore, base editing could be used to specifically introduce these mutations in an endogenous context without completely knocking out or overexpressing the mutant versions of these genes, more faithfully replicating the context in which they occur in leukemia, as recently shown in IDH mutant TF1 cells [ 105 ].…”