BCR::ABL1 Kinase N-lobe Mutants Confer Moderate to High Degrees of Resistance to Asciminib.

Secondary kinase domain mutations in BCR::ABL1 represent the most common cause of resistance to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia patients. The first five approved BCR::ABL1 TKIs target the ATP-binding pocket. Mutations confer resistance to these ATP-competitive TKIs and those approved for other malignancies by decreasing TKI affinity and/or increasing ATP affinity. Asciminib, the first highly active allosteric TKI approved for any malignancy, targets an allosteric regulatory pocket in the BCR::ABL1 kinase C-lobe. As a non-ATP-competitive inhibitor, the activity of asciminib is predicted to be impervious to increases in ATP affinity. Here we report several known mutations that confer resistance to ATP-competitive TKIs in the BCR::ABL1 kinase N-lobe that are distant from the asciminib binding pocket yet unexpectedly confer in vitro resistance to asciminib. Among these is BCR::ABL1 M244V, which confers clinical resistance even to escalated asciminib doses. We demonstrate that BCR::ABL1 M244V does not impair asciminib binding, thereby invoking a novel mechanism of resistance. Molecular dynamics simulations of the M244V substitution implicate stabilization of an active kinase conformation through impact on the -C helix as a mechanism of resistance. These N-lobe mutations may compromise the clinical activity of ongoing combination studies of asciminib with ATP-competitive TKIs.

Cysteine Oxidation in Proteins: Structure, Biophysics, and Simulation.

Cysteine side chains can exist in distinct oxidation states depending on the pH and redox potential of the environment, and cysteine oxidation plays important yet complex regulatory roles. Compared with the effects of post-translational modifications such as phosphorylation, the effects of oxidation of cysteine to sulfenic, sulfinic, and sulfonic acid on protein structure and function remain relatively poorly characterized. We present an analysis of the role of cysteine reactivity as a regulatory factor in proteins, emphasizing the interplay between electrostatics and redox potential as key determinants of the resulting oxidation state. A review of current computational approaches suggests underdeveloped areas of research for studying cysteine reactivity through molecular simulations.

Protomer alignment modulates specificity of RNA substrate recognition by Ire1.

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs-non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1's RNase activity, which is either stringently splice-site specific or promiscuous (Li et al., 2018). Here, we developed an assay that reports on Ire1's RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain's dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory, or High-Risk Leukemias: A Report from the LEAP Consortium.

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. SIGNIFICANCE: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations.See related commentary by Bornhauser and Bourquin, p. 1322.This article is highlighted in the In This Issue feature, p. 1307.

The Synthesis and Structural Requirements for Measuring Glucocorticoid Receptor Expression In Vivo with (±)-11C-YJH08 PET.

Noninvasive methods to study glucocorticoid receptor (GR) signaling are urgently needed to elaborate the complexity of GR signaling in normal physiology and human disorders, as well as to identify selective GR modulators to treat diseases. Here, we report evidence supporting translational studies with (±)-11C-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]-quinoline ((±)-11C-YJH08), a radioligand for PET that engages the ligand binding domain on GR. Methods: (±)-11C-YJH08 was synthesized by reacting the phenol precursor with 11C-methyl iodide. The biodistribution was studied in vivo. Specific binding was tested in vivo with adrenalectomy and ligand competition. A library of analogs was synthesized and studied in vitro and in vivo to understand the (±)-11C-YJH08 structure-activity relationship. Rodent dosimetry studies were performed to estimate the human-equivalent doses of (±)-11C-YJH08. Results: (±)-11C-YJH08 was synthesized by reaction of the phenolic precursor with 11C-methyl iodide, giving a radiochemical yield of 51.7% ± 4.7% (decay-corrected to starting 11C-methyl iodide). Specific binding was observed in many tissues, including the brain. An analysis of the (±)-YJH08 structure-activity relationship showed that (R)- and (S)-enantiomers are equally avid for GR by occupying discrete binding modes. A focused chemical screen revealed that the aryl fluoride motif on YJH08 is essential for high-affinity GR binding in vitro, high tissue uptake in vivo, and efficient passage across the blood-brain barrier. Lastly, we performed dosimetry studies on rodents, from which we estimated the human-equivalent doses of (±)-11C-YJH08 to be commensurate with the widely used 11C and 18F tracers. Conclusion: These studies reveal the molecular determinants of a high-affinity and high-selectivity ligand-receptor interaction and support the use of (±)-11C-YJH08 PET to make the first measurements of GR expression in human subjects.

ATP-Competitive Inhibitors Midostaurin and Avapritinib Have Distinct Resistance Profiles in Exon 17-Mutant KIT.

KIT is a type-3 receptor tyrosine kinase that is frequently mutated at exon 11 or 17 in a variety of cancers. First-generation KIT tyrosine kinase inhibitors (TKI) are ineffective against KIT exon 17 mutations, which favor an active conformation that prevents these TKIs from binding. The ATP-competitive inhibitors, midostaurin and avapritinib, which target the active kinase conformation, were developed to inhibit exon 17-mutant KIT. Because secondary kinase domain mutations are a common mechanism of TKI resistance and guide ensuing TKI design, we sought to define problematic KIT kinase domain mutations for these emerging therapeutics. Midostaurin and avapritinib displayed different vulnerabilities to secondary kinase domain substitutions, with the T670I gatekeeper mutation being selectively problematic for avapritinib. Although gatekeeper mutations often directly disrupt inhibitor binding, we provide evidence that T670I confers avapritinib resistance indirectly by inducing distant conformational changes in the phosphate-binding loop. These findings suggest combining midostaurin and avapritinib may forestall acquired resistance mediated by secondary kinase domain mutations. SIGNIFICANCE: This study identifies potential problematic kinase domain mutations for next-generation KIT inhibitors midostaurin and avapritinib.