Abstract 6499: Targeting BCR-ABL1-independent mechanisms of resistance in chronic myeloid leukemia

Abstract Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion gene in chronic myeloid leukemia (CML) have improved patient prognosis. While newer drugs have made inroads against BCR-ABL1-dependent TKI resistance, patients who develop resistance through pathways independent of BCR-ABL1 feature heterogeneous, poorly characterized molecular mechanisms and present a challenge for application of combination targeted therapy. To identify genes contributing to BCR-ABL1-independent resistance, we performed CRISPR/Cas9 genome-wide screens using Ba/F3 BCR-ABL1 cells cultured in the presence of DMSO, imatinib, and asciminib. After 10 days, cells were harvested and analyzed for gene-level enrichment/depletion of sgRNAs. Among candidate resistance genes (whose knockdown was enriched following TKI treatment), independent sgRNA guides for five genes were induced by lentiviral CRISPR/Cas9 knockout in Ba/F3 BCR-ABL1 cell line models for validation studies. Cell lines were evaluated for in vitro sensitivity to a panel of approved ABL1 TKIs and profiled against an expanded inhibitor panel spanning a range of drug targets. Expression levels of candidate resistance genes were compared by RNAseq in primary specimens from CML patients. CRISPR screening revealed varying subsets of genes enriched in TKI-treated cultures. Five candidate genes were selected for validation studies: Chic2, Stub1 and Pten from the imatinib-treated cells and Fbxo3 and Ptar1 from the asciminib-treated cells. In Ba/F3 BCR-ABL1 cell line models, knockout of each of these genes resulted in varying degrees of reduced sensitivity to a panel of ABL1 TKIs (2-45-fold increase in IC50 compared to wild-type cells). For example, knockout of Pten and Chic2 demonstrated increased IC50 values for imatinib of 12,714 and 914 nM, respectively, compared to 277 nM for wild-type cells. Profiling of cell lines using an expanded drug panel revealed differential sensitivities to inhibitors targeting multiple pathways. For example, Chic2, Stub1 and Fbxo3 knockout showed greater sensitivity (relative to wild-type cells) to inhibitors of WNT, proteasome, histone deacetylase, bromodomain, and/or nucleoside analog pathways. Lastly, analysis of RNASeq data highlighted differences in expression levels in patient samples. For example, expression levels of both Ptar1 and Pten were reduced in patients with BCR-ABL1-independent resistance to imatinib relative to newly diagnosed patients, and expression of Fbxo3 and Chic2 were decreased with disease progression (blast vs chronic phase). Taken together, our results identify genes implicated in tumor suppression (Pten), ubiquitination (Stub1, Fbxo3, Chic2), and post-translational modification (Ptar1) with contributing roles for BCR-ABL1-independent TKI resistance and map these to potential actionable pathways amenable to novel combination targeted therapy approaches. Citation Format: Mark Pusung, Christopher A. Eide, Jessica Gibbs, Daniel Bottomly, Haijiao Zhang, Brian J. Druker. Targeting BCR-ABL1-independent mechanisms of resistance in chronic myeloid leukemia abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6499.