Session: 632. Chronic Myeloid Leukemia: Clinical and Epidemiological: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research, Clinical Research, Health outcomes research
Methods: BCR::ABL1 transcripts were detected using Q-RT-PCR and clinical targeted RNA sequencing assays with anchored multiplex PCR and amplicon-based enrichment. DNA sequencing with Oxford Nanopore MinION identified breakpoints in ABL1 and BCR. BaF3 and K562 cell lines were engineered to express various BCR::ABL1 isoforms, including BCR::ABL1b13a2, BCR-::ABL1b13a2/T315I, BCR::ABL1b13a2/T315M, BCR::ABL1b6a3, BCR::ABL1b6a3/T315I, BCR::ABL1b6a3/T315M, and BCR::ABL1b6a3. These cell lines were subjected to dose-dependent proliferation assays with type-I (bosutinib, dasatinib, VX680, and BIRB796), type-II (imatinib, nilotinib, ponatinib, and ribastinib), and type-IV (asciminib) inhibitors. The new chimeric protein structures, with and without gatekeeper mutations, were modeled using Swiss-Model.
Results. An 74 yo patient with CML harboring the BCR::ABL1b13a2 rearrangement was initially treated with imatinib, and subsequently dasatinib due to GI toxicity. He progressed to blast phase (BP), B-acute lymphoblastic leukemia (B-ALL). Subsequently patient developed resistance by acquiring T315I mutation. Patient was switched to ponatinib and asciminib combination treatment that resulted in deep molecular remission (down to 0.0125%, BCR::ABL1b13a2/T315I). After eight months of treatment patient developed and succumbed to disease. Targeted RNA sequencing of the bone marrow sample revealed a novel BCR::ABL1 transcript from the rearrangement of BCR exon 6 and ABL1 exon 3 (BCR::ABL1b6a3) with persistent T315I mutation, while the BCR::ABL1 b13a2/T315I transcript was absent. Long read DNA sequencing confirmed the novel DNA rearrangement with breakpoints in ABL1 intron 3 and BCR intron 6. Structural modeling suggested that deletion of critical SH3 domain residues, which stabilize the SH2-kinase-linker at the kinase N-lobe, may cause hyperactivation by locking the SH2 domain at the N-terminus of the kinase domain. Biochemical analysis of BCR::ABL1b6a3 from the HEK293T and BaF3 cells revealed a 4-5 hold kinase activity than BCR::ABL b13a2, confirming in silico structural prediction. and growth factor-independent proliferation. Dose-dependent inhibitor assays revealed pan-resistance to type-II (imatinib, nilotinib, ponatinib, and ribastinib) and type-IV (asciminib) inhibitors, alone and in combination. Resistance conferred by the BCR-ABL1b6a3/T315I variant was 20-fold higher than BCR-ABL1b6a3, providing direct evidence for treatment failure.
Conclusions. We report a novel BCR::ABL1 rearrangement involving BCR exon 6 and ABL1 exon 3 following treatment with combination therapy of ponatinib and asciminib. This unique variant confers resistance to all known type-II and type-IV inhibitors, alone and in combination and may contribute to disease progression to blast phase. Interestingly, BCR::ABL1b6a3 showed increased sensitivity to type-I inhibitors, yet all clinical type-I inhibitors are ineffective against T315I and other gatekeeper variants. Therefore, developing gatekeeper-selective type-I inhibitor is crucial to overcome clinical resistance.
Disclosures: Brunner: Servier: Consultancy; AstraZeneca: Research Funding; Geron: Consultancy; Takeda Oncology: Consultancy, Research Funding; Rigel Pharmaceuticals: Consultancy; Novartis: Consultancy, Research Funding; Lava Therapeutics: Consultancy; Agios: Consultancy; BMS: Consultancy, Research Funding; i-Mab Biopharma: Consultancy; Keros Therapeutics: Consultancy.
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