A Novel BCR::ABL1 Rearrangement Harboring the Gatekeeper Mutation Drives Hyper-Kinase Activity Conferring Resistance to Ponatinib and Asciminib Combination Therapy
Authors: Nardi Valentina , Schwieterman Joshua , Kincaid Zachary , Brunner Andrew M. , Reyes Maria , Griffin Gabriel K. , Ansari Sekhu , Azam Mohammad 
Published: December 2024

Targeted inhibition of BCR::ABL1 by tyrosine kinase inhibitors (TKIs) has demonstrated remarkable clinical efficacy in chronic myeloid leukemia (CML) and Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). However, the emergence of resistant mutations undermines treatment response. These resistant mutants prevent drug binding either by steric blockade or by altering the conformational dynamics, called as conformational escape variants. Notably, the gatekeeper mutation (T315I) confers resistance through both mechanisms. To address this challenge, a combination therapy of ponatinib, which is effective against T315I, and asciminib, which stabilizes ABL1 in its inactive conformation, has demonstrated enhanced efficacy in patients with refractory or blast phase (BP) CML. Unexpectedly, resistance to this combination therapy has been observed. Here, we report the emergence of a novel BCR::ABL1 B6A3 rearrangement in a patient treated with ponatinib and asciminib combination, demonstrating pan-resistance to all clinical ABL1 inhibitors, both as monotherapy and in combination. This case underscores the need for ongoing surveillance and development of novel therapeutic strategies to overcome resistance mechanisms in BCR::ABL1-driven leukemias.
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.