Ewalt, Mark D

Abstract Early-phase clinical trials of Bruton’s tyrosine kinase (BTK) degraders have demonstrated efficacy in patients with BTK inhibitor-resistant chronic lymphocytic leukemia (CLL). How clinical resistance to BTK degraders arises is unknown. Here we sequenced serial CLL samples from patients enrolled in the phase I trials of zelebrudomide and bexobrutideg and observed recurrent expansion of preexisting BTK A428D mutations at relapse. Unlike previously studied BTK inhibitor resistance mutations, BTK A428D conferred pan-resistance to BTK inhibitors and degraders. In the absence of BTK-directed therapies, however, cells bearing BTK A428D exhibited a competitive disadvantage. A crystal structure of BTK A428D revealed that the mutant aspartate clashes with the adenine ring of ATP and the adenine-mimetic moiety of BTK inhibitors and degraders. Combining BTK degraders with venetoclax mitigated the expansion of BTK A428D. These results provide the molecular basis for clinical resistance to BTK degraders and will inform the development of next-generation BTK degrader therapies.

Abstract The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)–JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)–like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.