Pirtobrutinib targets BTK C481S in ibrutinib-resistant CLL but second-site BTK mutations lead to resistance

Abstract

Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKi) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, non-covalent BTK inhibitor with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and CLL patient cells we show that pirtobrutinib potently inhibits BTK-mediated functions including BCR signaling, cell viability and CCL3/CCL4 chemokine production in both BTK wild-type (WT) and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability and cytokine production. We employed longitudinal whole exome sequencing on two patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to non-covalent BTKi.

Curing CLL: one clone at a time

“…Two major conclusions emerge from the present article. 1 First, consistent with a prior report, 6 the authors provide data that BTK WT (wild-type BTK) B-cell lines were sensitive to pirtobrutinib and cBTKi. Pirtobrutinib was also effective in mitigating apical (BTK autophosphorylation) and downstream (extracellular-signal-regulated kinase [ERK] phosphorylation) signaling in BTK WT and BTK C481S HEK293 cells.…”

“…In contrast, the MEC-1 line overexpressing BTK WT , BTK C481S , or BTK C481R demonstrated inhibition of the BCR signaling cascade and biological consequences for a catalytically active C481S mutant. 6 Collectively these studies 1 , 6 preclinically establish pirtobrutinib’s functionality in BTK WT and BTK C481S cells.…”

“…In this issue of Blood Advances , Naeem et al 1 provide evidence that pirtobrutinib targets ibrutinib-resistant chronic lymphocytic leukemia (CLL) that harbors pathological Bruton’s tyrosine kinase (BTK) C481S-mutant. However, concomitant with disease progression (DP), non-C481 variants of BTK with or without gatekeeper T474 mutants have emerged, rendering pirtobrutinib ineffective in mitigating the B-cell receptor (BCR) signaling cascade.…”

Kinase-impaired BTK mutations are susceptible to clinical-stage BTK and IKZF1/3 degrader NX-2127

“…Although both covalent and noncovalent BTK inhibitors offer therapeutic benefits, it is now clear that acquired mutations in BTK outside of the C481 residue can confer resistance to noncovalent inhibitors and that a portion of these mutations also cause cross-resistance to certain covalent BTK inhibitors (5,(19)(20)(21)(22). Surprisingly, some BTK drug resistance mutations appear to reduce BTK Y223 autophosphorylation (5).…”

Mutations Detected in Real World Clinical Sequencing during BTK Inhibitor Treatment in CLL

“…Case #6 had a large clone of T474I with VAF of 71.8% at progression after 16 months of PIR treatment, but this patient already had this clone (T474I VAF 71.8%) 5 months prior to progression, although it had not been seen before PIR treatment. Detailed analyses of cases 1 and 5 were already presented in our previous paper [4].…”