Resistance to the Non-Covalent BTK Inhibitor Pirtobrutinib

Pirtobrutinib is a non-covalent BTK inhibitor (BTKi) designed to maintain activity despite the most common resistance mutation to covalent inhibitors, at BTK C481.

To investigate mechanisms of disease progression on pirtobrutinib, we evaluated the effect of pirtobrutinib in vitro on the BCR pathway in pre- and post-treatment patient samples (n=5), and on 2 of these patients, we performed whole exome sequencing longitudinally including prior to and at relapse on pirtobrutinib. Phylogenetics and subclonal dynamics associated with resistance were evaluated using the PhylogicNDT and Concerti tools. To investigate the impact of identified BTK mutations on BCR activation, we generated 6 single and 3 double mutants using site directed mutagenesis and expressed them in the BTK null DT40 B cell line.

We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, reduced CCL3/CCL4 chemokine secretion, as well as effective induction of apoptosis and inhibition of proliferation, in response to pirtobrutinib. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition and cytokine production, with reduced inhibition of proliferation and induction of apoptosis.

In WES analysis, patient #1 had 16 samples evaluated prior to, during, and at relapse on acalabrutinib, vecabrutinib and pirtobrutinib. Clonal analysis of samples collected during acalabrutinib shows steady selection of a clone harboring BTK p.C481S mutation with CCF 92% at relapse but then steadily decreases during pirtobrutinib treatment. Concerti's time-scaled phylogenetic tree shows the birth of a new clone containing the BTK gatekeeper mutation, p.T474I, during acalabrutinib treatment, which then grows rapidly under pirtobrutinib treatment, taking over nearly the entire cancer cell population and replacing the prior p.C481S clone. This complete clonal shift during pirtobrutinib treatment suggests that pirtobrutinib effectively inhibits the p.C481S clone, while the p.T474I gatekeeper clone is likely driving resistance in this patient. We also observed an additional gatekeeper clone BTK p.T474L develop at low levels, as well as another previously undescribed BTK mutation at p.M477I. Manual inspection showed that BTK mutations p.M477I and p.T474I are in cis and therefore in the same clone.

Patient #2 had 10 samples evaluated prior to, during and at relapse on ibrutinib and pirtobrutinib. During ibrutinib therapy, we observed a steady increase in a clone with TP53 p.S240G and SF3B1 p.K666N mutations, reaching CCFs >40% at relapse on ibrutinib. We also noted a significant increase in BTK p.C481R (CCF 33%), BTK p.C481S (CCF 11%) and TP53 p.R196* (CCF 5%) at progression on ibrutinib. Under pirtobrutinib treatment the clone carrying BTK p.C481R decreased to CCF 20%, while BTK p.C481S (28%) and TP53 p.R197* (35%) both increased. Concerti's phylogenetic tree captures the birth of a resistant clone, harboring BTK p.L528W which significantly increases to CCF 30% at progression on pirtobrutinib.

Functional characterization of the identified BTK mutations demonstrated that only T474I, T474L and C481S mutants showed adequate kinase activity, while all the other mutants including M477I, C48IR and L528W essentially lacked kinase activity as judged by phosphorylation at BTK Y223 and at PLCG2 Y753. As expected, the C481S variant was resistant to ibrutinib, but not to pirtobrutinib, while the T474I/L variants were sensitive to ibrutinib but resistant to pirtobrutinib. Interestingly, phosphorylation of AKT and ERK were retained downstream, even with mutations that failed to activate proximal BCR signaling. Furthermore, phosphorylation of AKT and ERK was also observed in the B7.10 cell line which lacks endogenous BTK, demonstrating significant activation of these pathways independent of BTK.

In this study, we demonstrate that ex vivo efficacy of pirtobrutinib declines as patients’ CLL starts to progress, in concert with the development of gatekeeper and alternative site BTK mutations that lead to resistance to pirtobrutinib. Interestingly, many of the second-site BTK mutations fail to activate BTK phosphorylation but are still associated with downstream activation of phospho-AKT and phospho-ERK; the mechanism of this activation remains to be elucidated.

Utro:IBM: Current Employment. Kim:Multiple Myeloma Research Foundation: Research Funding; LabCorp: Consultancy. Rhrissorrakrai:IBM: Current Employment. Levovitz:IBM: Current Employment. Davids:Adaptive Biotechnologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ascentage Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; TG Therapeutics: Consultancy, Research Funding; Eli Lilly and Company: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Research Funding; Novartis: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Research to Practice: Honoraria; Ono Pharmaceuticals: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem: Consultancy, Research Funding. Leshchiner:PACT Pharma Inc: Consultancy; ennov1 LLC: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Nord Bio, Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Nord Bio, Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Parida:IBM: Current Employment. Getz:MSMuTect: Patents & Royalties; SignatureAnalyzer-GPU: Patents & Royalties; MSIDetect: Patents & Royalties; MSMutSig: Patents & Royalties; Scorpion Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Founder; IBM: Research Funding; Pharmacyclics: Research Funding; POLYSOLVER: Patents & Royalties. Brown:Bristol-Myers-Squibb/Juno/Celgene: Consultancy; Eli Lilly: Consultancy, Research Funding; Sun: Research Funding; iOnctura: Consultancy; Hutchmed: Consultancy; Janssen: Consultancy; Grifols: Consultancy; Abbvie: Consultancy; Beigene: Consultancy, Research Funding; Pharmacyclics: Consultancy; MEI Pharma: Consultancy, Research Funding; TG Therapeutics: Research Funding; Catapult: Consultancy; SecuraBio: Research Funding; Genentech/Roche: Consultancy; Gilead: Research Funding; Acerta/ Astra-Zeneca: Consultancy; Pfizer: Consultancy.