Resistance to Noncovalent BTK Inhibitors Raises Questions Around Impact of Mutations in CLL

A small proportion of a subset of patients with chronic lymphocytic leukemia acquired mechanisms of genetic resistance to the novel noncovalent BTK inhibitor pirtobrutinib, according to results from a genomic analysis of patients in the phase 1/2 BRUIN trial.

A small proportion of a subset of patients with chronic lymphocytic leukemia (CLL) acquired mechanisms of genetic resistance to the novel noncovalent BTK inhibitor pirtobrutinib (LOXO-305), according to results from a genomic analysis of patients in the phase 1/2 BRUIN trial (NCT03740529).1,2

Nine of 55 patients examined developed resistance to pirtobrutinib. Investigators found that all 9 harbored BTK or PLCγ2 mutations. Resistance can arise through multiple mechanisms, including at residue C481, the binding site of covalent BTK inhibitors. C481S, specifically, is tied to resistance to these agents.

However, 7 patients developed newly acquired, non-C481 BTK mutations, including V416L, A428D, M437R, T474I, and L528W. These mutations, clustered in the kinase domain of BTK, conferred resistance to both noncovalent BTK inhibitors and certain covalent BTK inhibitors. The remaining 2 patients had persistence of PLCγ2 mutations that were identified at baseline.

“The exact mutation in BTK dictates which drug they might be resistant to,” study co-author Justin W. Taylor, MD, assistant professor, lab head/PI, Sylvester Comprehensive Cancer Center, University of Miami Health System, said in an interview with OncLive®. “For a long time, the only BTK mutations that we had to worry about were the C481 mutations. Now, we have other mutations, and each of them are slightly different.”

BTK inhibitors have become a staple in the treatment of multiple B-cell cancers, including CLL, in recent years, though acquired mutations and other factors can lead to drug resistance. The first generation of BTK inhibitors centered around covalent, or irreversible, agents. Next-generation noncovalent, or reversible, BTK inhibitors, such as pirtobrutinib, have been found to overcome these mutations and other sources of resistance.3

However, investigators do not yet understand new mechanisms of resistance surrounding noncovalent BTK inhibitors, prompting this genomic analysis of a subset of patients from the BRUIN trial.

BRUIN investigated pirtobrutinib in patients with relapsed or refractory B-cell cancers. Following the phase 1 dose-escalation study of pirtobrutinib monotherapy in 28-day cycles, patients were administered 200 mg of pirtobrutinib once daily in the phase 2 study.

Eligibility criteria for BRUIN included patients with CLL who had at least 2 prior lines of therapy, though this was later lowered to 1 prior line of therapy if it included a covalent BTK inhibitor. Responses were assessed by the investigators in accordance with the criteria from the 2018 International Workshop on Chronic Lymphocytic Leukemia.

Taylor and his colleagues conducted this correlative analysis as part of a separate biospecimen protocol using specimens obtained from patients who were enrolled on the BRUIN trial at Memorial Sloan Kettering Cancer Center. Investigators collected specimens before pirtobrutinib treatment and at the time of the occurrence of resistance to pirtobrutinib.

Investigators analyzed specimens from the 9 patients who developed resistance to pirtobrutinib using targeted next-generation sequencing with the MSK-IMPACT Heme/HemePACT platform. They also analyzed single-cell DNA sequencing with the Mission Bio Tapestri platform on specimens from 2 of the 9 patients.

To validate the resistance mutations, BTK and PLCG2 plasmids were transduced into TMD8 and OCI-LY10 cells. Cells expressing mutant or control alleles were used for competition assays, cell-viability assays, immunoblots, and intracellular calcium-release assays.

Of the 55 patients evaluable for the analysis, 12 had discontinued therapy because of progressive disease, 38 continued to receive therapy, and 5 had discontinued therapy for reasons other than disease progression. One of the 12 patients who developed disease progression was excluded from the analysis due to a short duration on treatment. Two patients did not have specimens available.

In the first 2 patients to experience disease progression of the 9 analyzed for pirtobrutinib resistance, sequencing revealed V416L and M437R mutations in the kinase domain of BTK outside the C481 residue that were not present at baseline. Additionally, mutations in the BTK kinase domain were observed at T474I and L528W in 2 and 4 patients, respectively. Notably, 1 patient had both T474I and L528W mutations at progression.

The 7 patients with newly acquired, non-C481 BTK mutations after progression on pirtobrutinib treatment had discontinued prior ibrutinib (Imbruvica) treatment due to disease progression. Four patients had preexisting BTK C481 mutations, and pirtobrutinib suppressed BTK C481 clones in 2 patients. Those patients developed new non-C481 BTK mutations after developing resistance to pirtobrutinib.

Investigators next conducted single-cell mutational analysis of BTK, PLCG2, and 29 additional genes commonly mutated in CLL on specimens collected from 2 patients before they received pirtobrutinib and at disease progression. Both patients had BTK L528W mutations identified at progression during pirtobrutinib treatment.

The BTK L528W mutation was not found in the baseline specimen for 1 patient but was discovered in 2 subclones after progression, which included a PLCγ2 mutation. In the other patient, pirtobrutinib suppressed the C481S mutation. However, investigators discovered L528W at progression, as well as pathogenic mutations that had previously coexisted with C481S.

The study authors noted that the V416L, A428D, M437R, and L528W mutations confer resistance to many noncovalent BTK inhibitors in clinical development, including nemtabrutinib (MK-1026; formerly ARQ 531), fenebrutinib, and vecabrutinib (SNS-062), plus many existing covalent BTK inhibitors.

Study authors also noted data from the mutational analysis suggest the need for new therapeutic approaches to overcome the newly described BTK inhibitor resistance mechanisms. However, they stressed the analysis focused on a small sample of patients who experienced resistance to pirtobrutinib.

“I do not want people to take home that these resistance mutations mean that [pirtobrutinib] is not going to be effective, because this was a subset of patients,” Taylor said. “The drug still is likely going to be very effective, just as ibrutinib has been very effective, even though we see resistance mutations [in those patients]. But these are things to be aware of, and now that we are doing more genetic sequencing of tumor samples, these might be seen in reports on patients.”


  1. Wang E, Mi X, Thompson MC, et al. Mechanisms of resistance to noncovalent Bruton's tyrosine kinase inhibitors. N Engl J Med. 2022;386(8):735-743. doi: 10.1056/NEJMoa2114110
  2. New England Journal of Medicine Study Reveals Potential Vulnerabilities in Emerging Cancer Therapy. University of Miami Health. Published February 24, 2022. Accessed March 30, 2022.
  3. Reiff SD, Mantel R, Smith LL, et al. The BTK inhibitor ARQ 531 targets ibrutinib-resistant CLL and Richter transformation. Cancer Discov. 2018;8:1300-1315. doi: 10.1158/2159-8290.CD-17-1409
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