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March 17, 2022 by Brittany Enzmann, PhD 2 min read

Publication Highlight: Mechanisms of Resistance to Noncovalent Bruton’s Tyrosine Kinase Inhibitors

Paper: Wang E., et al. Mechanisms of resistance to noncovalent Bruton’s tyrosine kinase inhibitors. N Engl J Med 2022; 386:735-743DOI: 10.1056/NEJMoa2114110 

Background: B-cell receptor signaling plays a role in B cell malignancies, such as chronic lymphocytic leukemia (CLL). The enzyme, Bruton’s tyrosine kinase (BTK) is a key component of B-cell antigen receptor signaling, and thus an important therapeutic target. The development of covalent (irreversible) Bruton’s tyrosine kinase inhibitors (BTKi), such as ibrutinib, has been a major advance in the treatment of patients with B-cell malignancies and patients who have been receiving those drugs have benefited from them. But a major challenge is that patients will eventually develop resistance to these drugs due to a mutation at the drug-binding site in the BTK enzyme at the cysteine 481 residue (C481).

This has led to the development of noncovalent (reversible) BTK inhibitors that do not require binding to C481. One such inhibitor currently in clinical trials, pirtobrutinib, is not susceptible to resistance through C481 mutations. However, the resistance mechanisms to this drug and other noncovalent BTKi are under investigation.

Question: What are the mechanisms of acquired resistance to the noncovalent BTKi, pirtobrutinib?

Methods: In this study, the team identified 9 of 55 CLL patients had acquired resistance to the noncovalent BTK inhibitor, pirtobrutinib. These patients provided peripheral blood samples for genomic analysis before treatment and at the time of disease progression. To identify resistance-associated mutations, the researchers performed bulk genomic sequencing on samples from the patients. While this method identifies the mutated genes, it does not reveal the co-occurrence of mutations within cells (clonal architecture) or how the subclones evolved during the treatment period.

To investigate the evolution of subclones during treatment, targeted single-cell DNA sequencing (scDNA-seq) was performed using Mission Bio’s Tapestri Platform on samples taken from 2 patients before treatment and at relapse. Single-cell DNA sequencing identified the clonal structure and evolution of relapsed patients over their period of treatment. This allowed the investigators to identify the co-occurring mutations within each subclone, providing a picture of how new subclones evolved from pre-existing ones.

Main Results: This study identified mutations in BTK outside of the cysteine 481 residue that emerged during treatment and caused resistance to pirtobrutinib. Bulk sequencing identified non-C481 mutations in BTK: A428D, T474I, V416L, M437R, and L528W. Mutations in phospholipase C gamma-2 (PLCγ2), a signaling molecule immediately downstream of BTK, were also found in the subclones at the pre-treatment timepoint.

The scDNA-seq analysis revealed that cells with some mutations were present prior to pirtobrutinib treatment and gave rise to other subclones that acquired mutations during treatment. For instance, one patient had a pre-existing PLCγ2 mutation that subsequently gave rise to a subclone with an acquired a BTK L528W mutation during treatment.

Conclusion: This study identified novel mechanisms of acquired resistance to pirtobrutinib, an investigational drug belonging to a new class of noncovalent BTK inhibitors that is currently in clinical development for the treatment of CLL and other B-cell malignancies. Identifying the subclones that emerge over time using scDNA-seq provides insight into the genetic basis of resistance and will inform future research.

Importantly, the authors found that the BTK mutations that conferred resistance to pirtobrutinib also conferred resistance to other noncovalent and covalent BTK inhibitors. This knowledge can be can be used to develop new therapeutic strategies that overcome these resistance mechanisms. 

Check out the paper here!


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