The development and regulatory approval of covalent Bruton tyrosine kinase (BTK) inhibitors changed the treatment landscape for chronic lymphocytic leukemia (CLL). Unfortunately, in many patients treated with BTK inhibitors, their disease will eventually develop resistance.
The currently available BTK inhibitors bind covalently to cysteine 481 (C481). Resistance to BTK inhibitors occurs predominantly by mutations in BTK at C481, affecting the binding ability of the drugs, or by mutations in the downstream target PLCG2.
Two studies published in Blood Advances explored possible mechanisms of resistance to BTK inhibitors to provide better options for patients.
Relevance of Gene Mutations
A study by Paolo Ghia, MD, of Università Vita-Salute San Raffaele in Milan, and colleagues looked at the frequency and relevance of BTK and PLCG2 mutations in association with resistance to the BTK inhibitor ibrutinib.1
Dr. Ghia and colleagues conducted a multicenter retrospective study of 98 patients with CLL who had been treated with ibrutinib: half relapsed after initial response and half were still responding after one year of treatment. The researchers used next-generation sequencing (NGS) to look at 13 CLL-relevant genes, including BTK and PLCG2.
“While the presence of BTK and/or PLCG2 mutations was a common finding in previously published smaller series, one-third of patients with CLL relapsing on ibrutinib in our cohort do not carry BTK [or] PLCG2 mutations, even with a 0.1% sensitivity,” Dr. Ghia said.
NGS showed that about two-thirds (65%) of the patients who relapsed had at least one hotspot BTK or PLCG2 mutation; these were also identified in 12% of responding patients.
Additionally, Dr. Ghia and colleagues identified “additional genomic aberrations and related mechanisms, such as del(8p), EGR2, and NF-κB pathway mutations that may be cooperating in determining progression on ibrutinib.”
Excluding BTK and PLCG2, the most commonly mutated genes at progression in patients who relapsed were TP53 (59%), ATM (29%), EGR2 (20%), SF3B1 (18%), NOTCH1 (16%), and BIRC3 (13%).
“In particular, BIRC3 and NFKBIE mutations were detected in cases without BTK mutations, while an enrichment of del(8p) was identified in relapsed cases carrying mutated BTK,” Dr. Ghia said. “These findings may prompt clinical trials exploiting biomarker-guided treatment strategies with novel inhibitors targeting differential signaling pathways based on the mutation pattern.”
The researchers used an extended capture-based panel and found that only BRAF and IKZ3 mutations had a predominance in patients who relapsed and who were enriched for del(8p). No difference in TP53 mutation burden was found for patients with BTK mutation compared with wild-type BTK.
Dr. Ghia said that the optimal use of this targeted NGS approach remains to be established before it can be applied in clinical practice.
Acquired BTK Mutations
In another study, Stephan Stilgenbauer, MD, of the Comprehensive Cancer Center in Ulm, Germany, and colleagues explored acquired BTK mutations associated with resistance to non-covalent BTK inhibitors.2 Using the mantle cell lymphoma (MCL) cell line, researchers implemented a long-term in vitro dose-escalation method to create resistance to ibrutinib and pirtobrutinib, vecabrutinib, nemtabrutinib, fenebrutinib, and RN-486.
They found that “mutations clustered in the protein kinase domain and affecting protein function are a general phenomenon associated with resistance to different non-covalent BTK inhibitors,” Dr. Stilgenbauer said.
Further, he noted, a number of next-generation, highly selective, and reversible BTK inhibitors are being developed as follow-up therapies to overcome resistance to covalent inhibitors, as well as for treatment of hematologic neoplasms in the first-line and relapsed or refractory settings.
“It is of high importance to characterize mechanisms that mediate resistance to these next generation BTK inhibitors to identify subsequent treatment strategies,” Dr. Stilgenbauer said.
To do that, he and colleagues used a long-term in vitro dose-escalation method to generate resistance to ibrutinib and several non-covalent BTK inhibitors using the REC-1 MCL cell line. Cell lines were analyzed using targeted NGS to identify acquired mutations to BTK and PLCG2.
All six lines treated with long-term ibrutinib gained the BTK C481F mutation. In those cell lines that were resistant to the non-covalent BTK inhibitors, six mutations in BTK and three mutations in PLCG2 were found.
Looking at patterns for specific inhibitors, the researchers found that four of six vecabrutinib-resistant lines acquired BTK G409R mutations and one acquired a PLCG2 L845F mutation. In cell lines resistant to fenebrutinib, three BTK mutations were found: L528S, G480R, and D539H. Pirtobrutinib-resistant cell lines acquired a BTK A428D mutation. RN-486-resistant cell lines acquired BTK G480R and V416L mutations.
“All of the newly identified mutations negatively affected the kinase activity of BTK,” Dr. Stilgenbauer said. “The novel BTK mutants showed cross-resistance to other covalent and non-covalent BTK inhibitors.”
Dr. Stilgenbauer noted that nemtabrutinib-resistant cell lines did not acquire BTK or PLCG2 mutations – indicating other mechanisms of resistance – and nemtabrutinib showed the highest efficacy across the different BTK mutant cell lines.
“Primary resistance might be observed in the context of single-agent non-covalent BTK inhibitor usage as follow-up treatment in cases harboring the kinase-domain BTK mutations,” Dr. Stilgenbauer said. “Hence, in diagnostics, it is essential to sequence either all exons or at least the kinase domain (Exon 13-19) of BTK in the context of progression under BTK [inhibitor] therapies.”
Based on additional experimental data, the researchers suggested combination treatments with BCL2 inhibitors in a clinical trial setting are warranted to understand whether such treatments would limit generation of resistance.
In the study, the researchers noted their results could be limited by the use of an in vitro system, but the “long-term dose-escalation REC-1 model has proven to be efficient in both recapitulating BTK mutations that have been previously reported (three out of five) in the context of in vivo pirtobrutinib resistance and in identifying novel mutations.”
Alternative signaling pathways may represent viable targets for overcoming resistance to non-covalent BTK inhibitors.
Any conflicts of interest declared by the authors can be found in the original articles.
References
- Bonfiglio S, Sutton L-A, Ljungström V, et al. BTK and PLCG2 remain unmutated in one third of patients with CLL relapsing on ibrutinib [published online, 2023 Jan 25]. Blood Adv. doi: 10.1182/bloodadvances.2022008821.
- Qi J, Endres S, Yosifov DY, et al. Acquired BTK mutations associated with resistance to non-covalent BTK inhibitors [published online, 2023 Jan 20]. Blood Adv. doi: 10.1182/bloodadvances.2022008955.
