Curing CLL: one clone at a time

Comment on Naeem et al, page 1929

In this issue of Blood Advances, Naeem et al¹ 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.

The BCR pathway is integral to survival, activation, proliferation, and mobilization of both normal and malignant B cells. An apical and pivotal node of the BCR pathway is BTK, which is successfully blocked by treatment with ibrutinib, an oral agent that has demonstrated clinical potency for overall and progression-free survival in patients with CLL. Importantly, patients belonging to a hitherto poor-prognoses group also benefited from this molecule^2,3 with limited untoward toxicity. Targeting BTK is a remarkable and revolutionary step in the treatment of CLL. The success of ibrutinib, a desire to reduce its adverse effects and increase its bioavailability, bolstered the synthesis and testing of more selective second-generation BTK inhibitors (BTKi): acalabrutinib and zanubrutinib.

A commonality of these BTKi is their covalent and hence irreversible binding to a cysteine (C481) residue. This exclusive tethering to C481 is also the culprit for the development of the most common treatment resistance mechanism (replacement of cysteine by an S or R amino acid). Although researchers have identified other substitutions of the C site, gatekeeper mutations of T474 in BTK, or the resistance variant PLCG2 in the BCR pathway, acquired C481S is the most prevalent and catalytically active mutation. This mutation collaborates with T474 variants to result in a super-resistant phenotype.⁴ The relentless presence of CLL cells in patients while undergoing chronic cBTKi-based therapy acts as a further catalyst for the development of mutant variants.

These observations and the development of aggressive disease due to pathological C481 mutations were instrumental in the design of noncovalent BTKi. Although many biosimilar drugs have been created and entered clinical use, pirtobrutinib is the most advanced ncBTKi. In the BRUIN trial, more than 130 patients with CLL⁵ were treated with pirtobrutinib. These patients had been previously treated with cBTKi and were either intolerant to it or had DP.

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. This inhibition was absent or was reduced after ibrutinib treatment. Because HEK293 are not driven by BCR, investigations of their biological effects is not feasible. 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.

Cell line data were strengthened by studies of primary CLL cells in which pirtobrutinib decreased phospho-ERK with a cBTKi-resistant variant.¹ Moreover, with acalabrutinib and ibrutinib BTK autophosphorylation decreased only by 10% and 50%, respectively, in 2 patients with a high (∼90%) BTK^C481S variant allele frequency. Although the number was limited, phospho-ERK, a hallmark signaling molecule, was decreased by pirtobrutinib in all 3 responding patients' cells. Another biomarker, plasma CCL4, rapidly declined within 3 months of pirtobrutinib use but increased at DP.¹ The CCL3/CCL4 chemokines exhibited similar alterations in 20 subjects with CLL.⁶ Collectively, these studies^1,6 demonstrated pirtobrutinib’s effectiveness in inhibiting BCR signaling in both patients with BTK^C481S and patients with BTK^WT CLL and the clinical potency in both cohorts was demonstrated in the BRUIN trial.⁵ 

The second major component of this article is the dissection of molecular events that occurred during DP in 2 patients receiving pirtobrutinib.¹ Compared to baseline, cells obtained at the time of DP were more resistant to apoptosis and had reactivated BCR signaling. In the first patient, acquisition of a multiclonal second site BTK mutation and a significant decline in BTK^C481S (from 95% to 2%) was observed after 12 months of therapy. In contrast, the gatekeeper BTK mutant increased from 5% to 99%, in addition to the acquisition of T474L (5%) and M477I (2%). In the second patient, the rate of the BTK L528W mutation increased from 2% to 30% after 8 months of pirtobrutinib. Although studied only in 2 patients, these data provide clinical evidence for the acquisition of alternative-site BTK mutations that lead to DP. These carefully curated, longitudinal sequencing data expand the knowledge that was elegantly reported for 7 patients with CLL in the BRUIN trial, in which the V416L, A428D, M437R, T474I, and L528W mutations clustered in the kinase domain were reported.⁷ 

Among the 26 patients from the BRUIN trial treated at MD Anderson, 50% had DP on pirtobrutinib.⁸ Notably, 85% of those who had DP had mutant BTK and 15% had wild-type BTK at the start of pirtobrutinib. The median time to progression was 14 cycles for the mutant cohort and 22 cycles for the wild-type cohort.⁹ These data implicate that the existence of any BTK mutation imposes an increased risk and velocity of progression during treatment with pirtobrutinib. However, not all patients with disease progression exhibited clonal BTK evolution, with the disappearance of the original clone. This suggests that other genetic or epigenetic alterations may be responsible for DP. Patients with DP while receiving pirtobrutinib have limited therapeutic options. Ex vivo pharmacological profiling of CLL cells at DP or during pirtobrutinib-based therapy showed that venetoclax and APR-246 (a glutathione modulator) were the most effective agents alone and in combination with BTKi.⁹ Several key therapeutic questions have emerged from these observations. Does the disease become more sensitive to cBTKi when the C481S clone is abolished? Should we start treatment with pirtobrutinib in patients who are BTKi-naïve? Should we test a combination of cBTKi and ncBTKi to prevent or postpone the development of pathological mutant BTK clones? Should pirtobrutinib be combined with venetoclax based on ex vivo profiling data and the clinical success of ibrutinib and venetoclax couplets?⁸ Should we become informed of the best regimen based on genomic analyses and pharmacological profiling?

The clinical success and failure of treatment with cBTKi followed by ncBTKi have opened a new era for CLL therapy (see figure). Treatment with these reversible and irreversible BTKi is accompanied by the development of novel clones, clonal expansion, and DP, necessitating innovative therapeutic strategies. Furthermore, to treat CLL, orthogonal approaches from a variety of perspectives are needed to identify the next cohort of drugs that can tackle new clones. Alternatively, combination strategies that target complementary and competing pathways may succeed in preventing development of mutant clones.¹⁰ 

Conflict-of-interest disclosure: V.G. has sponsored research agreements from Pharmacyclics, AbbVie, Acerta, Sunesis, and Loxo Oncology. The remaining authors declare no competing financial interests.