The enzyme Bruton tyrosine kinase (BTK) is concerned in B-cell signaling that promotes the migration, proliferation, and survival of neoplastic B-cells of sufferers with power lymphocytic leukemia (CLL) [1]. Consequently, medication that block the motion of BTK are extremely efficient and essentially have modified the usual of take care of sufferers with CLL because the therapeutic introduction of the primary BTK inhibitor (BTKi), ibrutinib, in 2013. Ibrutinib, and second-generation BTKi’s, resembling acalabrutinib and zanubrutinib, kind a covalent bond with BTK inside its kinase area at residue C481, inflicting everlasting inactivation of BTK and requiring the leukemia cell to synthesize new enzyme to revive B-cell signaling [2]. A nonsynonymous mutation in BTK at C481, resembling C481S, can block the capability of those medication to kind a covalent bond with BTK, thus conferring medical resistance to ‘covalent’ BTKi’s [3]. This stimulated improvement of ‘non-covalent’ BTKi’s, resembling pirtobrutinib or nemtabrutinib, which can be efficient in treating sufferers with CLL cells that harbor the BTK C481S mutation [4]. Nonetheless, sufferers subsequently had been famous to develop resistance to remedy with pirtobrutinib by means of acquisition of different nonsynonymous BTK mutations, e.g. T474I or L528W [5]. Moreover, different BTK mutations that confer medical resistance to covalent and/or noncovalent BTKi’s now have been recognized, together with V416L, A428D, and M437R [6,7,8]. This has stimulated curiosity in improvement of medication that may goal BTK for proteasomal degradation, the so-called “BTK-degraders” [9], that are presumed capable of mitigate the danger of drug resistance as a result of mutations in BTK [10].
Right here we describe a affected person who successively acquired resistance to every era of BTKi, together with a BTK-degrader, BGB-16673, which has demonstrated medical exercise in early part I medical trials [11]. He was 66 years outdated when recognized in 2014 with CLL that expressed an unmutated IGHV with 100% homology to IGHV1-69 and that reportedly had trisomy 12 as the only real cytogenetic abnormality detected by Fluorescence in situ Hybridization (FISH). As a result of fast illness development with a lymphocyte-doubling time of ≈4 months, he was handled with bendamustine and rituximab in 2015, reaching a medical full response (CR) by iwCLL standards [12]. As a result of relapsed progressive CLL in 2017, he introduced to us for remedy with obinutuzumab and venetoclax (after debulking with excessive dose methylprednisolone), once more reaching a medical CR. He remained on remedy with venetoclax till 2019 when he developed quickly progressive lymph node enlargement, for which he initiated ibrutinib, leading to full decision of his cumbersome lymphadenopathy. In 2021 he skilled symptomatic cardiac arrythmias, thought probably ibrutinib-related; consequently his remedy was switched to acalabrutinib. He maintained a superb medical response till 14 months later, when he once more developed progressive lymphadenopathy. Subsequent era sequencing (NGS) of his marrow aspirate, of which 20% had been CLL cells (07/07/2022, Desk 1), revealed a mutation in BTK c1442G>C at a variant allelic frequency (VAF) of three.1%, leading to BTK p.C481S. Additionally famous was a mutation in TP53 c.730G>A at a VAF of 10%, leading to TP53 p.G244S and a fancy karyotype with del(17p) (Desk 1). He initiated remedy with an inhibitor of the δ/γ isoforms of phosphoinositide-3-kinase, duvelisib, which once more resulted in full decision of his lymphadenopathy. Nonetheless, due to opposed results of remedy, he discontinued duvelisib and initiated remedy with pirtobrutinib. His illness was properly managed on pirtobrutinib for 7 months, after which era he once more developed quickly progressive lymphadenopathy. NGS of his marrow aspirate, of which 42% had been CLL cells (09/12/2023, Desk 1), revealed a particular subclone of CLL harboring a mutation in BTK c.1421C>T at a VAF of 19%. This mutation resulted in BTK p.T474I, which prior research discovered confers resistance to pirtobrutinib [6]. Additionally detected was the beforehand recognized mutation in TP53 c.730G>A at a VAF of 35% and related advanced karyotype, however not the mutation in BTK c1442G>C (Desk 1). After this evaluation, he discontinued pirtobrutinib and enrolled right into a medical research, by which he was handled with the BTK-degrader, BGB-16673. After initially experiencing a marked discount in cumbersome lymphadenopathy, he got here off this research after 4 months of remedy due to quickly progressive illness. Subsequent to his coming off-study, NGS of his marrow aspirate, of which 67% had been CLL cells (02/15/2024, Desk 1), revealed yet one more distinctive subclone of CLL cells with a brand new mutation in BTK c.1283C>A at a VAF of 28%, leading to BTK p.A428D, however with out detectable BTK c1442G>C or BTK c.1421C>T, whereas sustaining the identical mutation in TP53, particularly c.730G>A, at a VAF of 62% and identical advanced karyotype. These findings assist a mannequin of selective emergence of distinct subclones throughout his successive remedy with every sort of BTKi from cells harboring the identical mutation in TP53, particularly c.730G>A and complicated karyotype as famous in earlier samples. Remarkably there didn’t look like substantial evolution within the karyotypic complexity famous within the cytogenetics or FISH analyses carried out on samples collected on 07/22/2019, 07/07/2022, 09/12/2023, and 02/15/2024, which respectively had been obtained earlier than and after remedy with obinutuzumab, venetoclax, and successive BTKi’s (Desk 1 and Supplementary Fig. 1 and Supplementary Desk 1). In any occasion, this case highlights the challenges of focusing on BTK in some sufferers with CLL.
The BTK A428D mutation is situated within the ATP-binding interface of BTK and considerably reduces its capability to bear autophosphorylation at Y223 or phosphorylation of its downstream substrate, phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) [7, 8]. Nonetheless, upon B-cell receptor ligation the mutant BTK A428D, together with different catalytically inactive BTK mutants, e.g. BTK V416L or L528W, nonetheless can allow launch of inositol-1-phosphate and supply for an enhanced Ca2+ flux in response to anti-µ stimulation in cells which have such mutant BTK compared with cells with wildtype BTK [6, 8]. As such, these BTK mutant proteins can allow downstream signaling resulting in activation of AKT, ERK, and NF-κB upon B-cell receptor ligation that isn’t inhibited by pirtobrutinib [6, 8, 9, 13]. Testomony to that is the discovering of the BTK A428D mutation in leukemia cells of sufferers who develop remedy resistance to pirtobrutinib [6]. Furthermore, this mutation additionally might impair the sensitivity to different non-covalent inhibitors in medical improvement, together with ARQ-531, fenebrutinib, and vecabrutinib [6], in addition to confer resistance to covalent BTKi’s [7]. However, the BTK A428D mutation was not detected within the CLL cells of this affected person till he developed resistance to the BTK degrader, BGB-16673.
This can be because of the selective stress utilized by BTK-degrader remedy. In work introduced on the 2023 European Hematology Affiliation Assembly, Feng and colleagues used BGB-16673 to deal with numerous TMD8-derived lymphoma cell strains, every bearing both wildtype BTK or any one of many reported mutated BTK’s which might be related to BTKi resistance (e.g. V416L, A428D, M437R, T474I, T474L, M477I, C481S, C481F, C481Y, or L528W) [14]. They reported that BGB-16673 had a nanomolar IC50 and effected BTK protein degradation in every of those TMD8 lymphoma cell strains, apart from these with the BTK A428D mutation, suggesting that BGB-16673 interacts with BTK at or round A428. It’s noteworthy that the crystal construction of the BTK interacting “hook” of one other BTK-degrader in medical improvement, particularly NX-2127, reveals an in depth drug interface with the BTK A428 residue [9, 15]. Modeling of a BTK A428D mutation locations a negatively charged aspartic acid rather than the hydrophobic aspect chain of alanine throughout the binding pocket of NX-2127 to BTK (Fig. 1). This implies that CLL cells with BTK A428D additionally could also be immune to NX-2127, as they already are recognized to be with both non-covalent or covalent BTKi’s [6]. Consequently, the 2 BTK degraders furthest superior in medical trials doubtlessly might choose for CLL cells with BTK A428D which might be immune to all accepted BTKi’s.
A Crystal construction of wildtype BTK with residues that had been discovered mutated highlighted in yellow. B BTK construction highlighting in orange the three modeled resistance mutation variants clustered within the BTK kinase area. C The hook area (in pink) of the BTK degrader NX-2127 certain to wildtype BTK in proximity to the BTK A428 residue (in yellow). D Modeled BTK resistance mutation A428D with the aspartic acid aspect chain (in orange) contained in the binding pocket for NX-2127.
In abstract, focusing on BTK has profoundly modified the face of CLL remedy over the previous decade. Iterative advances within the cat and mouse recreation of resistance and redesign have moved BTK inhibitors from covalent to non-covalent and now focused protein degraders. Nonetheless, opposite to the presumption that protein degraders could also be impervious to mutations in BTK, we now current medical proof {that a} mutation within the kinase area of BTK can confer illness resistance to a BTK degrader presently in medical trials. Furthermore, we anticipate that sufferers with CLL cells that harbor the A428D mutation in BTK might show refractory to remedy with both BGB-16673 or NX-2127.
