BTK T474I

One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 co-mutations. While common BTK C481 mutations were observed with both treatments, patterns of mutation and co-mutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W, A428D) in this patient population.

For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor...ONO-7018 and tirabrutinib were orally administered to the mice twice a day... ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

Pts received 1 or more cBTKi: ibrutinib (n=44, 90%), acalabrutinib (n=10, 20%) or zanubrutinib (n=1, 2%). Whether similar resistance patterns would manifest if pirtobrutinib was utilized prior to cBTKi treatment remains uncertain. Figure 1.

We performed whole exome sequencing on matched tumor and normal samples from 19 CLL patients treated with BTK inhibitors (Ibrutinib, Ibr, 42% (8/19); Acalabrutinib, Acala, 21% (4/19) and Pirtobrutinib, Pirto, 37% (7/19))...The ibr cohort had no prior BTKi therapy whereas 1 patient in the acala cohort had received spebrutinib, another cBTKi...The approach of combining WES data with growth pattern modeling can help unravel the complexities of tumor evolution and drug resistance for the different classes of BTKi. Data on comparative clone growth rates will be presented at the meeting.

The majority of IR DP with BTK/PLCG2 mutations (6/8), were 100% homologous to germline IGHV unmutated, suggesting an inherent higher risk disease profile as a risk factor for acquiring BTKmt with prolonged BTKi therapy. BTK/PLCG2 mutations were enriched amongst very late progressors with 6/8 pts progressing shortly after stopping therapy at 72 mo., which is later than previously reported for relapsed/refractory CLL pts.

Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL including that with BTK mutations conferring BTKi resistance. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al., 2022).

Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib...In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib... After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years.

BTK Cysteine 481 substitution is known to contribute to cBTKi acquired resistance to ibrutinib, acalabrutinib, and zanubrutinib. Despite this cohort representing the first relapsing CLL patients from BRUIN and presenting with frequent baseline BTK mutations, response to pirtobrutinib was high, with an ORR of 83%, and substantial clearance of BTK C481 clones. At progression, the majority of pts (56%) either acquired non-BTK mutations or did not acquire any resistance mutations in this targeted panel, suggesting alternative resistance mechanisms. A smaller group of patients (44%) displayed emergence of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations.

We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib...In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S, PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment including known resistance mutations p.C481R, p.T474I and p.C481S...For example, BTK p.L528W in patient R001 increased in frequency more than 1000-fold (from CCF 0.02% to 35%), and p.T474I in patient R002 increased in CCF from 0.03% to 4.2% (more than 100-fold). Our data illuminates the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

The C481S and C481R mutations eliminated the anti-proliferative effects of covalent inhibitors ibrutinib, acalabrutinib, and zanubrutinib, whereas the V416L, T474I, and L528W mutations had variable effects on the covalent inhibitors. By contrast, the non-covalent inhibitors pirtobrutinib, vecabrutinib, and fenebruitnib maintained activity against C481S but displayed partially reduced potency against the C481R mutation and dramatically reduced potency against the V416L, T474I, and L528W mutations...These degrader molecules may also have utility in earlier lines of therapy due to their ability to suppress scaffold-mediated BTK signaling. Phase 1a/b trials of NX-5948 and NX-2127 in patients with relapsed or refractory B-cell malignancies are ongoing (NX-5948: NCT05131022; NX-2127: NCT04830137).

Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL, including BTK inhibitor resistant disease. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) [7].

73 patients had only one BTKi (ibrutinib (IBR), 64; acalabrutinib (ACA), 9). 12 pts had multiple BTKis, 8 with two drugs with IBR first followed by ACA (Nf3, 37.5%), vecabrutinib (Nf1, 12.5%), and PIR (Nf4, 50.0%); and 4 with three or more drugs... Our retrospective report summarizes mutations detected during BTKi treatment and shows that BTK L528W can occur during both covalent and non-covalent BTK inhibitor therapy. Four of six patients who progressed on PIR had T474 mutations. In addition, our results may suggest that activating mutations in RAS/RAF/MAPK pathway are related to BTKi resistance.

main study Substudy patients treated with VenR (n = 194) patients treated with BR (n = 195) patients retreated with VenR (n = 25) Baseline characteristics mean age, years (SD) 63.9 (10.5) 64.4 (9.6) 65.8 (8.3) Number of prior cancer therapy, n (%) 1 111 (57.2) 117 (60.0) 0 (0.0) 2 58 (29.9) 43 (22.1) 20 (80.0) ≥3 25 (12.9) 35 (17.9) 5 (20.0) del(17p) and/or TP53 mutation (aCGh), n (%) mutated 53 (27.3) 55 (28.2) 14 (56.0) unmutated 104 (53.6) 98 (50.3) 9 (36.0) unknown 37 (19.1) 42 (21.5) 2 (8.0) Genomic complexity, n (%) n = 48 n = 46 n = 20 3–4 34 (70.8) 29 (63.0) 3 (15.0) ≥5 14 (29.2) 17 (37.0) 8 (40.0) iGhV, n (%) n = 180 n = 180 n = 23 mutated 53 (29.4) 51 (28.3) 2 (8.7) unmutated 123 (68.3) 123 (68.3) 21 (91.3) unknown 4 (2.2) 6 (3.3) 0 (0.0) efficacy results median follow-up, months 85.7 85.7 33.4 Best oRR, % 93.3 67.7 72.0 umRD at eoCT of main study, n (%) 121 (62.4) 26 (13.3) 16 (64.0) umRD at eoCT of substudy, n (%) N/a N/a 8 (32.0) umRD at eoT of main study, n (%) 83 (70.3)* N/a 14 (56.0) umRD at eoT of substudy, n (%) N/a N/a 0 (0.0) median pFS, months (95% Ci) 54.7 17.0 23.3 (52.3, 59.9) (15.5, 21.7) (15.6, 24.3) 3-year oS rate, % (95% Ci) 88.4 78.9 53.1 (83.8, 93.0) (72.8, 84.9) (25.1, 81.0) *patients who completed 2 years of Ven without pD (n = 118) aCGh: array comparative genomic hybridization; BR: bendamustine- rituximab; Ci: confidence interval; del(17p): deletion in chromosome 17p; eoCT: end of combination treatment; eoT: end of treatment; iGhV immu- noglobulin heavy chain gene; oRR: overall response rate; oS: overall sur- vival; pD: progressive disease; pFS: progression free survival; SD: standard deviation; TP53: tumor protein p53; umRD: undetectable minimal residual disease; Ven(R): venetoclax-(rituximab) cBTKi pre-treated CLL pts treated with pirtobrutinib monotherapy in the phase 1/2 BRUIN trial (NCT03740529) who subsequently developed disease progression (PD) were included in this analysis...Pts received one or more of the following cBTKi: ibrutinib (n = 44, 90%), acalabrutinib (n = 10, 20%), or zanubrutinib (n = 1, 2%)... Pts who progressed on pirtobrutinib showed clearance of BTK C481 clones and the emergence or outgrowth of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations, as well as other VUS. Many acquired BTK mutations were shown to pre-exist at baseline at low VAF, reflecting emergence on prior cBTKi. Importantly, these baseline kinase domain BTK mutations did not preclude pirtobrutinib efficacy.

Novel, non-C481 BTK mutations have been described, including BTK L528W, which has been reported in patients treated with ibrutinib, zanubrutinib, and pirtobrutinib (Maddocks, JAMA Oncol. This is the largest dataset characterizing the incidence and patterns of BTK and PLCG2 mutations in patients with CLL treated with ibrutinib. BTK C481 mutations are the most frequently occurring mutations. PLCG2 mutations occur across the gene at a low incidence per locus (<5%).

Inhibition of BTK by covalent BTK inhibitors (BTKi), such as ibrutinib, acalabrutinib, and zanubrutinib, have revolutionized the management of CLL and other B cell malignancies. BGB-16673 is a potent inhibitor against tumors expressing wildtype and clinical-relevant BTK mutations. In addition, it is superior to ibrutinib and LOXO-305 equivalent for more durable anti-tumor activities and less metastasis. Absolute lymphocyte count, Tumor model, Lymphoma, Mouse model

Pts received one or more of the following cBTKi: ibrutinib (n=44, 90%), acalabrutinib (n=10, 20%), or zanubrutinib (n=1, 2%). Pts who progressed on pirtobrutinib showed clearance of BTK C481 clones and the emergence or outgrowth of non-C481 clones, particularly gatekeeper T474 and kinase-impaired L528W mutations, as well as other VUS. Many acquired BTK mutations were shown to pre-exist at baseline at low VAF, reflecting emergence on prior cBTKi. Importantly, these baseline kinase domain BTK mutations did not preclude pirtobrutinib efficacy.

In pre-clinical safety studies, NX-2127 demonstrates an acceptable safety profile. NX-2127 is currently in phase 1 clinical trials (NCT04830137) for hematological malignancies.

"Twenty-three pts were treated with standard of care brexucabtagene autoleucel (CART)... The clinical NGS-based assay for MCL pts at our institution has proven to have prognostic significance. Resistant MCL pts exhibit a preponderance of mutations involving TP53, epigenetic modifier and chromatin regulator genes. Comprehensive risk stratification of pts early on and during therapy is very useful and improves patient care, helps design next generation clinical trials and improves our understanding of the biology of resistant MCL in the BTKi and CART era."