Abstract 2850: NX-5948 promotes selective, sub-nanomolar degradation of inhibitor-resistant BTK mutants

Abstract

Small molecule kinase inhibitors have revolutionized the treatment of hematological malignancies by suppressing signaling pathways essential for tumor cell survival. Bruton’s Tyrosine Kinase (BTK) inhibitors are widely used in the clinic for treatment of patients with B cell malignancies. Acquired resistance mutations, however, can reduce or eliminate their efficacy and represent a growing challenge. Mutations at C481 dramatically reduce the binding of covalent BTK inhibitors, whereas other clinically-observed mutations such as V416L, T474I, and L528W reduce or eliminate the activity of next-generation non-covalent inhibitors. Some of these mutations abolish BTK kinase activity while retaining intact BCR signaling and BTK-dependent growth, indicating that mutant BTK elicits scaffold-mediated signaling essential for malignant B cell survival. To assess the impact of resistance mutations on the activity of BTK inhibitors, we generated a DLBCL line (TMD8) harboring BTK-C481S, V416L, T474I, or L528W point mutations. The C481S mutation eliminated the anti-proliferative effects of covalent inhibitors ibrutinib, acalabrutinib, and zanubrutinib, while the V416L, T474I, and L528W mutations dramatically reduced the activity of pirtobrutinib, vecabrutinib, and fenebrutinib. L528W largely abolished the activity of ibrutinib and zanubrutinib, whereas V416L substantially reduced the activity of acalabrutinib. This variability in BTK inhibitor sensitivity to resistance mutations complicates treatment decisions for patients who relapse on BTK inhibitors, necessitating agents that can more broadly target resistance mutations. Here we assessed the activity of NX-5948, a heterobifunctional degrader molecule that induces the targeted degradation of BTK. We used surface plasmon resonance to evaluate the binding of NX-5948 to WT and mutant (C481S, T474I, V416L and L528W) BTK proteins. NX-5948 binds potently to BTK WT, C481S and T474I with single-digit nanomolar affinities, but loses potency against the V416L (14-fold) and L528W (33-fold) mutants. Despite this reduction in binary binding affinity, NX-5948 induces sub-nanomolar degradation of all mutant forms of BTK and potently suppresses expression of activation markers and proliferation in TMD8 cells harboring these mutations. We propose that the positive cooperativity that NX-5948 induces between BTK and the E3 ligase cereblon contributes to its potent and sustained degradation activity against BTK resistance mutants. We also assessed the selectivity of NX-5948 by global proteomics and observed exquisite selectivity across cell types and conditions. The exceptional potency, selectivity, and activity of NX-5948 against BTK mutants warrant its investigation in clinical settings that develop diverse inhibitor resistance. A phase 1a/b trial of NX-5948 for patients with relapsed or refractory B-cell malignancies is ongoing (NCT05131022). Citation Format: Mark Andrew Noviski, Nivetha Brathaban, Ratul Mukerji, Stephanie Yung, Jordan Ye, Hugo Bousquet, Mateo Sanchez Garcia de los Rios, Brandon Bravo, Jiwen Chen, Paul Auger, Jeffrey Mihalic, Hao Lu, Cristiana Guiducci, Gwenn Hansen. NX-5948 promotes selective, sub-nanomolar degradation of inhibitor-resistant BTK mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2850.

Optimizing BTK Inhibition in Waldenström Macroglobulinemia

“…48 In these cases, small molecule-induced protein degradation may allow for continued targeting of BTK. 49 The novel agent NX-5948 is a chimeric targeting molecule that contains a BTK hook linked to a cereblon harness and allows for ubiquitylation and proteasomal degradation of BTK. 50 Preclinical studies have shown successful degradation of BTK, even in models with BTK C481S mutations.…”

Strategies for overcoming resistance to Bruton's tyrosine kinase inhibitor zanubrutinib

“…82 Both PROTACs were efficient in C481S mutated cells, NX-5948 was proven to induce also degradation of other forms of mutated BTK (T474I, V416L and L528W). 83,84 Expectedly, NX-2127 degraded BTK also in patients who have acquired BTK resistance mutations as a result of prior BTK inhibitor therapies. 35…”

“…Moreover, NX‐2127 possesses dual degradation mechanism, and besides BTK can target other clinically validated proteins: transcription factors Aiolos (IKZF3) and Ikaros (IKZF1) regulating T‐cells function 82 . Both PROTACs were efficient in C481S mutated cells, NX‐5948 was proven to induce also degradation of other forms of mutated BTK (T474I, V416L and L528W) 83,84 . Expectedly, NX‐2127 degraded BTK also in patients who have acquired BTK resistance mutations as a result of prior BTK inhibitor therapies 35…”