Abstract A04: Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma
Authors: Montoya Skye , Wang Eric , Bourcier Jessie , Chaudhry Sana , Totiger Tulasigeri , Pardo Alejandro , Pardo Gabriel , Affer Maurizio , Jahn Jacob , Mato Anthony , Abdel-Wahab Omar , Taylor Justin 
Published: September 2022

Abstract
Bruton’s tyrosine kinase (BTK) is a target for multiple generations of covalent (irreversible) and non-covalent (reversible) inhibitors due to its critical role in the proliferation and survival of B-cell malignancies. Similar to drug resistance mechanisms in other cancers, resistance to covalent BTK inhibitors (BTKi) in chronic lymphocytic leukemia (CLL), such as ibrutinib, arise through on-target BTK mutations at the BTK C481 residue (the binding site of ibrutinib), which allow escape from BTK inhibition. Non-covalent BTKi represent a new avenue to overcome resistance to the clinically approved covalent BTKi; however, we have recently discovered mechanisms of resistance to non-covalent BTK inhibition in patients with CLL (Wang, Xi, Thompson, Montoya et al NEJM 2022). We used bulk and single cell genomic analyses that identified a series of acquired BTK mutations in a cohort of CLL patients that relapsed on the phase I/II clinical trial of pirtobrutinib. We discovered mutations (BTK V416L, A428D, M437R, T474I, L528W) that occur at critical residues within the catalytic kinase domain of BTK and conferred resistance to both non-covalent and covalent BTKis. Based on structural models of BTK, we observed that these mutations physically impede drug binding, and disrupt the normal kinase activity of BTK but can, upon B-cell receptor stimulation, sustain AKT, ERK, and NF𝜅B signaling as well intracellular Ca2+ release in the presence of pirtobrutinib. In addition, CITE-seq analyses of 53,722 cells from this cohort identified pre-existing leukemic and immune cell states associated with development of resistance in patients. These findings identify novel mechanisms to engender clinical resistance to non-covalent BTKis, including contribution of the immune microenvironment to response to BTK inhibition. Together our data suggest that mutations at the BTK kinase domain may alter conformation of BTK’s non-kinase protein interaction domains thus allowing BTK to be used as a scaffold for other signaling molecules to phosphorylate phospholipase C gamma 2 (PLC𝛾2), the direct downstream target of BTK. We have performed mass spectrometry phosphoproteomics to evaluate potential signaling molecules that are active and could bypass BTK in catalytically inactive BTK mutant cells. Given the above, we decided to target BTK’s non-kinase function by investigating targeted protein degraders of BTK using BTK-degrading protein targeting chimeras (PROTACS). The immediate advantages of using protein degraders are (1) Binding may occur at any site of the target protein, and (2) PROTACS can act catalytically to bind to and degrade multiple target proteins. Our studies have shown that several of the BTK mutants that engendered clinical resistance to covalent and non-covalent BTKi are sensitive to these degraders, elucidating an exciting alternative therapeutic approach for patients who become resistant to BTKi therapies. We are continuing to test the efficacy of BTK PROTAC degraders in overcoming acquired resistance mechanisms in CLL as well as other B-cell lymphomas.
Citation Format: Skye Montoya, Eric Wang, Jessie Bourcier, Sana Chaudhry, Tulasigeri Totiger, Alejandro Pardo, Gabriel Pardo, Maurizio Affer, Jacob Jahn, Anthony Mato, Omar Abdel-Wahab, Justin Taylor. Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A04.