Characterization of LP-118, a Novel Small Molecule Inhibitor of Bcl-2 and Bcl-Xl in Chronic Lymphocytic Leukemia Resistant to Venetoclax

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia, which is characterized by the accumulation of mature CD19+CD5+ B cells that evade apoptosis by upregulating anti-apoptotic BH3 protein, B cell lymphoma protein 2 (Bcl-2). Venetoclax, a first-in-class Bcl-2 inhibitor has transformed therapy for CLL. However, with continuous administration of venetoclax, patients often acquire resistance via mutations at or near the drug binding pocket at G101 in Bcl-2 (Blombery et al, Cancer Discov 2019, Lucas et al, Blood, 2020). In some patients, the acquisition of a mutation in tumor suppressor protein TP53 or the upregulation of anti-apoptotic family members including Bcl-xL has been shown to drive resistance to venetoclax. In particular, a switch to Bcl-xL dependency provides the rationale for dual inhibition of Bcl-2/-xL.

We performed BH3 profiling on treatment naïve and venetoclax relapsed/refractory (R/R) primary CLL cells to investigate the dependency of several anti-apoptotic proteins and their contributions to cell survival. Primary CLL cells were incubated with Bcl-xL specific HRK peptides, Mcl-1 specific MS-1 peptides, Bcl-2 interacting BAD peptides, or BIM peptides which interact with all anti-apoptotic members. We observed that the majority of these cells responded to both BIM and BAD peptides indicating the dependency on anti-apoptotic protein Bcl-2. However, in some samples, including venetoclax R/R primary CLL cells, showed dependency on other anti-apoptotic proteins including Bcl-xL as indicated by a depolarization from the HRK peptide.

We subsequently performed pre-clinical investigations of LP-118, a novel small molecule inhibitor of Bcl-2, more potent than venetoclax and with added advantage of selectively binding to Bcl-xL while minimizing the platelet toxicity. LP-118 was rationally designed to have an enzymatic IC ₅₀ for Bcl-xL at 10.1 nM which was in-between venetoclax (62.2 nM) and navitoclax (2.9 nM) to prevent the on-target effects of platelet toxicity. Since most venetoclax- R/R patients have mutation in Bcl-2 and/or have increased dependency on Bcl-xL, we hypothesized that these patients would benefit from the additional inhibition of Bcl-xL.

To test this hypothesis, treatment naïve and venetoclax R/R primary CLL cells were incubated with LP-118, venetoclax, or navitoclax continuously for 18 hours at concentrations ranging from 0.1 nM to 10 nM. Apoptosis was measured through Annexin V/Tetramethylrhodamine methyl ester perchlorate (TMRM) staining followed by flow cytometry. We found that treatment naive CLL cells were more sensitive to LP-118 than venetoclax and navitoclax with IC ₅₀ values at 0.5nM, 10 nM, and >10 nM, respectively. Venetoclax R/R CLL cells were 50% killed by 1 nM LP-118 and were not sensitive to either venetoclax or navitoclax. To investigate the mechanism of LP-118 induced cell death, we used intracellular flow cytometry and observed pore-forming pro-apoptotic protein Bak transforming to an active conformation as early as 8 hours after treatment in venetoclax R/R and treatment naive CLL cells. Furthermore, after 12 hours of treatment we observed a release of cytochrome c indicating that cells were going through apoptosis.

To determine whether cells harboring the Bcl-2 Gly101Val mutation respond to LP-118, we transfected RS4; 11 cells with plasmids containing the mutation. We treated these mutant cells for 72 hours and found that while these mutant cells were not sensitive to venetoclax, they responded to LP-118 with IC ₅₀ of 20 nM. Furthermore, LP-118 induced quicker and earlier bak transformation and cytochrome C release in these mutant cells than venetoclax. To further evaluate the efficacy of LP-118, we tested this compound in a RS4; 11 xenograft model. We treated mice daily via oral gavage and observe that 100 mpk of LP-118 significantly decreases overall tumor growth and increases survival compared to 100 mpk of venetoclax (p=0.0004).

Collectively, our in vitro studies demonstrated preclinical efficacies of LP-118 in primary CLL samples and Gly101Val mutant cells. LP-118 is highly potent in venetoclax resistant patient samples and cell lines. Additional in vivo work has further confirmed the efficacy of LP-118 in mutant cell lines. This work justifies continued preclinical and clinical work with this agent, and a phase 1 first in human study will begin soon in patients with relapsed hematologic malignancies (NCT04771572).