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

Abstract

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). Disclosures Tan: Guangzhou Lupeng Pharmaceutical Company Ltd.,: Current Employment. Chen: Newave Pharmaceutical Inc.: Current Employment. Anthony: Newave Pharmaceutical Inc.: Current Employment. Chen: Newave Pharmaceutical Inc.: Current Employment. Shen: Guangzhou Lupeng Pharmaceutical Company Co. Ltd.,: Current Employment. Byrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria. Woyach: AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

Bcl-2 pathway inhibition in solid tumors: a review of clinical trials

“…LP-118 is an oral selective Bcl-2/Bcl-xL inhibitor and it is the newest of all drugs we have included in this study. Its special feature is that its anti Bcl-xL activity is adjusted to the minimum so that the risk of thrombocytopenia is limited [ 49 ]. We report only one recruiting clinical trial of LP-118 as a single agent in patients with lymphoma or solid tumors and the results are expected in the forthcoming years (Table 2 ).…”

New era for myelofibrosis treatment with novel agents beyond Janus kinase‐inhibitor monotherapy: Focus on clinical development of BCL‐XL/BCL‐2 inhibition with navitoclax

“…Other BCL‐X L inhibitors are currently in phase 1 clinical trials but have notable differences from navitoclax. LP‐118 is a BCL‐2/BCL‐X L inhibitor that binds BCL‐X L with an enzymatic 50% inhibitory concentration (IC 50 ) of 10.1 nM, which is significantly less potent than navitoclax at 2.9 nM; however, in contrast to navitoclax, LP‐118 also has very high affinity for BCL‐2 (Table 1 summarizes the current BCL‐X L inhibitors) 33 . In contrast to the oral delivery of navitoclax, AZD0466 is an intravenously administered BCL‐2/BCL‐X L inhibitor that also has a higher affinity for BCL‐2 34 .…”

“…LP-118 is a BCL-2/BCL-X L inhibitor that binds BCL-X L with an enzymatic 50% inhibitory concentration (IC 50 ) of 10.1 nM, which is significantly less potent than navitoclax at 2.9 nM; however, in contrast to navitoclax, LP-118 also 1 summarizes the current BCL-X L inhibitors). 33 In contrast to the oral delivery of navitoclax, AZD0466 is an intravenously administered BCL-2/BCL-X L inhibitor that also has a higher affinity for BCL-2. 34 Finally, DT2216 is specific to BCL-X L that functions by degrading its target, has shown encouraging preclinical activity to date, and has been given fast-track designation to treat relapsed/refractory peripheral and cutaneous Tcell lymphoma.…”

No time to die? Intrinsic apoptosis signaling in hematopoietic stem and progenitor cells and therapeutic implications

“…It can be assumed that combined inhibition of two or more antiapoptotic BCL-2 proteins will have synergistic effects. Indeed, this was shown in vitro for combined inhibition of BCL-2 and MCL-1 in AML [39][40][41][42], B-ALL [43 & ], CLL [44], or mantle cell lymphoma [45] as well as for inhibition of both BCL-2 and BCL-X L in CLL [46], or B-ALL [43 megakaryopoiesis. In contrast, MCL-1 is only essential for survival of immature progenitors but not mature blood cells.…”