Page 87 - Haematologica Vol. 110 - January 2025

ARTICLE - LP-118: a promising treatment for CLL
J. Ravikrishnan et al.
of the BCL2 family, including myeloid cell leukemia-1 (MCL1) through copy number gain or gene upregulation,7,10 and B-cell lymphoma-extra-large (BCLXL) by protein overexpression.11-13 Patients resistant to venetoclax can develop multiple resis- tance mechanisms simultaneously due to clonal evolution.5,8 These resistance mechanisms suggest that targeting multiple pro-survival BCL2 family members would be of clinical benefit. Despite being attractive targets for venetoclax-resistant CLL, targeting MCL1 or BCLXL has had limited clinical utility due to on-target cardiac toxicity14-16 or platelet toxicity,17,18 respec- tively, with the latter causing dose-limiting thrombocytopenia. However, a recent trial combining venetoclax with low-dose navitoclax showed efficacy in patients with relapsed refrac- tory acute lymphoblastic leukemia (ALL) without evidence of thrombocytopenia, suggesting that modulating BCLXL inhibition may allow successful therapeutic targeting.19 Here, we characterize and demonstrate the preclinical efficacy of a new selective BCL2 inhibitor with moderate BCLXL targeting, LP-118, designed to minimize platelet toxicity and bypass various venetoclax resistance mechanisms. Most notably, we show that LP-118 is more potent than venetoclax in various BCL2-dependent models and has preclinical efficacy in vene- toclax-resistant CLL. Therefore, LP-118 has the potential to treat patients with CLL, including those with venetoclax-re- lapsed disease, justifying the ongoing phase I trial.
Methods
Drug treatments and cell lines
LP-118 was provided for all studies by Newave Pharmaceuti- cal Inc. Navitoclax (#S1001), venetoclax (#S8048), A-1331852 (#S7801), and Z-VAD-FMK (#S7023) were purchased from Selleckchem. Cell lines source and maintenance are detailed in the Online Supplementary Methods.
Biochemical
BCL2 tagged with 6XHIS (BPS Bioscience) and BCLXL (R&D systems) were pre-incubated with compounds for 15 minutes. BAK BH3 peptide labeled with anti-GST antibodies labeled with TAMRA and Tb-cryptate (CisBio) were incubated for one hour (hr) and read on an Analyst HT multimode plate reader (Molecular Devices).
Computational modeling
Docking of LP-118 (PubChem, ID 146663563) on murine BCL2 (UniProt, P10417) or human BCL2 (UniProt, P10415) was mod- eled using SiteMap module of Schrodinger Maestro Suite. BCL2 mutations (G101V, D103E) were modeled using Muta- genesis in Pymol. Docked molecules were ranked based on GlideScore.
Cytotoxicity assays
Studies and patient samples were used with approval and review by the Ohio State University Institutional Review
Board with written consent from patients. BH3 profiling was performed as previously described by Letai et al.20 and run on a CytoFLEX LX (Beckman Coulter). Primary CLL cells were treated with the indicated compounds and for the indicated timepoints, in suspension or co-cultures. For cytotoxicity assays, primary CLL cells were stained with Annexin V-FITC (Leinco), and TMRM (Thermo Fisher) or propidium iodide (PI) (Leinco) to examine apoptosis on the Gallios flow cytometer (Beckman Coulter). Primary CLL cells and RS4;11 cells were treated and stained with near-IR live / dead staining (Thermo Fisher), fixed and permeabilized with BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences), 0.125 μg of primary BAK (TC100) antibody (Enzo, Cat. #BML-SA298-0050) and Dylight 488 conjugated goat anti-mouse secondary anti- body (Thermo Fischer, Cat. #35502), and analyzed on Gallios (Beckman Coulter) flow cytometer.
Mouse models
All experiments using animal models were carried out in accordance with the guidelines established by the Ohio State University and the Institutional Animal Care and Use Committee. Adoptive transfer of CD5+CD19+ cells carried out from spleens of an Eμ-TCL1 mouse into C57Bl6 mice. At 10% of CLL cells in peripheral blood, mice were dosed daily via oral gavage. Female SCID mice (CB17/Icr-Prkdcscid/IcrIcoCrl, Charles River) were engrafted with 1x107 RS4;11 cells, injected subcutaneously on the right flank. At 100-150 mm3 tumor volume, mice were drugged orally for 28 days. Tumors were measured twice a week then weekly after Day 63. NOG-F and NOG-M mice (Taconic, Albany, NY, USA) were engrafted with 1x107 OSU-CLL cells intravenously. After four days, mice were treated once a day via oral gavage and tracked for overall survival. Early removal criteria are described in the Online Supplementary Methods.
Statistical analysis
Experiments with continuous variables were analyzed using mixed effect model, accounting for observational dependen- cies across treatment conditions, group comparisons, and trend tests IC50 obtained through non-linear mixed effect modeling. Skewed data such as cell counts were normalized by log transformation. Other data were analyzed following the scales demonstrated on the figures (subtracting DMSO or standardizing over DMSO). Survival data were analyzed by log rank test. Data were analyzed in SAS 9.4 (SAS Institute, Cary, NC, USA).
See the Online Supplementary Methods for more details. Results
LP-118, a second generation novel BCL2 inhibitor with rationally designed BCLXL inhibition
LP-118 is a BCL2 potent inhibitor with moderate BCLXL in- hibition, rationally designed to provide improved antitumor
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