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4084 LP-118, a Selective Bcl-2 Inhibitor with Tuned Bcl-Xl Activity, Causes Myeloid Differentiation and Cell Death in Acute Myeloid Leukemia (AML)

Program: Oral and Poster Abstracts
Session: 616. Acute Myeloid Leukemias: Investigational Therapies, Excluding Transplantation and Cellular Immunotherapies: Poster III
Hematology Disease Topics & Pathways:
Research, apoptosis, Translational Research, Non-Biological therapies, drug development, Therapies, Biological Processes
Monday, December 12, 2022, 6:00 PM-8:00 PM

Kristin L Koenig, MD1, Colin Brame2*, Steven Sher3*, Larry Beaver, BS1*, Casey B. Cempre, B.S.1*, Katie Williams, MS1*, Matthew Purcell4*, Bonnie Harrington, DVM, PhD5*, Yi Chen, PhD6*, Felai Tan, PhD, MD7*, Stephen P. Anthony, DO6, Yu Chen, MD, PhD6*, Yue Shen, PhD6*, John C. Byrd, MD8 and Rosa Lapalombella, PhD9

1Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH
2The Ohio State University, Columbus
3Division of Hematology, Department of Internal Medicine, THE OHIO STATE UNIVERSITY, COLUMBUS, OH
4The Ohio State University, Columbus, OH
5Charles River Laboratories, Wilmington
6Newave Pharmaceutical Inc., Pleasanton, CA
7Newave Pharmaceuticals Inc., Pleasanton, CA
8University of Cincinnati College of Medicine, Cincinnati, OH
9Department of Internal Medicine, Division of Hematology, College of Medicine, The Ohio State University, Columbus, OH

Introduction

Targeted therapy against bcl-2 has proven efficacious in AML, notably with bcl-2 inhibitor venetoclax. Bcl-2 is one of several proteins in the bcl-2 protein family of anti-apoptotic proteins. Primary and adaptive resistance to venetoclax-induced apoptosis is common and can be due to increased levels of other bcl-2 family proteins besides bcl-2, notably bcl-xL and mcl-1. LP-118, a next generation oral, highly potent, selective bcl-2 inhibitor with tuned bcl-xL activity, has demonstrated preliminary efficacy in venetoclax relapsed/refractory (R/R) acute lymphocytic leukemia cell lines.

Methods

Cell-based viability assays (MTS) were performed in primary AML cells and AML cell lines. Colony forming unit (CFU) assays were performed on primary AML cells and CD34+ stem cells from healthy donor umbilical cord blood. An in vivo study was conducted in a MOLM-13 AML mouse model (Charles River Laboratories; 8-week-old male NGC mice were injected with AML cells day 1, started treatment day 4). Immunoblot studies were performed to measure on-target activity of LP-118 and bcl-2 family proteins. Flow cytometry and cytospins were performed on primary AML cells and AML cell lines to test for maturation.

Results

A 24-hour (hr) continuous exposure of LP-118 against AML cell lines demonstrated growth inhibition in all except OCI-AML3 and U937 (also resistant to venetoclax), with an IC50 in the nM range of 2.2 in MOLM-13 (venetoclax IC50 9), 3.5 in MV4-11, 0.75 in HL-60 (venetoclax IC50 2), 86.4 in K562, 7.8 in KG-1 (venetoclax IC50 30), and 4.3 in Kasumi-1 (venetoclax IC50 20). A 72-hr continuous exposure of LP-118 against primary AML cells demonstrated growth inhibition with an IC50 of 12.9uM. Venetoclax R/R primary AML cells were sensitive to LP-118 between 5 and 100uM after 72-hr continuous exposure. Primary AML cells also exhibited a 63% decrease in CFUs at 10uM dose from vehicle, while CD34+ stem cells maintained colony forming capability by CFU assay. Induction of apoptosis was observed with caspase-3 and PARP cleavage on immunoblot in AML cell lines and primary AML cells.

Relative baseline levels of bcl-2 family proteins by immunoblot did not correlate with AML cell line sensitivity or resistance to LP-118. Following 4- and 24-hr exposure to LP-118 in MOLM-13, MV4-11, and Kasumi-1 cell lines, BCL-2, BCL-xL, MCL-1, and BCL-W protein levels were not increased relative to vehicle by immunoblot, but BCL2A1 levels increased 3-fold. Similar results were seen following a 24-hour exposure to LP-118 in primary AML cells.

Myeloid differentiation was demonstrated in primary AML cells (n=3) and AML cell lines MOLM-13 (n=2) and Kasumi-1 (n=6) by flow cytometric detection of CD11b expression after 48- and 72-hr continuous exposure to LP-118, as well as, morphologically on cytospin images. CD11b was also detected after 48-hr continuous venetoclax exposure on Kasumi-1 cells. Limiting-cell RNA-sequencing is ongoing to identify differentially expressed genes following LP-118-induced myeloid differentiation.

In a MOLM-13 mouse model, single-agent LP-118 (50mg/kg PO daily) had a statistically significant median overall survival (mOS) of 24 days compared to control (20 days), but venetoclax (100mg/kg daily for 3 days then weekly) did not (mOS 21 days). There was no significant difference in OS between LP-118+azacitidine (5mg/kg IP days 1-5 of 21-day cycle) and venetoclax+azacitidine. After 60 days, the median OS of LP-118+gilteritinib (15.24 mg/kg PO daily) was not reached and showed increased OS compared to azactidine+gilteritinib and venetoclax+azacitidine. By splenic histopathology, blasts are notably reduced in mice treated with azacitidine+gilteritnib, but spleens are effaced by neoplastic blasts in all other treatment groups.

Conclusions

LP-118 shows effective cell killing in multiple AML cell lines and primary samples with various molecular and cytogenetic profiles, including a patient sample with a PTPN11 mutation, against which venetoclax is known to be ineffective. Compared to venetoclax, LP-118 is effective at lower IC50 values in AML cell lines and is active in venetoclax R/R primary AML samples. LP-118 also proves efficacious in vivo, exhibiting improved OS and decreased disease burden. Finally, LP-118 induces myeloid differentiation, to our knowledge a novel mechanism of action of bcl-2/bcl-xL inhibition in AML. These data support the recently initiated AML clinical trial (NCT04771572).

Disclosures: Koenig: Hairy Cell Leukemia Foundation: Research Funding. Chen: Newave Pharmaceuticals: Current Employment. Tan: Newave Pharmaceuticals: Current Employment. Anthony: Newave Pharmaceuticals: Current Employment. Chen: Newave Pharmaceuticals: Current Employment. Shen: Newave Pharmaceuticals: Current Employment. Byrd: Newave: Consultancy; Syndax: Consultancy; Novartis: Consultancy; AstraZeneca: Consultancy; Kura: Consultancy; Vincerx: Consultancy, Current equity holder in private company, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Pharmacyclics: Research Funding; Zencor: Research Funding; Janssen: Consultancy; Ohio State University: Patents & Royalties; AbbVie: Consultancy; Trillium: Consultancy.

*signifies non-member of ASH