Lisaftoclax (APG-2575) Is a Novel BCL-2 Inhibitor with Robust Antitumor Activity in Preclinical Models of Hematologic Malignancy

B-cell lymphoma 2 (BCL-2) proteins control programmed cell death (apoptosis), and dysregulation of these proteins can promote tumor cell survival. The BCL-2 family comprises antiapoptotic proteins [e.g., BCL-2, myeloid cell leukemia 1 (MCL-1), BCL‒extra-large (BCL-xL)] that bind to and sequester proapoptotic family members, such as BCL-2‒like protein 11 (BIM), BCL-2 homology domain 3 (BH3)-interacting domain death agonist (BID), BCL-2–associated X and K proteins (BAX/BAK), Noxa, and p53 upregulated modulator of apoptosis (1–3). Small-molecule inhibitors of BCL-2 proteins enhance cancer cell apoptosis across many hematologic malignancies, such as chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), acute myeloid leukemia (AML), myelodysplastic syndrome, Waldenström macroglobulinemia (WMG), and multiple myeloma (4–9).

Structural insights into interactions between proapoptotic and antiapoptotic proteins culminated in the discovery and clinical development of the first BH3 mimetic: navitoclax (ABT-263; ref. 10). Despite clinical efficacy among patients with advanced cancer, many patients who received navitoclax experienced high-grade thrombocytopenia driven by on-target BCL-xL–mediated platelet inhibition (11, 12).

Subsequently, BH3-mimetic venetoclax (ABT-199; refs. 13, 14) was developed to bind more selectively to BCL-2 and became the first FDA-approved BCL-2–selective small-molecule inhibitor, initially in patients with CLL (and subsequently AML). However, many individuals who receive venetoclax experience hematologic toxicities and tumor lysis syndrome (15–17), which warrants a 5-week dose ramp-up (according to US product labeling) that may pose both logistical challenges for some patients as well as a burden on the healthcare system.

Therefore, we designed lisaftoclax (APG-2575) as a novel, potent, oral BCL-2–specific inhibitor. We herein describe preclinical characterization of lisaftoclax across a range of hematologic malignancies, focusing on the promising antitumor activity and potential underlying mechanisms of action for this new BCL-2 inhibitor.

In NOD/SCID NOD.CB17-Prkdc^scid/J or NOD/SCID/γ (NSG) NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ mice, a patient-derived xenograft (PDX) tumor model was developed using a specimen of fresh primary tumor tissue collected posthumously (under an approved protocol by the Mayo Clinic Institutional Review Board) from a patient with aggressive WMG. The PDX mouse model was established as previously reported (18–20) and per Mayo Clinic Institutional Animal Care and Use Committee approval and institutional guidelines.

BCL-2 in the crystal structure between BCL-2 and a navitoclax analog (PDB ID 4LVT; ref. 13) was extracted to construct the binding modes of lisaftoclax with BCL-2. Ionization states of the amino acids in BCL-2 were calculated and hydrogen atoms added using the protonate 3D module in the Molecular Operating Environment (MOE) program (21) by setting the pH to physiologic 7.0. The structure of lisaftoclax was also prepared using MOE and docking simulation performed using the Genetic Optimisation for Ligand Docking program (version 5.6.2; refs. 22, 23). We performed the template docking protocol by imposing distance constraints (1.5–3.5 Å and a force constant of 5.0 kcal/mol/Ų) between 7 atoms on navitoclax and atoms on BCL-2 (D137, W141, R143, G191, V89, L94, and S102). The center of the binding site was set at G142 in BCL-2, and the radius of the binding site was 13 Å. Default parameters and the GoldScore fitness function were used in the docking calculations. The top-ranked pose of lisaftoclax was selected as the predicted binding model.

Recombinant human BCL-2, BCL-xL, and MCL-1 protein were prepared as previously described (24). Specifically, an isoform 2 construct of human BCL-2 with an N-terminal 6xHis tag (a polyhistidine tag) was used. Human BCL-xL constructs with internal deletions of amino acids 45–85 and a C-terminal truncation of amino acid residues 212–233 were cloned into a pHis-TEV vector (a modified pET vector). An MCL-1 fragment encoding amino acid residues 171–327 was also cloned into a pHis-TEV vector.

Fluorescein-labeled BIM (amino acids 81–106), BAK (amino acids 72–87), and BID (amino acids 79–99) peptides, named as Flu-BIM, Flu-BAK, and Flu-BID, respectively, were used as fluorescent probes in fluorescence polarization (FP) assays for BCL-2, BCL-xL, and MCL-1, respectively. For each reaction, 1 nmol/L of Flu-BIM and 2 nmol/L of Flu-BAK or Flu-BID (plus increasing concentrations of BCL-2, BCL-xL, or MCL-1) were added to a final volume of 125 μL in assay buffer (100 mmol/L potassium phosphate, pH 7.5; 100 μg/mL bovine γ-globulin; 0.02% sodium azide; Invitrogen, with 0.01% Triton X-100; 4% DMSO). Reactants were mixed and incubated at room temperature for 1 hour, with gentle agitation to assure equilibrium.

All FP measurements were performed using Microfluor 1 96-well, black, round-bottom plates (Thermo Fisher, M33089) on an Infinite M1000 plate reader (Tecan US, Inc; Morrisville, NC). Polarization values in millipolarization units were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Equilibrium dissociation constants (Kd) were then calculated using Prism 5.0 software (GraphPad Software, San Diego, CA) by fitting sigmoidal dose-dependent FP increases as a function of protein concentration. By monitoring total FP values of mixtures composed of fluorescent probes at fixed concentrations and proteins with increasing concentrations to full saturation, we determined mean (SD) Kd values of Flu-BIM to BCL-2 (0.55 ± 0.15 nmol/L), Flu-BAK to BCL-xL (4.4 ± 0.8 nmol/L), and Flu-BID to MCL-1 (6.9 ± 0.9 nmol/L).

Inhibition constants (Ki) for binding of lisaftoclax and venetoclax to BCL-2, BCL-xL, and MCL-1 were determined from competitive binding experiments in which serial dilutions of the BCL-2 inhibitors were added to 96-well plates containing fixed concentrations of fluorescent probes and proteins in each well. Mixtures of 5 μL of the tested inhibitors in DMSO and 120 μL of preincubated protein/probe complexes in the assay buffer were added to assay plates and incubated at room temperature for 2 hours with gentle agitation. Final concentrations of the protein and probe (respectively) were 1.5 nmol/L and 1 nmol/L for the BCL-2 assay, 10 nmol/L and 2 nmol/L for the BCL-xL assay, and 20 nmol/L and 2 nmol/L for the MCL-1 assay. Negative controls containing only protein/probe complexes (equivalent to 0% inhibition) and positive controls containing only free probe (equivalent to 100% inhibition) were included in each assay plate.

FP values were measured as described above. Half-maximal IC₅₀ values were determined by nonlinear regression fitting of the competition curves. Ki values of competitive inhibitors were calculated using a previously published equation (25) based on measured IC₅₀ values, Kd values of probes to proteins, and concentrations of proteins and probes in the competitive assays. Ki values were also calculated using a previously described equation (26).

To assess the kinetics of BCL-2 complex disruption by lisaftoclax, we performed cellular uptake assays on RPMI8226 and KMS-11 cells. Cancer cell lines and culture conditions are summarized in Supplementary Table S1. RPMI8226 and KMS-11 cells were cultured to confluence on a 24-well plate, then incubated with fresh culture medium containing 1 μmol/L lisaftoclax or venetoclax at 37°C.

At predefined time points, culture media were removed and cells washed 3 times with ice-cold PBS. Cell lysis buffer was then added, followed by centrifugation to collect the supernatant as a cell lysate. A total of 50 μL of cell lysate was added with 1,000 μL of acetonitrile containing tolbutamide 5 ng/mL [internal standard (IS)]. The mixture was vortexed for 2 minutes and centrifuged at 13,000 rpm for 10 minutes, and the supernatant transferred to vial inserts and injected into the liquid chromatography/tandem mass spectrometry (LC/MS-MS) system for analysis. Total protein concentration in the cell lysate was quantitated using a Pierce BCA Protein Assay Kit (Thermo Fisher, 23227) according to the manufacturer instructions. Cellular concentrations of lisaftoclax and venetoclax were normalized to protein concentration and expressed as pmol drug/mg protein.

The LC/MS-MS analysis was conducted using an ExionLC HPLC system (AB Sciex LLC, Framingham, MA) coupled with an atmospheric pressure ionization 5500 LC/MS-MS system (AB Sciex). HPLC separation was achieved using an Agilent C-18 column (5 μmol/L, 2.1 mm × 50 mm) (Agilent Technologies, Santa Clara, CA). Mobile phases contained acetonitrile, water, and formic acid (A, 5:95:0.1 by volume; B, 95:5:0.1 by volume), and the flow rate was 0.5 mL/min. The MS system was operated at electrospray ionization-positive ion mode and ion detection performed in the multiple-reaction-monitoring mode. Mass transition of m/z 882.3→638.3, 868.3→636.3, and 271.1→172.0 were monitored for lisaftoclax, venetoclax, and IS, respectively. The ion spray voltage and ionization temperature were set at 5,500 V and 500°C, respectively. Instrument parameters, including curtain gas, Gas 1, and Gas 2 (auxiliary gas), were set at 40, 55, and 55 psi, respectively. Compound parameters, including declustering potential and collision energy, were set at 58 and 41 V, 99 and 38 V, and 80 and 18 V for lisaftoclax, venetoclax, and IS, respectively. Data acquisition and processing were performed using Analyst software version 1.6.3 (AB Sciex).

A calibration curve was generated for sample quantitation. Samples were prepared by mixing a series of working solutions of lisaftoclax or venetoclax with blank cell lysate matrix containing IS, followed by the same processing procedure as described above. The calibration curve was obtained by plotting the peak area ratio of lisaftoclax or venetoclax to IS against the corresponding standard concentrations. Linear regression range was used, and the range of linearity was 1 to 3,000 nmol/L for each agent. Quality-control samples at low (3 nmol/L), medium (240 nmol/L), and high (2,400 nmol/L) concentrations were prepared independently in the same way, with acceptable accuracy (85%–115%).

Six- to 8-week-old male mice (founder group) were maintained and pretreated with ibuprofen (4.5 mL/water pouch). We implanted ∼5 mm of tumor tissue subcutaneously, along with 200 μL of Matrigel (Corning; Glendale, AZ), and monitored tumors daily. When xenograft tumors reached a volume of ∼500 to 1,500 mm³, mice were humanely euthanized and tissue fragments transplanted to new mice in order to expand xenograft tissue. Tumor tissue was also maintained in liquid nitrogen in a freezing medium containing 10% FBS, 5% DMSO, and 200 mmol/L trehalose in RPMI1640 medium. Male mice were chosen to sex match the index patient from whom the tumor was derived.

To test the effect of lisaftoclax, tumors extracted from the founder group were implanted subcutaneously into 60 mice (∼5 mm tumor tissue/mouse) and randomized to 4 groups of 8 each on Day 5 for treatments with (i) vehicle (60% phosphatidylcholine concentrate with propylene glycol [Phosal 50], 30% polyethylene glycol 400, and 10% ethanol); (ii) lisaftoclax (100 mg/kg); (iii) ABT-199 (100 mg/kg); and (iv) the Bruton tyrosine kinase (BTK) inhibitor ibrutinib (25 mg/kg; Selleck Chemicals; Houston, TX). From Day 5, all mice received daily treatment, and tumor volumes were measured by caliper every third day using the following formula:

where length represents the largest tumor diameter, and width represents the perpendicular tumor diameter. Percent treatment/control (T/C %) or percent regression (regression %) values were calculated using the above formula. Survival curves were recorded and compared within different treatment groups. Mice were observed for survival and euthanized when tumor burden reached 10% of body weight.

Values are expressed as mean ± SEM for normally distributed data and median ± range for non-normally distributed data. Data were analyzed for significance using Prism 6.0, according to paired or unpaired t tests, Mann–Whitney U tests, and/or one-way analysis of variance, with a two-tailed P = 0.0500. Overall survival was determined by Kaplan–Meier analysis. Other detailed protocols are reported in the Supplementary Materials and Methods.

All data associated with this study are present in the paper or Supplementary Materials. The authors agree to make additional data supporting the results or analyses presented in their paper available from the corresponding author (Y. Zhai) upon reasonable request.

Studies were conducted to identify new compounds with high affinity of binding to BCL-2 and excellent pharmacokinetic profiles in vivo. These investigations resulted in a series of novel analogs, ultimately showing lisaftoclax to be a highly potent, selective, orally active BCL-2 inhibitor (Fig. 1A).

There are two key structural differences between lisaftoclax (APG-2575) and the reference BCL-2 inhibitor venetoclax (ABT-199; Fig. 1B). At site 1, the dimethyl group was replaced by a cyclobutyl ring, and at site 2 the tetrahydro-2H-pyran group was replaced by 1,4-dioxan. Structure–activity relationship studies established that modifications at sites 1 and 2 were feasible, allowing lisaftoclax to still possess potent binding affinity for BCL-2, exhibit cellular activity in BCL-2–dependent cell lines, and demonstrate an acceptable pharmacokinetic profile in animals. In competitive FP binding assays, lisaftoclax had a very similar profile of binding to BCL-2, BCL-xL, and MCL-1 as compared with venetoclax, with a Ki < 0.1 nmol/L for BCL-2, a weaker affinity of binding to BCL-xL, and a much weaker affinity of binding to MCL-1 (Supplementary Fig. S1).

In vitro cellular viability activity of lisaftoclax was examined in BCL-2–dependent cell lines RS4;11, HL-60, and MV-4–11. IC₅₀ values of lisaftoclax in these cells were 3.6 nmol/L (RS4;11), 2.4 nmol/L (HL-60), and 1.9 nmol/L (MV4;11; each comparable with or lower than venetoclax; Fig. 1C). Despite its nanomolar binding affinity to BCL-xL, lisaftoclax showed minimal effects on cell viability in the SKLU-1 cell line, which depended on BCL-xL antiapoptotic protein for survival, as demonstrated by the cell line's sensitivity to BCL-xL–specific inhibitor WEHI-539 (27) and BCL-2/BCL-xL inhibitor ABT-263 (Fig. 1D, bottom; ref. 11). Consistent with its weak affinity of binding to MCL-1, lisaftoclax also had no effect on the viability of NCI-H23 cells, which were MCL-1–dependent, and cell viability was inhibited by MCL-1 inhibitor A1210477 (Fig. 1E, bottom; ref. 28).

CLL-patient–derived primary samples were more sensitive to lisaftoclax (vs. venetoclax), with IC₅₀ values that were 2-fold lower for lisaftoclax after 4 hours of exposure to each BCL-2 inhibitor (Fig. 2A, left; P = 0.05; Supplementary Table S2A). Additional CLL samples also showed more potent inhibition of cellular viability by lisaftoclax (P = 0.0022 vs. venetoclax; Fig. 2A, right; Supplementary Table S2B). In cellular proliferation assays, the median IC₅₀ of lisaftoclax in multiple myeloma cell lines was 3.6 μmol/L (vs. 4.4 μmol/L for venetoclax), and the median IC₅₀ in WMG cell lines was also lower (i.e., lisaftoclax more potent; 3.0 vs. 7.0 μmol/L; P = 0.0027). In primary human multiple myeloma cells, the median IC₅₀ was ≥2 times lower for lisaftoclax (1.33 μmol/L) compared with venetoclax (3.01 μmol/L; P = 0.0030; Fig. 2B; Supplementary Table S3). In an extended panel of 30 hematologic cancer cell lines, lisaftoclax alone showed activity that was generally equivalent to venetoclax (Fig. 2C; Supplementary Table S4).

To elucidate the mechanism of action of lisaftoclax, we implemented an advanced electrochemiluminescence assay (ECLA) that quantitates protein–protein complexes formed between proapoptotic and antiapoptotic BCL-2 family members. When treated with either lisaftoclax or venetoclax, RS4;11 [acute lymphoblastic leukemia (ALL)] and Toledo (DLBCL) cell lines, demonstrated a decrease in BCL-2:BIM but not BCL-xL:BIM complex in cell lysates (Fig. 3A). The latter complex was in fact increased by lisaftoclax and venetoclax, probably due to the binding of BIM liberated from BCL-2. Coimmunoprecipitation (Co-IP) assays using SU-DHL-4 (another DLBCL cell line) or Toledo cells confirmed the dose-dependent and selective disruption of BCL-2:BIM complex by lisaftoclax and venetoclax (Fig. 3B). Such rapid, time-dependent disruption of BCL-2:BIM complex by lisaftoclax in RS4;11 cells (by 2–6 hours) coincided with increased apoptosis in these cells (Fig. 3C).

We then investigated the ability of lisaftoclax to disrupt BCL-2:BIM complex formation in multiple myeloma and WMG models using ECLA. Similar to findings in RS4;11 and Toledo cells, we observed rapid and time-dependent disruption of BCL-2:BIM complexes in KMS-11 and BCWM.1 (WMG) cell lines. Lisaftoclax reduced BCL-2:BIM complexes by more than 50% within 12 hours [P = 0.0020 (KMS-11) and P = 0.0022 (BCWM.1)], with less than 10% complex remaining by 24 hours (P = 0.0032 and P = 0.0015, respectively vs. vehicle; Fig. 3D).

Further assessment of lisaftoclax was conducted by direct mitochondrial exposure using four cell lines with enforced expression of human BCL-2 (O-BCL-2), BCL-xL (O-BCL-xL), MCL-1 (O-MCL-1), or BFL-1/A1 (O-BFL-1/A1), as well as one additional line with homozygous deletions of both BAX and BAK [O-double knockout (DKO); ref. 29]. Lisaftoclax induced cytochrome c release from O-BCL-2 mitochondria at a concentration that was 2 orders of magnitude lower than in O-BCL-xL mitochondria (Fig. 3E). Lisaftoclax had no effect on cytochrome c release in O-MCL-1 or O-BFL-1/A1 mitochondria and did not trigger cytochrome c release in O-DKO (BAX/BAK DKO) mitochondria. Cellular toxicity assays demonstrated that lisaftoclax had greater activity in killing O-BCL-2 cells (vs. O-BCL-xL, O-MCL-1, and O-BFL1), but this activity was abolished in O-DKO and other cells (Fig. 3E).

In RPMI8226 (multiple myeloma) cells, cellular uptake essays showed that lisaftoclax entered tumor cells rapidly, with intracellular concentrations reaching a plateau in 2 hours that was sustained for the 6-hour incubation (Fig. 4A). Given that lisaftoclax entered RPMI8226 cells more rapidly, 1.44- to 1.61-fold higher intracellular concentrations were reached at each time point as compared with venetoclax (P < 0.0500). A similar pattern was observed with KMS-11 (multiple myeloma) cells (Fig. 4A), with intracellular lisaftoclax concentrations that were 1.46- to 1.92-fold higher (P < 0.0500 vs. venetoclax).

Consistent with the rapid cellular uptake of lisaftoclax, release of cytochrome c into the cytoplasm triggered by lisaftoclax was stronger compared with venetoclax, as indicated by immunoblot analysis (Supplementary Fig. S2A). Significantly higher downstream caspase-3/7 (proapoptotic) activity was further demonstrated with lisaftoclax in multiple myeloma (P = 0.0022) and WMG cell lines (P = 0.00230; each vs. venetoclax; Fig. 4B). Median lisaftoclax-induced apoptosis was also significantly higher (vs. venetoclax) in multiple myeloma (72.92% vs. 37.04%; P = 0.0022) and WMG (60.20% vs. 50.00%; P = 0.0411) cell lines (Supplementary Fig. S2B).

Characterization of lisaftoclax was further performed in primary tumor cells derived from patients with multiple myeloma or WMG. Mitochondrial outer membrane permeabilization (MOMP), a key biomarker for initiation of apoptosis, was significantly increased in cells exposed to lisaftoclax, represented by a decrease in mitochondrial depolarization (Fig. 4C). Compared with venetoclax, apoptosis resulting from lisaftoclax treatment was increased and significantly higher in primary tumor cells derived from patients with multiple myeloma (61.68% vs. 53.28%; P = 0.0015) or WMG (73.84% vs. 58.00%; P = 0.0043; Fig. 4D).

Western blot analysis of BCL-2 family proteins revealed that levels of proapoptotic proteins BIM and Noxa were rapidly increased by lisaftoclax (Fig. 4E; Supplementary Fig. S2C and S2D). In a time-course analysis of KMS-11 and BCWM.1 cells, BIM significantly increased within 2 hours after lisaftoclax treatment onset (P < 0.0001 vs. vehicle for each) and decreased by 6 hours. Treatment with lisaftoclax also significantly increased Noxa expression within 1 to 6 hours (KMS-11 P = 0.0004; BCWM.1 P = 0.0027 vs. vehicle). MCL-1 levels increased modestly in KMS-11 but not in BCWM.1 cells (Supplementary Fig. S2E and S2F). As a measure of the role of BIM in initiating apoptosis, more BIM was observed to be accumulated in mitochondria of KMS-11 cells after exposure to lisaftoclax (vs. vehicle or venetoclax; Fig. 4F).

Intrigued by the ability of lisaftoclax to trigger the mitochondrial apoptotic signaling cascade, we assessed its potential impact on mitochondrial bioenergetics by measuring oxygen consumption rate (OCR). Maximal respiration, which was achieved when cells were treated with the mitochondrial uncoupler carbonyl cyanide-4-phenylhydrazone (FCCP), was significantly lower in KMS-11 cells exposed to lisaftoclax (P = 0.0027 vs. venetoclax; P = 0.0023 vs. vehicle; Fig. 4G). Nonmitochondrial respiration (P = 0.0035) and spare respiratory capacity (P = 0.0150) were also significantly decreased in cells exposed to lisaftoclax (vs. vehicle). These changes resulted in a significant reduction in mitochondrial ATP production within lisaftoclax-treated cells (P = 0.0035 vs. vehicle; Fig. 4H), supporting the ability of lisaftoclax to induce damage to other mitochondrial functions when initiating apoptosis.

In a BCL-2–dependent RS4;11 xenograft murine model of ALL (Fig. 5A), tumor growth inhibition was noted after oral administration of lisaftoclax 6.25 mg/kg once daily by Day 15 (T/C 60%), peaking at a 100-mg/kg dose (T/C 4%), without apparent changes in body weight (Fig. 5B). A Toledo xenograft model of DLBCL further confirmed that tumor growth inhibition was proportional to lisaftoclax dose, with the greatest inhibition at 100 mg/kg (Fig. 5C).

A combination of lisaftoclax with anti-CD20 antibody rituximab in OCI-LY8 (DLBCL) tumor-bearing mice achieved better responses than either single agent, delaying tumor regrowth (Supplementary Fig. S3A). Also, in the OCI-LY8 model, a ternary combination of lisaftoclax, bendamustine, and rituximab (BR) yielded sustained tumor regression and longer duration of response [6/6 mice, 100% complete response (CR)], based on the modified Response Evaluation Criteria in Solid Tumors for animal studies (30), when compared with antitumor effects with lisaftoclax alone or BR dual combination (Fig. 5D). Further, in vivo target engagement of lisaftoclax on BCL-2 was tested in a xenograft model of AML (OCI-AML-3). Tumor tissues collected from lisaftoclax-treated tumor-bearing mice on Day 12 demonstrated a significant reduction of the BCL-2:BIM complex compared with vehicle control (Supplementary Fig. S3B), even in this insensitive model, because of high MCL-1 protein expression (31).

Using a 100-mg/kg dose, we next investigated the effect of lisaftoclax in a multiple myeloma xenograft model (KMS-11). Tumor burden was significantly reduced in the lisaftoclax-treated group by Day 20, as measured by bioluminescent signal intensity from Luc-labeled tumor cells (total flux; P = 0.0010 vs. vehicle), though there was no statistical difference between lisaftoclax- and venetoclax-treated mice (Fig. 5E). Lisaftoclax was well tolerated, with no significant decrease in body weight. Kaplan–Meier analysis demonstrated significantly longer survival in mice treated with lisaftoclax (median survival 25 vs. 21 days with vehicle control; P < 0.0500; Supplementary Fig. S3C). IHC analysis of staining intensity from resected lisaftoclax-treated tumors showed decreased Ki67 but significantly increased cleaved caspase-3 (P = 0.0220 vs. vehicle; Fig. 5F; Supplementary Fig. S3D).

In a WMG xenograft model (BCWM.1), a T/C ratio of 25.12% (with no apparent toxicities or weight loss) was evident after lisaftoclax treatment by Day 15. Median survival was longer in mice receiving lisaftoclax (29 days) compared with vehicle (21 days; P = 0.0190). Increased IHC staining intensity of cleaved caspase-3 (P = 0.0025 vs. vehicle) was observed in tumor tissues, confirming enhanced apoptosis in BCWM.1 tumor cells from lisaftoclax-treated mice (Supplementary Fig. S3E–S3G).

In addition to human cancer cell line-derived xenografts, a PDX model was established from a patient with ibrutinib-resistant WMG (Fig. 5G and H). Lisaftoclax was administered for 29 days to closely mimic one cycle of patient treatment. Mice exposed to lisaftoclax showed significantly lower tumor burden (T/C 17.56%) compared with venetoclax (P = 0.0002) or the BTK inhibitor ibrutinib (P = 0.0003; Fig. 5G). Correspondingly, mice treated with lisaftoclax showed significantly greater survival (63 days) compared with venetoclax (36 days; P = 0.002) or the other groups (vehicle 34 days; ibrutinib 38 days; Fig. 5H).

In an RS4;11 xenograft model, mice received one oral dose of lisaftoclax or venetoclax 6.25 mg/kg before tumor samples were collected for pharmacodynamic analysis. Cleaved caspase-3 was induced rapidly after 4 hours and peaked at 24 hours of lisaftoclax treatment. The increase in cleaved caspase-3 was accompanied by induction of poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, indicating comparable apoptosis-inducing activity triggered by lisaftoclax or venetoclax (Fig. 6A).

Pharmacokinetic analysis was carried out in similar models of RS4;11-bearing mice receiving one oral dose of lisaftoclax 6.25 mg/kg, 25 mg/kg, or 100 mg/kg, and venetoclax 25 mg/kg as a control. Dose-dependent increases in lisaftoclax were observed in both plasma and tumors. Mice dosed with lisaftoclax 100 mg/kg had a plasma C_max of 7,803.3 ng/mL and AUC_0-last of 123,401.2 hr•ng/mL (Supplementary Table S5). Lisaftoclax and venetoclax 25 mg/kg showed similar elimination half-life (t_1/2) values in plasma: 5.2 to 6.0 and 6.3 hours, respectively. In tumors, the t_1/2 ranged from 13.2 to 48.1 hours with the three lisaftoclax doses, as compared with 18.1 hours with venetoclax 25 mg/kg (Fig. 6B; Supplementary Table S5).

To evaluate the effect of lisaftoclax on functional hematopoiesis, bone marrow samples were collected from normal inbred BALB/c (H2d) female mice and seeded in a 96-well plate for treatments with DMSO (vehicle control) and increasing concentrations of lisaftoclax and venetoclax. After 24 and 72 hours of treatment, viability analysis showed that IC₅₀ values for lisaftoclax were approximately 20 μmol/L and 10 μmol/L, respectively. On the basis of IC₅₀ values, cells were similarly sensitive to lisaftoclax and venetoclax at both 48- and 72-hour time points (Supplementary Fig. S4).

Further assessments were conducted in male and female CD-1 mice that were orally administered lisaftoclax at 100, 300, and 1,000 mg/kg daily for 28 days. Mild-to-moderate hematologic effects related to lisaftoclax were observed only in mice administered 1,000 mg/kg/day, with fewer findings noted in animals receiving 100 or 300 mg/kg/day (Supplementary Table S6A). The changes included mildly lower red cell mass in animals administered 1,000 mg/kg/day; mildly lower mean corpuscular hemoglobin (MCH) and MCH concentration in males administered ≥300 mg/kg/day and females administered ≥100 mg/kg/day; and a mildly-to-moderately lower lymphocyte count, resulting in a lower white blood cell count, in animals administered ≥ 100 mg/kg/day.

Similar to the hematology test results, only a few minor, potentially lisaftoclax-related findings were noted in the bone marrow smears of male mice administered 1,000 mg/kg/day. The findings included a mildly higher total erythroid count, which was due to a higher metarubricyte count, and a mildly lower lymphocyte count (Supplementary Table S6B).

It has been challenging to develop BCL-2 inhibitors for cancer therapy because of the inherent difficulty in disrupting intracellular, protein–protein interactions of the BCL-2 family proteins with high precision. Certain inhibitors have been reported to antagonize BCL-2 function in preclinical models, including those that decrease BCL-2 protein level; however, they do not possess features of BH3 mimetics. Currently, there is only one BCL-2 inhibitor (a BH3 mimetic) approved by the FDA. BH3 profiling has recently emerged as a functional platform to determine if a BCL-2 inhibitor is a bona fide BH3 mimetic (29). This methodology enabled us to measure BCL-2 family protein–protein interactions, which determine mitochondrial priming and the threshold of cells committed to apoptosis (6, 32, 33). On the basis of this unique mechanism of action, it has been suggested that BH3-mimetic BCL-2 inhibitors need to rapidly reach a mitochondrial threshold to trigger apoptosis; their effects are thus driven by the rate of cell penetration and maximum concentration (C_max; ref. 3).

On the basis of structure–activity relationship studies, we have discovered lisaftoclax, a new BCL-2 inhibitor with high binding affinity and selectivity for the BCL-2 protein. Relative to more sensitive approaches such as homogeneous time-resolved fluorescence, which combines fluorescence resonance energy transfer with time-resolved measurement, the competitive FP binding assay demonstrated only a five-fold difference in affinity between BCL-2 and BCL-xL for lisaftoclax or venetoclax, which may be due to a limitation of the assay (26). Consistent with mechanisms of action ascribed to true BH3 mimetics, we have used BH3 profiling and cell death assays to demonstrate that lisaftoclax triggers cytochrome c release and apoptosis in BCL-2–dependent cell lines in a BAX/BAK–dependent manner. Knockdown of BAX and BAK completely blocks cytochrome c release and apoptosis in tumor cells treated with lisaftoclax. This mode of action coincides with disruption of BCL-2:BIM complexes. Once liberated from BCL-2, BIM can trigger BAX/BAK activation to increase MOMP, leading to apoptosis within 1 to 6 hours. Sample analysis using cancer cell lines and mouse xenograft models have shown that on-target engagement by lisaftoclax for the BCL-2:BIM complex underlies its antiproliferative and overall antitumor activities. A range of B-cell lymphoid cancers are sensitive to lisaftoclax, although there is some variation across the panel of cancer cells tested (as would be expected), with CLL and certain types of DLBCL (OCI-LY1), ALL (RS4;11), FL (DOHH2), and AML (MOLM-13) cells being the most sensitive.

Although lisaftoclax and venetoclax share a similar mechanism of action, the magnitudes of their anti-BCL-2 effects may differ. Treatment with lisaftoclax led to stronger caspase 3/7 activation and a greater extent of apoptosis-inducing activity in multiple myeloma and WMG cell lines and patient-derived primary samples. These features of lisaftoclax can likely be attributed to: (i) more rapid cellular uptake, with a higher accumulation in multiple myeloma cells (vs. venetoclax); (ii) more potent disruption of the BCL-2:BIM complex; (iii) stronger induction (by lisaftoclax) of prodeath BH3-only protein BIM and its translocation from the cytosol to the mitochondria; and/or (iv) more marked increases in levels of prodeath BH3-only protein Noxa (with lisaftoclax), which were not changed significantly by venetoclax. In addition to these cellular and molecular disparities between lisaftoclax and venetoclax, a PDX model derived from a patient with an aggressive form of WMG refractory to ibrutinib therapy illuminated a potentially clinically relevant distinction. Treatment with lisaftoclax markedly inhibited WMG tumor growth, leading to significantly prolonged survival in the model. In contrast, venetoclax displayed minimal antitumor activity. The biological mechanism underlying this difference is currently under investigation.

Lisaftoclax showed dose-dependent systemic and tumor tissue exposure in an RS4;11 xenograft model, with a slightly higher C_max than that of venetoclax but with a similar plasma t_1/2. The minimal effective dose of lisaftoclax was 6.25 mg/kg, which led to caspase-3 activation and PARP-1 cleavage as early as 4 hours and peaked at 24 hours after lisaftoclax treatment. While lisaftoclax demonstrated single-agent activity in an RS4;11 xenograft model, combination treatments revealed more pronounced tumor growth inhibition in a DLBCL xenograft model (OCI-LY8). When combined with rituximab, or rituximab and bendamustine, lisaftoclax exhibited a longer duration and deeper response (i.e., CR) compared with single or double agents, suggesting that combination therapies combining lisaftoclax with standard-of-care treatments may be more effective in certain types of cancers, such as lymphoma with heterogeneous dependencies on oncogenic or antiapoptotic proteins other than BCL-2.

The key mechanism of action of lisaftoclax is to disrupt the BCL-2 complex and antagonize its antideath function to resume apoptosis in cancer cells. Initiation of apoptosis requires activation of BAX/BAK with oligomerization for pore formation, leading to MOMP. Besides the canonical pathways central to the BCL-2 family of proteins, we also uncovered that lisaftoclax treatment reduced both mitochondrial and nonmitochondrial respiratory capacity, and decreased mitochondrial ATP production, as examined in multiple myeloma cells. Further, lisaftoclax induced more marked reductions in mitochondrial function compared with venetoclax in multiple myeloma and/or WMG cells. These findings suggest a wider cellular stress response triggered by damaged mitochondrion due to lisaftoclax and are aligned with a recent report showing that the electron transport chain complex was altered by venetoclax in T cells (34), in order to augment antileukemic activity via increased generation of reactive oxygen species.

Preliminary toxicity assessments of normal mouse hematology and hematopoiesis, both in vitro and in vivo, suggest that normal bone marrow tolerates lisaftoclax well. Low lymphocyte counts in peripheral blood and bone marrow samples after lisaftoclax treatment is likely related to the pharmacology of BCL-2 inhibitors, which would inhibit B cells and subsets of T cells (35). The high metarubricyte count observed in bone marrow smears of organisms treated with lisaftoclax may represent an early compensatory response to the lower red cell mass or a shift to mature erythroid precursors, but the exact mechanism was not determined. It is worth noting that hematology and bone marrow differential findings exhibited reversibility at the end of another 28-day recovery phase.

In conclusion, we have discovered a bona fide BH3 mimetic, BCL-2–selective inhibitor with potential biological and therapeutic relevance in several preclinical models of hematologic malignancies, including CLL, ALL, DLBCL, multiple myeloma, and WMG. Moreover, lisaftoclax treatment overcame BTK inhibitor resistance and inhibited tumor growth in a PDX model established from a patient with aggressive WMG that relapsed after ibrutinib. (For a summary of our working model, see Supplementary Fig. SS5.) Our findings show that lisaftoclax triggers mitochondria-mediated, BAX/BAK–dependent apoptosis in cell-based and xenograft models, with broad effects on mitochondrial functions.

Our findings should be considered in the context of potential study limitations. Although we have demonstrated that lisaftoclax confers stronger apoptosis-inducing and antitumor activity (vs. venetoclax) in multiple myeloma, WMG, and CLL models, activities of these BCL-2–selective inhibitors are equivalent in other models such as AML. Additional studies are warranted to further differentiate the agents in cancer-type or condition-specific models. Consistent with these encouraging preclinical results, several phase Ib and phase II clinical trials have been initiated and are currently ongoing to evaluate lisaftoclax in hematologic cancers as monotherapy or in combination with an anti-CD20 antibody or BTK inhibitor (NCT03537482; NCT04260217).

J. Deng was a full-time employee of and/or equity shareholder in Ascentage Pharma Group Inc. A. Paulus reports grants from Ascentage and personal fees from Ascentage during the conduct of the study; nonfinancial support and other support from Alpha2 Pharmaceuticals outside the submitted work. J. Chen reports grants from Ascentage during the conduct of the study, as well as a patent for BCL-2 inhibitors issued, licensed, and with royalties paid from Ascentage. L. Bai reports patents for US 8691184, US 9096625, and US 9403856 issued, licensed, and with royalties paid from Ascentage. D. McEachern reports a patent for WO2012103059A2 issued, licensed, and with royalties paid from Ascentage, a patent for WO/2006/050447 issued, a patent for WO/2006/099193 issued, and a patent for WO2012103059 issued, licensed, and with royalties paid from Ascentage. C.Y. Yang reports a patent for WO2012103059A2 issued, licensed, and with royalties paid from Ascentage, a patent for WO/2006/050447 issued, a patent for WO/2006/099193 issued, and a patent for US20120189539 issued, licensed, and with royalties paid from Ascentage. J. Ryan reports personal fees from Zentalis Pharmaceuticals outside the submitted work, as well as a patent for BH3 Profiling issued, licensed, and with royalties paid from Zentalis Pharmaceuticals. A. Letai reports personal fees from Zentalis Pharmaceuticals, Dialectic Therapeutitics, and Flash Therapeutics, as well as personal fees from Anji Onco outside the submitted work; in addition, A. Letai has a patent for BH3 profiling owned by Dana-Farber pending, issued, licensed, and with royalties paid from Zentalis and Dialectic. S. Ailawadhi reports grants from Ascentage during the conduct of the study; grants from GSK, AbbVie, Xencor, Janssen, Cellectar, Amgen, Pharmacyclics, and BMS; personal fees from Sanofi and Takeda; and personal fees from Beigene outside the submitted work. D. Yang reports personal fees from Ascentage during the conduct of the study; has patents issued for US8557812, US 163805, US20090092684A1, US7432304B2, US7354928B2, US20060084647A1, and US20040214902A1; is a board member within the funding institution; and owns stock/equity in Ascentage (HK: 6855). S. Wang reports grants, personal fees, and other support from Ascentage during the conduct of the study; grants, personal fees, and other support from Oncopia Therapeutics; grants and other support from Roivant Sciences; grants and personal fees from Proteovant Therapeutics; other support from Debiopharm; and personal fees from American Chemical Society outside the submitted work. In addition, S. Wang has a patent 10,829,488 issued, licensed, and with royalties paid from Ascentage; and is a member of the Board of Directors of Ascentage and a paid consultant of Ascentage. A. Chanan-Khan reports other support from Ascentage during the conduct of the study and outside the submitted work. In addition, A. Chanan-Khan has a patent for AT101 issued and licensed to Alpha2; and serves as the Chairman and CEO of Alpha2, which has a codevelopment agreement with Ascentage for their molecule AT-101. No disclosures were reported by the other authors.

J. Deng: Conceptualization and study design, data acquisition and interpretation, writing–original draft, review, editing, and approval; and investigation (led preclinical research, including mechanism of action, target engagement and apoptosis assay). A. Paulus: Conceptualization and study design, data acquisition and interpretation, writing–original draft, review, editing, and approval; and investigation (led the preclinical study of multiple myeloma [MM] and Waldenström macroglobulinemia [WM]). D.D. Fang: Conceptualization and study design, data acquisition and interpretation, writing–review, editing, and approval; and investigation (led preclinical research, including xenograft efficacy and pharmacokinetic and pharmacodynamic studies). A. Manna: Performed animal experiments and ECLA; data acquisition and interpretation; writing–review, editing, and approval; and investigation (MM and WM preclinical study). G. Wang: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (led preclinical research, including in vitro and in vivo pharmacology studies). H. Wang: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (preclinical research of pharmacokinetics study). S. Zhu: Data acquisition, interpretation, writing–review, editing, and approval; and investigation (preclinical research of cellular uptake in MM). J. Chen: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay). P. Min: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (led preclinical research, including in vitro and in vivo pharmacology studies). Y. Yin: Data acquisition, interpretation, writing–review, editing, and approval; and investigation (preclinical research on mechanism of action and target engagement). N. Dutta: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (led the MM and WM preclinical study and performed the coimmunoprecipitation and drug synergy testing). N. Halder: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (performed protein kinetic studies). G. Ciccio: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (assistance in establishing the patient-derived xenograft [PDX] model). J.A. Copland III: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (provided critical scientific oversight in establishing the patient xenograft model). J. Miller: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (assistance in establishing the PDX model). B. Han: Data acquisition for seahorse experiments, functional hematopoiesis in lisaftoclax-treated MM and CLL patient analysis; writing–review, editing, and approval. L. Bai: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). L. Liu: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). M. Wang: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). D. McEachern: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). S. Przybranowski: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). C.Y. Yang: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). J.A. Stuckey: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (discovery chemistry, FP binding assay, and cell viability assays). D. Wu: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (provided the first set of chronic lymphocytic leukemia [CLL] ex vivo studies). C. Li: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (provided the first set of chronic lymphocytic leukemia [CLL] ex vivo studies). J. Ryan: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (provided unique BH3 profiling in a panel of cell lines demonstrating lisaftoclax as a true BH3 mimetic). A. Letai: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (provided unique BH3 profiling in a panel of cell lines demonstrating lisaftoclax as a true BH3 mimetic). S. Ailawadhi: Data acquisition and interpretation, writing–review, editing, and approval; and investigation (led the MM and WM patient sample study study). D. Yang: Conceptualization and study design, data acquisition and interpretation, writing–review, editing, and approval. S. Wang: Conceptualization and study design, data acquisition and interpretation, writing–review, editing, and approval; and discovery investigation (performed discovery chemistry, fluorescence polarization [FP] binding assay, and cell viability assays). A. Chanan-Khan: Conceptualization and study design, data acquisition and interpretation, writing–review, editing, and approval; and investigation (led the MM and WM patient sample study study). Y. Zhai: Conceptualization and study design, data acquisition and interpretation, writing–review, editing, and approval.

We thank colleagues of Ascentage CMC and Analytic Center for providing lisaftoclax. The experiments and analyses carried out in the Mayo Clinic were supported in part by the Daniel Foundation of Alabama (to A. Chanan-Khan), the Predolin Foundation (to A. Chanan-Khan), the Mayo Clinic Cancer Center (CA015083, to A. Chanan-Khan), and the Mayo Clinic Multiple Myeloma SPORE (P50 CA186781–01A1, to A. Paulus). We thank Dr. Laura Lewis-Tuffin for her technical and scientific support for use of the Attune NxT Flow Cytometer and BD FACS Aria II cell sorter in the Mayo Florida Cellular Imaging and Flow Cytometry Shared Resource. Assistance in manuscript research and preparation was provided by Mr. Gutkin and Drs. Tao, Pathak, Ogba, and Fletcher (all with Ascentage). Preparation of this study report was informed by Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for preclinical research (Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010;8:e1000412). This study and its report were supported by Ascentage Pharma Group Corp Ltd.

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