Lisaftoclax in Combination with Alrizomadlin Overcomes Venetoclax Resistance in Acute Myeloid Leukemia and Acute Lymphoblastic Leukemia: Preclinical Studies

Hematologic malignancies can be treated with several drug combinations, including targeted therapies; however, development of resistance is a major challenge (1). For adult patients with chronic lymphocytic leukemia (CLL) and certain subpopulations with acute myeloid leukemia (AML), approval of B-cell lymphoma (BCL)-2–selective inhibitor venetoclax has enhanced treatment. Beyond these approved indications, the efficacy of BCL-2 inhibitors alone or in combination with other therapeutics is being evaluated in many other cancer types, with promising clinical outcomes in patients with Waldenström macroglobulinemia and multiple myeloma (2–7).

In the management of AML, chemotherapy has been a treatment mainstay, opening avenues for targeted pharmacologic innovation. Although basic and translational research has largely elucidated the molecular landscape of AML and enhanced treatment outcomes, most patients experience primary treatment resistance and/or disease relapse, with fatal consequences (8). A large U.S. epidemiologic study showed that the 4-week mortality rate in AML among adults was as high as 45%, with a particularly dismal prognosis in elderly patients (9).

As observed with most targeted therapies, the clinical benefit of venetoclax is hampered by intrinsic and acquired drug resistance (10). Inactivation of tumor suppressor p53 and upregulation of other antiapoptotic BCL-2 family proteins [mainly myeloid cell leukemia-1 (MCL-1) and BCL–extra-large (BCL-xL)] is among the most common mechanisms exploited by cancer cells to escape BCL-2–mediated apoptosis, along with loss of proapoptotic BCL-2–associated X protein (BAX; ref. 11).

Apart from these issues, BCL-2 gene mutations have recently emerged as a de novo mechanism conferring resistance to BCL-2 inhibition in patients with CLL (12, 13). The most frequent mutations occur in genes encoding the BH3 domain of the BCL-2 protein, including G101V, D103E, and moieties adjacent to the BH3 domain (e.g., V156D), interfering with binding of BCL-2 to pro-death proteins or BH3 mimetics. Thus, an emerging unmet medical need is to develop agents or regimens that effectively overcome drug resistance mediated by these mechanisms.

As a key regulator of apoptosis, p53 functions upstream in the apoptotic cascade, through both direct effects at the mitochondrial membrane and transcriptional activation of proapoptotic BCL-2 family molecules, including BCL-2–like protein 11 (BIM), p53 upregulated modulator of apoptosis (PUMA), BAX, and BCL-2 antagonist/killer 1 (BAK; refs. 14–16). The prevailing balance of the equilibrium of interactions between proapoptotic and antiapoptotic proteins (BCL-2, BCL-xL, and MCL-1) determines whether tumor cells live or die (17). In addition, mouse double minute 2 (MDM2) homolog regulates p53 activity, blunting p53-mediated tumor suppression (18).

In addition to its direct effects on cancer cells, alrizomadlin can regulate the tumor cell immune microenvironment and augment T-lymphocyte–mediated immunity. In preclinical models, targeting of the p53–MDM2 axis by alrizomadlin augmented T-cell MDM2, stabilizing T-cell signal transducer and activator of transcription 5 (STAT5), activating CD8⁺ T-cell–mediated antitumor immunity, and inducing an IFNγ signature (19). Cross-talk between BCL-2– and MDM2-mediated apoptosis pathways may also play a role in regulating cell survival. To date, no MDM2 inhibitor has been approved for cancer treatment.

In preclinical studies of AML, treatment that cotargeted BCL-2 and MDM2–p53 apoptotic pathways synergistically enhanced antitumor effects. The combination of venetoclax and MDM2 inhibitor idasanutlin increased antitumor effects and induced synthetic lethality in T53 wild-type (TP53^WT) AML cell lines and xenograft models (20–22). Mechanistic studies demonstrated that p53 activation promoted degradation of antiapoptotic protein MCL-1, and that BCL-2 inhibition shifted outcomes of p53 activation from G₁ arrest to apoptosis. Ultimately, p53 activation and BCL-2 inhibition reciprocally overcome tumor cell apoptosis resistance (20–22). A phase Ib study evaluating venetoclax combined with MDM2 inhibitor idasanutlin in elderly patients with relapsed/refractory or previously treated secondary AML suggested that this binary regimen had a manageable safety profile and encouraging antileukemic activity (23). Among 49 evaluable patients, the antileukemic response rate was 41%. Additional clinical investigations have been initiated to evaluate therapies that inhibit both MDM2 and BCL-2, in AML and/or myelodysplastic syndrome (23).

The chief aim of the present study was to evaluate whether the combination of two novel, targeted agents—clinical-stage BCL-2 inhibitor lisaftoclax (APG-2575; refs. 24–26) and MDM2/p53 inhibitor alrizomadlin (APG-115; refs. 27–30)—could overcome BCL-2 mutation-driven resistance to approved BCL-2 inhibitor venetoclax in patients with AML.

Human AML cell lines MV-4–11 and MOLM-13 were purchased from ATCC (Manassas, VA). Human AML cell line OCI-AML-3 and acute lymphoblastic leukemia (ALL) cell line RS4;11 were sourced from Cobioer (Nanjing, China). MV-4–11 cells were propagated in Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY; catalog no. C12440500BT) supplemented with 10% FCS (AUSGENEX, Loganholme, QLD, Australia; catalog no. FBSSA500-S). Other cell lines were propagated in RPMI1640 medium (Gibco; catalog no. C11875500BT) containing 10% FCS. All experiments utilized genetically authenticated, microbial-free cells in their exponential phases of growth.

Lisaftoclax and alrizomadlin were disssoved in DMSO (Sigma, St. Louis, MO; catalog no. D8418) and further diluted in cell culture media for in vitro experiments. For in vivo studies, alrizomadlin was suspended in 0.2% hydroxypropyl methylcellulose (Sigma; catalog no. H7509–25G) and lisaftoclax dissolved in 40% polyethylene glycol 400 (PEG 400, Sigma; catalog no. 91893–1L-F) and 60% Phosal 50 PG (phosphatidylcholine concentrate; Germany Lipoid GmbH, Germany; catalog no. 368315–31700201006). Both agents were administered once daily or every other day via oral gavage in a volume of 10 mL/kg.

According to previously reported protocols (31), venetoclax-sensitive RS4;11 cells were exposed to gradually increasing venetoclax concentrations over a period of a few months until they became resistant at 10 μmol/L. The resulting cells were named RS4;11^199R cells.

To generate RS4;11 cells harboring mutated BCL-2, lentiviral vectors carrying BCL-2 mutants (including G101V, D103E, G101V/D103E, and V156D) in frame with a Flag (polypeptide protein epitope) tag were electroporated into RS4;11 cells. Transfected cells were selected with puromycin for cells stably expressing mutant BCL-2. Expression of BCL-2 mutants was confirmed by western blot analysis using an anti-Flag antibody.

Bone marrow aspirates were obtained from patients with relapsed or refractory AML. Written informed patient consent was obtained from all patients in accordance with the Declaration of Helsinki, and protocols were reviewed and approved by an institutional Ethics Committee.

To extract primary AML cells, we processed bone marrow aspirates by removing erythrocytes using a Cell Lysis Solution (Promega, Madison, WI; catalog no. A7933), and isolated mononuclear cells were washed twice with PBS. To assist in verifying an AML diagnosis, we prepared single-cell suspensions and stained them for cell surface markers, human anti-CD45 (BD Biosciences; catalog no. 555485) and human anti-CD33 antibody (BD Biosciences; catalog no. 562854); these steps were taken before plating cells for further analysis. Primary AML cells were then assessed by flow cytometry using an Attune NxT Flow Cytometer (Thermo Fisher Scientific).

where (Dχ)1 and (Dχ)2 represent the concentration of each drug alone to exert χ% effect, while (D)1 and (D)2 signify concentrations of the two drugs in combination to elicit the same effect. A CI value of less than 1 indicates synergism; 1, additivity; and greater than 1, antagonism (32). In the current combination study, cellular proliferative data were further analyzed with CI < 0.1 labeled as 5⁺ for a very strong synergistic effect; CI between 0.1 and 0.3 labeled as 4⁺ for a strong synergistic effect; and CI between 0.3 and 0.7 labeled as 3⁺ for a medium synergistic effect.

For cell-cycle analyses, AML cells were exposed to alrizomadlin, lisaftoclax, or a combination of both agents for 24 hours. After treatment, cells were harvested and stained with the FITC BrdUrd Flow Kit (BD Biosciences; catalog no. 51–2354AK) according to the manufacturer's instructions. A total of 20,000 events were acquired and proportions of cells in each phase of the cell cycle calculated using FlowJo™ software v10.4.2 (BD Biosciences, San Jose, CA).

Apoptosis analyses were conducted as previously described (29). In brief, AML cells were treated in triplicate for 24 hours and subjected to flow cytometry analyses using the Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (BD Biosciences; catalog no. 556547). Results were expressed as percentages of Annexin V⁺ cells that were apoptotic.

Western blot analyses were also performed as previously described (30). The following primary antibodies were used in the experiments (each from Cell Signaling Technology, Danvers, MA): anti-p53 (catalog no. 2524S), anti-p21 (catalog no. 2947S), anti-BAX (catalog no. 5023S), anti-MCL-1 (catalog no. 94296S), anti-caspase-3 (catalog no. 9665S), anti-cleaved caspase-3 (catalog no. 9664S), anti-cleaved PARP-1 (catalog no. 9532S), anti-BCL-2 (catalog no. 4223S), anti-BCL-xL (catalog no. 2764S), anti-PUMA (catalog no. 4976S), anti-β-actin (catalog no. 4970S), and anti-Flag (catalog no. 14793S).

Streptavidin-coated 96-well plates (MSD, Rockville, MD; catalog no. L15SA-2) were used to immobilize biotin-labeled anti-BIM antibody (Abcam, Cambridge, UK; catalog no. ab32158). Duplicated protein samples (50 μg) were subsequently added to each well and plates incubated for 1 hour at room temperature with rotations at 600 rpm to pull down BIM. After three washes with a wash buffer (1 × PBS with 0.5% Tween-20), sulfo-tagged anti–BCL-2 antibody (Thermo Fisher Scientific, Waltham, MA; catalog no. MA5–11757) was added to each well and the reactants incubated with rotations at 600 rpm for 1 hour at room temperature. After three further washes with buffer, we added 150 μL of MSD GOLD read buffer A (MSD; catalog no. R92TC-1) to each well and measured electroluminescence signal intensity on an MSD Sector S 600 plate reader.

In vivo antitumor studies, including tumor size and body weight assessments, were performed in cell- and patient-derived xenograft (CDX/PDX) mouse models as previously described (29). CDX studies were conducted in the GenePharma animal facility (Suzhou, China) with approvals of protocols and experimental procedures by the GenePharma Institutional Animal Care and Use Committee (IACUC). PDX experiments were conducted at Crown Bioscience Inc. (Jiangsu, China) with approval by its IACUC. Animals were acclimatized to the environment for at least 3 days, then transferred to a temperature- (20°C to 26°C) and humidity-controlled (40%–60%, relative humidity) specific pathogen–free room with a 12-hour light/12-hour dark cycle during the experimental period. Animals were housed in cages, with no more than five mice per cage. All animals had free access to sterile drinking water and food.

To establish subcutaneous CDX models, we subcutaneously injected OCI-AML-3 (1 × 10⁶) and MV-4–11 (5 × 10⁶) cells into the right flanks of 6- to 8-week-old NOD/SCID mice. Similarly, RS4;11, RS4;11^199R, RS4;11^G101V-Flag, and RS4;11^V156D-Flag cells (1 × 10⁷ with Matrigel) were subcutaneously injected into SCID beige mice. When tumors reached a mean volume of approximately 100 to 150 mm³, mice were randomly grouped on the basis of body weight and tumor volume and treated with vehicle or the indicated active agents.

To establish a systemic MOLM-13 AML model, we intraperitoneally pretreated 6- to 8-week-old female NOD/SCID mice with cyclophosphamide 150 mg/kg for 2 consecutive days. Luciferase-labeled MOLM-13 cells (1 × 10⁷) were then intravenously injected through the tail vein. After 3 days, tumor-bearing mice were randomized to each treatment and monitored periodically for bioluminescence intensity (i.e., tumor burden) with an IVIS Lumina II imaging system (PerkinElmer, Waltham, MA). Mice were also assessed daily for development of hind-limb paralysis, abdominal swelling resulting from disease progression, and/or more than 20% loss in body weight. These safety indices served as humane endpoints.

Antitumor studies in a systemic AM7577 AML PDX model were performed at Crown Bioscience. The genotype of this model was described in a previous study (25). Briefly, this PDX model harbors FLT3-ITD, TP53^WT, IDH2^R140Q, DNMT3A^R882H, NPM1^ins, and CEBPA^ins. Two million (2 × 10⁶) AM7577 cells suspended in 0.1 mL sterile PBS were injected into 6- to 8-week-old female NOD/SCID mice through the tail vein. After 28 days, when the average proportion of human CD45⁺ and CD33⁺ cells (i.e., tumor burden) in the peripheral blood reached approximately 0.5%, tumor-bearing mice were randomized to four groups and treated as indicated. Proportions of human CD45⁺ and CD33⁺ cells in the peripheral blood of each mouse were monitored weekly by flow cytometry.

After treatment, we collected liver, spleen, and bone marrow tissue samples for IHC staining of human CD45. Cells from peripheral blood, spleen, and bone marrow of two femurs were also collected and stained for FITC-conjugated human CD45 (BioLegend, San Diego, CA; catalog no. 304038) and APC-conjugated human CD33 (BioLegend, catalog no. 303408) antibodies. After incubation, cells were washed twice with PBS and subjected to flow cytometry analysis on the Attune NxT Flow Cytometer (Thermo Fisher). Data were analyzed using FlowJo software and reported as percentages of total viable human CD45⁺/CD33⁺cells.

IHC analyses were performed at Crown Bioscience. In brief, bone marrow, spleen, and liver tissues were formalin-fixed, paraffin-embedded (FFPE). FFPE blocks were sectioned into 4-μm thick sections with a manual rotary microtome. Tissue sections were deparaffinized and rehydrated using 100% xylene and decreasing concentrations of ethanol solutions. Rehydrated tissue sections were incubated with monoclonal mouse anti-human CD45 antibody (Dako/Agilent Technologies, Carpinteria, CA; catalog no. GA751), followed by sequential incubation with bond polymer refine detection (Leica Biosystems, Nussloch, Germany; catalog no. DS9800), which resulted in a brown precipitate at the antigen site. All stained sections were scanned with a panoramic digital slide scanner (3DHISTECH, Budapest, Hungary). High-resolution images of whole sections were generated and further analyzed.

Plasma and tumor concentrations of lisaftoclax and alrizomadlin were analyzed by quantitative LC/MS-MS as described previously (30). Briefly, quantitative LC/MS-MS analysis was conducted using an Exion HPLC system (AB Sciex, Framingham, MA) coupled to an API 5500 mass spectrometer (AB Sciex) equipped with an API electrospray ionization source. The Phenomenex Titank phenyl-Hexyl column (50 mm × 2.1 mm, 5-μm particle size) was used to achieve high-performance liquid chromatography separation. The injection volume was 2 μL and the flow rate was kept constant at 0.5 mL/min. Chromatography was performed with mobile phase A, acetonitrile: water: formic acid (5:95:0.1, in volume) and B, acetonitrile: water: formic acid (95:5:0.1, in volume). The mass spectrometer was operated at electrospray ionization positive ion mode (ESI⁺) for lisaftoclax and alrizomadlin. The results were presented as dot plots with each dot representing a sample.

Data are presented as the mean ± SD or SEM of the indicated number of biological replicates in each experiment. One-way ANOVA followed by Games-Howell post-test was applied to assess the statistical significance of differences between multiple treatment groups. All data were analyzed using SPSS version 19.0 (IBM, Armonk, NY). Prism version 9 (GraphPad Software Inc., San Diego, CA) was used for graphic presentations.

All data associated with this study are present in the paper or Supplementary Appendix. Raw data will be provided upon request from the corresponding author.

The antiproliferative activity of combined treatment with lisaftoclax and alrizomadlin was first evaluated in three TP53^WT human AML cell lines, including OCI-AML-3, MV-4–11, and MOLM-13. Although either lisaftoclax or alrizomadlin alone displayed variable antiproliferative activity, their combination consistently exerted strong synergistic activity in all three AML cell lines (Fig. 1A). Compared with vehicle control, lisaftoclax or alrizomadlin induced apoptosis in all three cell lines. Compared with effects with each agent alone, lisaftoclax combined with alrizomadlin exhibited strong and statistically significant synergy in inducing apoptosis (Fig. 1B and C).

Antileukemic effects of lisaftoclax and alrizomadlin were evaluated in three CDX models derived from MV-4–11, OCI-AML-3, and MOLM-13 cell lines. In the subcutaneous MV-4–11 CDX model, lisaftoclax was associated with a T/C value of 55.1% (Supplementary Fig. S1A, top), as compared with 67.1% after a pulsed high dose of alrizomadlin. Antitumor effects were potentiated to a T/C value of 9.1% for lisaftoclax with alrizomadlin, which was statistically significantly lower than with vehicle control (P < 0.001) or alrizomadlin treatment (P < 0.05). The synergy ratio of 4.06 is consistent with synergistic antileukemic activity for the combination. Among six animals in the combination group, the ORR was 83% and DCR 100%, including five mCRs and one mSD. No significant loss of mouse body weight was observed in any treatment group (Supplementary Fig. S1A, bottom). Taken together, these results suggest that combined treatment with lisaftoclax and alrizomadlin exhibits strongly synergistic antileukemic effects in the mouse MV-4–11 CDX model.

The subcutaneous AML CDX model was established from OCI-AML-3 cells, which are resistant to venetoclax because of intrinsic upregulation of MCL-1 (35). Owing to rapid growth of xenografts in this model, experiments were terminated on day 15. Lisaftoclax alone showed limited antitumor activity, with a T/C value of 61.69% on day 15 (Supplementary Fig. S1B, top). Alrizomadlin exerted potent antitumor activity, with a T/C value of 37.84% on day 15.

Paralleling findings in AML cell lines, lisaftoclax combined with alrizomadlin caused significant synergistic in vivo antitumor activity, with a T/C value of 8.13% and a corresponding synergy ratio of 2.85. In addition, no significant loss of mouse body weight was observed in any treatment group (Supplementary Fig. S1B, bottom). Pharmacokinetic analysis revealed that in comparison with the corresponding single agents, the combination treatments with lisaftoclax and alrizomadlin caused no significant change in systemic or local exposures of each agent, indicating a low risk of drug–drug interactions (Supplementary Fig. S2).

The combined benefit of lisaftoclax and alrizomadlin was further confirmed in a difficult-to-treat, disseminated, systemic AML CDX model derived from luciferase-labeled MOLM-13 cells. Compared with the vehicle control group, treatment with lisaftoclax exerted limited antileukemic activity according to changes in tumor burden (Supplementary Figures S1C and S1D). However, treatment with lisaftoclax improved animal survival, with a median overall survival time of 24 days compared with 19 days in the control group (Supplementary Fig. S1E). Compared with vehicle, pulsed high-dose treatment with alrizomadlin also reduced tumor burden and extended median survival to 34.5 days (Supplementary Fig. S1C–S1E). Notably, treatment with the combination led to no detectable tumor burden until day 19, with a median overall survival of 63 days (Supplementary Fig. S1C–S1E).

In the PDX model, human AML tumor burden was assessed by the proportion of human CD45⁺/CD33⁺ cells in peripheral blood and other key organs of mice bearing human primary AML cell xenografts (AM7577). Compared with vehicle control, only lisaftoclax (and not alrizomadlin) decreased tumor burden in peripheral blood throughout the study (Fig. 2A). Treatment with both agents exerted substantial antileukemic activity, without tumor burden in peripheral blood (Fig. 2A). Tumor burdens (i.e., mean percentages of human CD45⁺/CD33⁺ cells) in peripheral blood were 50.42% in the control, 22.56% in the lisaftoclax, 45.53% in the alrizomadlin, and 0.04%, in the combination group.

Cotargeting of BCL-2 by lisaftoclax and MDM2 by alrizomadlin also significantly decreased tumor burden in the spleen and bone marrow (Fig. 2B). Further, IHC analysis showed that single-agent lisaftoclax, but not alrizomadlin, reduced tumor burden in the liver (as illustrated by IHC; Fig. 2C and D). Lisaftoclax combined with alrizomadlin significantly decreased tumor burden in the liver, spleen, and bone marrow compared with either agent alone or control (Fig. 2C and D). Spleen weights were also significantly reduced by lisaftoclax plus alrizomadlin compared with either treatment alone or vehicle control (Fig. 2E).

Using the AML PDX model, we confirm that the combination of lisaftoclax and alrizomadlin significantly reduced tumor burden in bone marrow, spleen, and liver, suggesting that it may eradicate minimal residual disease in AML, with more durable clinical responses.

Putative mechanisms underlying the synergistic activity of lisaftoclax combined with alrizomadlin were investigated in OCI-AML-3 AML cells and xenografts. Compared with controls, treatment with alrizomadlin significantly upregulated p53 and p21 protein levels on western blots, suggesting that it activates p53/p21 signaling pathways (Fig. 3A; Supplementary Fig. S3). Alrizomadlin, alone or combined with lisaftoclax, also downregulated antiapoptotic protein MCL-1 compared with control. Moreover, combined treatment increased tumor cell apoptosis, as evidenced by upregulated BAX and cleaved caspase-3 and PARP-1 proteins, which are hallmarks of apoptosis. The foregoing results were also observed in OCI-AML-3 xenografts (Fig. 3B). Finally, lisaftoclax combined with alrizomadlin also downregulated antiapoptotic BCL-xL (Fig. 3B; Supplementary Fig. S3).

We further investigated the effect of lisaftoclax combined with alrizomadlin on BCL-2:BIM, a key protein–protein complex that determines tumor cell life or death. Although alrizomadlin alone had no significant effect, lisaftoclax (alone or combined with alrizomadlin) disrupted (i.e., decreased) the BCL-2:BIM complex (Fig. 3C). These findings confirm the on-target activity of lisaftoclax as a bona fide BH3 mimetic.

In summary, alrizomadlin treatment plays an important role in achieving synergistic antileukemic activity when combined with lisaftoclax. By activating p53/p21 signaling pathways, alrizomadlin upregulates proapoptotic BAX and downregulates both antiapoptotic MCL-1 and BCL-xL, hence priming AML cells to apoptosis via BCL-2 inhibition. In contrast, the combination treatment did not further enhance disruption of the BCL-2:BIM complex, suggesting that synergy is not mediated through the protein–protein complex.

Next, we interrogated TP53^WT BCL-2–driven human ALL cell line RS4;11 to determine if combined treatment can overcome venetoclax resistance. First, antitumor activity of the combination was investigated in a CDX model derived from parental RS4;11 cells as the baseline. As anticipated, administration of lisaftoclax, but not alrizomadlin, exerted significant antitumor activity (Fig. 4A). Lisaftoclax combined with alrizomadlin achieved synergy, with a T/C value of 1%. The corresponding synergy ratio of 19.02 is indicative of synergistic antileukemic activity, and the ORR was 100%.

Compared with the parental RS4;11 line, venetoclax-resistant tumor cells have increased MCL-1, and decreased BAX, expression (Fig. 4B). RS4;11^199R cells also had reduced BCL-2:BIM and BCL-2:PUMA complexes, along with increases in BCL-xL:BIM, BCL-xL:PUMA, and MCL-1:BIM complexes (Fig. 4C). Consequently, the RS4;11^199R CDX model had low sensitivity to venetoclax treatment even with increased doses (50 mg/kg to 100 mg/kg; Fig. 4D). Of potential interest, RS4;11^199R CDX tumors had a slight response to alrizomadlin, with a T/C value (77.91%) suggesting that RS4;11^199R cells may partially depend on the MDM2-p53 pathway for survival. Accordingly, the combination of lisaftoclax and alrizomadlin yielded significant tumor growth inhibition, with a T/C value of 31.61% (Fig. 4D).

Several factors contribute to resistance against BCL-2–targeted therapies, including upregulation of MCL-1, loss of BAX, a shift from BCL-2 to BCL-xL, and an increase in MCL-1–constituted protein–protein complexes (31, 35–37). BCL-2 mutations may also confer resistance to BCL-2 inhibition in some patients (12, 13). We introduced major clinically relevant BCL-2 mutations (i.e., G101V, D103E, V156D, G101V-D103E) into parental RS4;11 cells. In these genetically engineered cells, expression of Flag-tagged BCL-2 mutants was confirmed by western blot analyses (Supplementary Fig. S4A). In general, higher expression of total BCL-2 was detected by a BCL-2 antibody, indicating a combination of endogenous BCL-2 WT and Flag-tagged BCL-2 mutant proteins (e.g., as seen in G101V BCL-2 mutant cells).

Engineered to overexpress WT BCL-2 gene, RS4;11 WT cells remained sensitive to venetoclax and lisaftoclax, with IC₅₀ values lower than 40 nmol/L (Supplementary Fig. S4B). In contrast, RS4;11 cells carrying various BCL-2 mutants became resistant to the treatments, as evidenced by at least a 10-fold increase in IC₅₀ values in cells carrying the BCL-2^V156D mutation. Cells harboring BCL-2^G101V/D103E double mutations were the most resistant, with IC₅₀ values greater than 10 μmol/L (i.e., a 250-fold increase). In addition, the BCL-2^G101V and BCL-2^G101V-D103E mutant cells displayed substantially reduced BCL-2:BIM complex compared with BCL-2^WT cells, leading to a higher apoptotic threshold (Supplementary Fig. S4C). Although BCL-2^D103E mutant cells exhibited a higher BCL-2:BIM signal, BCL-2:BIM complexes were more difficult to disrupt in the presence of the BCL-2^D103E mutation, conferring resistance to BH3 mimetic inhibitors (38).

Lisaftoclax combined with alrizomadlin synergistically inhibited proliferation of RS4;11 cells carrying BCL-2^G101V, BCL-2^D103E, or BCL-2^V156D mutation in vitro (Fig. 5A; Supplementary Fig. S5). The combination synergistically induced cellular apoptosis, as evidenced by an increase in Annexin V⁺ signal in RS4;11 cells carrying BCL-2^G101V (Fig. 5B). As a single agent, alrizomadlin treatment led to cell-cycle arrest at G₀–G₁ (Fig. 5C), while lisaftoclax resulted in a higher proportion of sub-G₁ apoptotic cells. Hence, the combination reduced S phase and increased G₂–M phase, leading to increased accumulation in the sub-G₁ phase (Fig. 5C).

In CDX models derived from either BCL-2^G101V– or BCL-2^V156D–mutant cells, lisaftoclax exhibited no or limited activity, alrizomadlin slightly more potent activity, and the combination synergistic antitumor activity (Fig. 5D). Mechanistically, treatment with alrizomadlin most likely mitigated lisaftoclax-driven MCL-1 upregulation and enhanced BAX and PUMA expression (Fig. 5E). In summary, our findings suggest that lisaftoclax combined with alrizomadlin can overcome BCL-2 mutation–driven venetoclax resistance in preclinical models.

The primary AML samples derived from 2 patients with different molecular/mutational and karyotype profiles who had experienced treatment failure on venetoclax combined with hypomethylating agent azacytidine, were treated with lisaftoclax in combination with alrizomadlin ex vivo. Patient clinical characteristics are presented in Supplementary Table S1. The primary AML cells were treated for 24 to 48 hours with lisaftoclax and alrizomadlin, alone or combined with serial dilutions of each drug. According to the proportion of human CD45⁺ and CD33⁺ primary cells, cell viability in AML patient 01 (CD45⁺/CD33⁺ cells proportion was 99.92%) was measured using CellTiter-Glo assays, and cellular viability of AML patient 02 (CD45⁺/CD33⁺ cells proportion was 76%) was evaluated by flow cytometry. The results showed that lisaftoclax or alrizomadlin can inhibit the viability of primary AML cells from patient 01, and indeed, synergies (CI < 1.0,) were observed (Fig. 6A). In AML patient 02, dose-dependent induction of apoptosis was observed after treatment with lisaftoclax or alrizomadlin for 24 or 48 hours, according to the overall drug combination dose–response matrix pattern. In addition, the average synergy score calculated on the basis of the Loewe model (39, 40) in SynergyFinder application (https://synergyfinder.fimm.fi) for combined lisaftoclax and alrizomadlin was 13.66 at 24 hours and 13.28 at 48 hours (Fig. 6B), indicating synergistic effects of these two agents. Taken together, we have shown that combined lisaftoclax and alrizomadlin reduces cell viability and augments programmed cell death in primary AML specimens from patients with venetoclax-resistant AML (Fig. 6).

Intrinsic and acquired resistance to BCL-2 inhibitors may compromise treatment responses in both approved and additional indications. Resistance to BCL-2 inhibition may stem from upregulation of antiapoptotic proteins MCL-1 and BCL-xL (11, 35), loss of BAX (41), and/or gain of BCL-2 gene mutations (12, 38). These mutations affect genes encoding the inside of (or adjacent to) the BH3 domain, weakening binding of BH3 mimetics such as venetoclax to BCL-2.

One plausible pharmacologic approach to overcome such resistance is to develop a new generation of BCL-2 inhibitors that target specific BCL-2 mutants. Accomplishing this objective is time consuming, and newly developed therapeutics may apply only to a fraction of patients with corresponding mutations, largely limiting their clinical utility. Alternatively, a rational combination treatment with clinical-stage agents that attenuates resistance factors and restores BAX expression may be a more viable strategy to address this urgent unmet medical need.

Cotargeting BCL-2 and MDM2-p53 apoptotic pathways may enable synergistic proapoptotic effects, which have recently garnered attention in cancer therapy. In particular, blocking an alternative escape pathway with one agent, while targeting the key oncogenic driver pathway with another, culminates in cancer cell death. Synergistic effects resulting from coinhibition of BCL-2 and MDM2-p53 have been demonstrated in preclinical settings (20, 22) and are currently under clinical evaluation (42). However, it remains largely unknown whether combined therapies targeting these pathways can overcome venetoclax resistance.

Lisaftoclax (APG-2575) and alrizomadlin (APG-115) are under active clinical development, as monotherapy or combination therapy, in various hematologic and solid malignancies (NCT02935907, NCT03611868, NCT04275518, NCT03781986, NCT03913949, NCT03537482, NCT04215809, and NCT04260217). Lisaftoclax is a potent and orally bioavailable BH3 mimetic BCL-2 inhibitor that selectively binds to BCL-2 with a high affinity (Ki < 0.1 nmol/L), restoring tumor cell apoptosis by liberating pro-death proteins (especially BIM) from protein–protein complexes (24, 25). Alrizomadlin is a potent MDM2 inhibitor that binds to human recombinant MDM2 protein with high affinity (IC₅₀ = 3.8 ± 1.1 nmol/L), reactivates the p53 tumor-suppressor pathway, and induces apoptosis in cancer cells (29). Preliminary clinical antitumor activity and favorable safety profiles of both agents make it feasible to cotarget BCL-2 and MDM2 apoptogenic pathways in patients with hematologic malignancies.

In our research, combination of the BCL-2 inhibitor lisaftoclax with the MDM2 inhibitor alrizomadlin exerts strongly synergistic antiproliferative, apoptogenic, and antitumor activity in both cancer cell lines, as well as CDX and PDX models of venetoclax-sensitive and venetoclax-insensitive human AML and ALL. The synergy is likely driven by upregulation of proapoptotic protein BAX and downregulation of antiapoptotic proteins (i.e., MCL-1, BCL-xL), biasing the prevailing balance toward cell death. In addition to its effect on the mitochondrial apoptotic pathway, lisaftoclax augments p53-driven cell-cycle regulation, enhancing accumulation of sub-G₁ apoptotic cells.

We also developed venetoclax-resistant cellular and xenograft models that mimic clinical resistance. In venetoclax-induced resistant RS4;11 cells, our studies demonstrated upregulation of antiapoptotic protein MCL-1 and downregulation of proapoptotic protein BAX (11, 41). RS4;11 cells exhibited increases in MCL-1 and BCL-xL complexes, including MCL-1:BIM, MCL-1:PUMA, BCL-xL:BIM, and BCL-xL:PUMA, suggesting that formation of these complexes may play an important role in conferring venetoclax resistance. Collectively, our results provide new insights into putative mechanisms underlying venetoclax resistance.

Strikingly, the combination of lisaftoclax and alrizomadlin overcame venetoclax resistance in venetoclax-induced or genetically engineered cellular and CDX and PDX models, as well as primary (ex vivo) samples from patients with AML. Our study evaluated ex vivo primary samples derived from patients with R/R AML that failed to respond to BCL-2 inhibitor venetoclax treatment. Treatment with another BCL-2–selective inhibitor (lisaftoclax) in combination with MDM2-p53 inhibitor alrizomadlin, synergistically reduced tumor cell viability and proliferation. These findings echo work by Andreef's research team, who demonstrated that dual targeting of BCL-2 (using lisaftoclax) and MDM2 (using alrizomadlin), potentiated apoptosis in acquired venetoclax-resistant AML cells and prolonged survival in murine models (43, 44).

Studies have demonstrated that TP53-mutant AML cells are less sensitive to approved BCL-2 inhibitor venetoclax. One putative mechanism to explain this phenomenon is that TP53 increases expression of endogenous apoptogenic BH3 mimetic proteins. These include Noxa, PUMA, and BIM. Conversely, loss of TP53 could decrease expression of these proteins and hence limit release of BCL-2 in response to the BH3 mimetics, culminating in a reduction of BAX/BAK activation underlying tumor cell programmed cell death (45, 46).

Alrizomadlin may also confer immunomodulatory effects while targeting the MDM2-p53 axis by augmenting T-cell MDM2, stabilizing T-cell STAT5, while also promoting CD8⁺ T-cell survival and function, favorably polarizing tumor-associated macrophages and inducing an IFNγ signature (19). These potential immune-based benefits of alrizomadlin are under active investigation.

To date, lisaftoclax or alrizomadlin monotherapy has been well tolerated in clinical trials (24, 27). Also warranting further attention in the future are clinical data on progression-free and overall survival, as well as potential mechanisms of action underlying the combination regimen in AML. We report that combined treatment of lisaftoclax and alrizomadlin restored BAX expression and downregulation of MCL-1, which were major contributors to antitumor activity observed in this setting. For the first time to our knowledge, we demonstrate that co-targeting BCL-2 and MDM2-p53 pathways overcomes BCL-2 (and other) mutant-driven venetoclax resistance via multiple potentially clinically relevant mechanisms.

In conclusion, drug resistance is a perennial problem with targeted therapeutics and other treatments for AML, and previous researchers have pointed toward targeting downstream apoptosis machinery as a promising approach to elicit cell death while minimizing resistance, including the fact that BCL-2 inhibition may reciprocally overcome resistance to MDM2-p53 blockade by biasing cellular responses from pro-survival arrest at G₁ to apoptosis (20). Our data offer support for synergistic effects of BCL-2 inhibitor lisaftoclax when combined with MDM2-p53 inhibitor alrizomadlin across multiple sensitive and resistant (intrinsic or acquired) AML and ALL preclinical models. Uniquely (to our knowledge), our study also revealed evidence of potential synergistic clinical benefits when primary (ex vivo) samples from patients with venetoclax-resistant AML showed reduced cellular proliferation and viability when treated with BCL-2 inhibitor lisaftoclax and MDM2-p53 inhibitor alrizomadlin. These findings provide a sound rationale to overcome such resistance in the clinic. Our studies provide a novel and potentially viable combination treatment strategy to benefit patients with hematologic malignancies, especially disease that has relapsed or become refractory to venetoclax therapy.

Y. Zhai reports other support from Ascentage Pharma outside the submitted work. D.D. Fang reports being a full-time employee of the listed company on the article when the work was performed. J. Deng reports being a full-time employee of Ascentage Pharma Group when the relevant study and manuscript were conducted and prepared. K. Zhang reports being a full-time employee of Ascentage Pharma Group when the study and manuscript were conducted and prepared. D. Yang reports personal fees from Ascentage Pharma Group Corp Ltd. (Hong Kong) during the conduct of the study; in addition, D. Yang has a patent for US8557812 issued, a patent for US163805 issued, a patent for US20090092684A1 issued, a patent for US7432304B2 issued, a patent for US7354928B2 issued, a patent for US20060084647A1 issued, and a patent for US20040214902A1 issued. In addition, Dr. Yang is a board member within the funding institution; he also owns stock/equity in Ascentage Pharma Group International (HK: 6855). No disclosures were reported by the other authors.

Y. Zhai: Conceptualization, supervision, funding acquisition, investigation, visualization, methodology, writing–original draft, writing–review and editing. Q. Tang: Investigation, methodology, writing–original draft, project administration. D.D. Fang: Data curation, investigation, methodology, writing–original draft. J. Deng: Data curation, investigation, methodology, writing–original draft, writing–review and editing. K. Zhang: Methodology, writing–review and editing. Q. Wang: Methodology, writing–review and editing. Y. Yin: Investigation, methodology, writing–review and editing. C. Fu: Investigation, methodology, writing–review and editing. S.-L. Xue: Conceptualization, supervision, funding acquisition, investigation, visualization, methodology, writing–review and editing. N. Li: Supervision, funding acquisition, visualization, methodology, writing–review and editing. F. Zhou: Investigation, methodology, writing–review and editing. D. Yang: Supervision, funding acquisition, investigation, visualization, methodology, writing–review and editing.

This study and its report were supported by Ascentage Pharma Group Corp Ltd. (Hong Kong). We thank Ascentage CMC and Analytic Center colleagues for synthesizing the studied compounds and providing quality control. Ashutosh K. Pathak, MD, PhD, MBA, FRCP (Edin.), Stephen W. Gutkin, Ndiya Ogba, PhD, and Paul Fletcher, PhD, with Ascentage Pharma provided further substantive input in manuscript research and preparation. Preparation of this study report was informed by Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for preclinical research (47).

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