Evading apoptosis is a hallmark of cancer that is achieved by altering the dynamic binding interactions between pro-survival (BCL-2, BCL-XL, BCL-W, MCL-1, BFL-1) and pro-death (e.g., BAX, BAK, BAD, BIM, NOXA, PUMA) BCL-2 family members. BCL-2 itself plays a dominant role in survival of malignant hematologic cells; a dependency which has been exploited therapeutically as exemplified by the established clinical activity of BCL-2 inhibitors in patients with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), multiple myeloma (MM), and acute myeloid leukemia (AML) (Fowler-Shorten et al., 2024). ABBV-453, a next-generation BCL-2 inhibitor, was designed for enhanced molecular and biological properties to improve upon the patient experience relative to available therapies. Specifically, incorporation of macrocyclic elements into the hot-spot binding regions of BCL-2 afforded a constrained molecule with high potency, selectivity and durable in vivo activity at low doses. In vitro, ABBV-453 possesses sub-nM affinity for BCL-2 (KI = 0.228 nM) and high selectivity over family members BCL-XL and MCL-1. The high affinity and selectivity of ABBV-453 translates to potent cell killing activity human tumor cell lines dependent upon BCL-2, including the acute lymphocytic leukemia (ALL) cell line RS4;11 (EC50 = 4.7 nM in the presence of 10% human serum), and greatly decreased activity in human tumor cell lines dependent on BCL-XL (Molt-4 EC50 = 2.85 µM) and MCL-1 (H929 EC50 >10 µM) for survival. Moreover, the cellular activity of ABBV-453 is on-target and mechanism-based as demonstrated by the disruption of BCL-2:BIM complexes and the rapid activation of the intrinsic apoptosis signaling pathway (as evidenced by caspase-3-7 activation and dissipation of the inner mitochondrial membrane potential) and the caspase dependent externalization of annexin-V at the plasma membrane of ABBV-453 treated tumor cells. Activity of ABBV-453 monotherapy was compared to other clinically relevant BCL-2 inhibitors in an in vivo subcutaneous xenograft model of ALL. ABBV-453 demonstrated superior growth inhibition of the RS4;11 xenograft compared to both sonrotoclax or lisaftoclax at equivalent doses and schedule. ABBV-453 has the potential to be the best-in-class next-generation BCL-2 inhibitor and is actively being investigated in phase 1 clinical trials in relapsed or refractory (R/R) MM (ClinicalTrials.gov ID, NCT05308654) and R/R CLL/SLL (NCT06291220).

B7H3, an immune checkpoint protein, inhibits immune cell response and is overexpressed in various malignancies, correlating with poor prognosis. B7H3 is selectively highly expressed in acute myeloid leukemia (AML) blast but not in normal hematopoietic stem cells (Tyagi et al., Blood, 2022). BCL-XL has emerged as a promising target in AML, particularly in patients who have progressed from venetoclax (VEN)-based therapy, as their cancer cells shift dependency from BCL-2 to BCL-XL (Zhang et al. Nat Cancer, 2020). Our study investigated the preclinical activity of ABBV-155 (mirzotamab clezutoclax; ABBV), a novel antibody-drug conjugate targeting B7H3 with a selective BCL-XL inhibitor payload. We hypothesized that ABBV could selectively deliver BCL-XL inhibitor to B7H3-expressing AML cells, inducing apoptosis alone or in combination with the BCL-2 inhibitor venetoclax. We first tested B7H3 expression in four AML patient-derived xenograft (PDX) models and found high expression in all samples (56%-97%). Sample #3912018, with the highest B7H3 expression and multiple mutations (FLT3-ITD, DNMT3A, IDH1, KIT, NPM1), was selected for in vivotesting. NSG aged 6-8 weeks mice were intravenously injected with PDX cells (1e6 cells per mouse) following sublethal irradiation (250 cGy). Upon detecting circulating leukemia cells by hCD45 flow cytometry (41 days post-transplant), mice were randomized into four groups (8 mice per group) to receive vehicle, ABBV (10 mg/kg, i.p., weekly for 4 weeks), VEN (50 mg/kg, p.o., 5 days per week for 4 weeks), or a combination of ABBV and VEN. Tumor burden and treatment safety were monitored weekly via hCD45% in peripheral blood and mice body weight, respectively. After the last dose, 3 mice per group were euthanized for biomarker analysis (pharmacodynamic study), while the remaining 5 mice received a second treatment cycle after a 2-week break, continuing to monitor tumor burden and survival. All treatments were well-tolerated, with less than 10% weight loss after 4 weeks. Compared to the vehicle group, both ABBV and VEN inhibited leukemia burden, with the combination achieving the best inhibition after 3 weeks of treatment (average circulating hCD45%: 19.4%, 11.1%, 10.2%, and 0.9% for vehicle, ABBV, VEN, and combination, respectively). In the pharmacodynamic study, only the combination group significantly (p=0.0021) inhibited tumor burden, as reflected by spleen weight (average: 0.448, 0.600, 0.660, and 0.077 g for vehicle, ABBV, VEN, and combination). The combination largely eliminated leukemia in bone marrow, liver, and spleen, indicated by undetectable hCD45% (<1%) compared to leukemia infiltration (>95%) in other groups. Analysis of BCL-2 family complex showed that VEN dissociated BCL-2/BIM complexes, increasing BCL-XL/BIM and MCL-1/BIM complexes. ABBV dissociated BCL-XL/BIM complexes, increasing BCL-2/BIM complexes. The combination completely disrupted both BCL-2/BIM and BCL-XL/BIM complexes, supporting the hypothesis that maximal release of BH3-only proteins from two pro-survival proteins leads to highly efficacious AML clearance. During the treatment break, leukemia burden increased rapidly in all groups except the combination. The second cycle treatment failed to inhibit progression in single-agent cohorts. On the contrary, mice in the combination treatment cohort survived, achieving significantly better survival (p=0.0382) with median overall survival of 122 days compared to 77, 69.5, and 77 days for vehicle, ABBV, VEN, respectively. In summary, our study demonstrates the ABBV effectively dissociates the BCL-XL/BIM complex in a venetoclax-refractory AML PDX model. The combination targeted engagement of BCL-XL with B7H3-ADC and of BCL-2 with VEN significantly inhibits leukemia burden and prolongs survival in AML PDX models through synergistic release of BH3-only proteins. These findings suggest a promising therapeutic strategy for AML, particularly in cases resistant to venetoclax-based treatments.

Ferroptosis is a regulated non-apoptotic cell death process characterized by iron-dependent lipid peroxidation. This process has recently emerged as a promising approach for cancer therapy. Peroxidation of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) is necessary for the execution of ferroptosis. Ferroptosis is normally suppressed by glutathione peroxidase 4 (GPX4), which reduces lipid hydroperoxides to lipid alcohols. Some evidence indicates that GPX4 may be a useful target for drug development, yet factors that govern GPX4 inhibitor sensitivity in vivo are poorly understood. We find that pharmacological and genetic loss of GPX4 function was sufficient to induce ferroptosis in multiple adherent ("2D") cancer cell cultures. However, reducing GPX4 protein levels did not affect tumor xenograft growth when these cells were implanted in mice. Furthermore, sensitivity to GPX4 inhibition was markedly reduced when cells were cultured as spheroids ("3D"). Mechanistically, growth in 3D versus 2D conditions reduced the abundance of PUFA-PLs. 3D culture conditions upregulated the monounsaturated fatty acid (MUFA) biosynthetic gene stearoyl-CoA desaturase (SCD). SCD-derived MUFAs appear to protect against ferroptosis in 3D conditions by displacing PUFAs from phospholipids. Various structurally related long chain MUFAs can inhibit ferroptosis through this PUFA-displacement mechanism. These findings suggest that growth-condition-dependent lipidome remodeling is an important mechanism governing GPX4 inhibitor effects. This resistance mechanism may specifically limit GPX4 inhibitor effectiveness in vivo .

Overexpression of the antiapoptotic protein B-cell lymphoma-extra large (BCL-X-L) is associated with drug resistance and disease progression in numerous cancers. The compelling nature of this protein as a therapeutic target prompted efforts to develop selective small-molecule BCL-X-L inhibitors. Although efficacious in preclinical models, we report herein that selective BCL-X-L inhibitors cause severe mechanism-based cardiovascular toxicity in higher preclinical species. To overcome this liability, antibody-drug conjugates were constructed using altered BCL-X-L-targeting warheads, unique linker technologies, and therapeutic antibodies. The epidermal growth factor receptor-targeting antibody-drug conjugate AM1-15 inhibited growth of tumor xenografts and did not cause cardiovascular toxicity nor dose-limiting thrombocytopenia in monkeys. While an unprecedented BCL-X-L-mediated toxicity was uncovered in monkey kidneys upon repeat dosing of AM1-15, this toxicity was mitigated via further drug-linker modification to afford AM1-AAA (AM1-25). The AAA drug-linker has since been incorporated into mirzotamab clezutoclax, the first selective BCL-X-L-targeting agent to enter human clinical trials.

Supplemental Table 2: Navitoclax EC50s in breast cancer cell lines in the presence or absence of MCL-1 siRNA.

Supplementary Fig. S2 from ABT-263 and rapamycin act cooperatively to kill lymphoma cells <i>in vitro</i> and <i>in vivo</i>

Down regulation of Bcl-XL by MS417 and ABBV-075

supplemental figure legends

Supplementary Fig. S1 from ABT-263 and rapamycin act cooperatively to kill lymphoma cells <i>in vitro</i> and <i>in vivo</i>

Tumor environmental factors such as hypoxia or the presence of prosurvival cytokine such as IL-6 does not significantly change cellular sensitivity to ABBV-075

Supplemental Figure 3: MCL-1 dependency dictates cellular response to the CDK9 inhibitor flavopiridol and correlates with kinetics of death resulting from MCL-1 siRNA treatment.

Supplemental Table 1: Viability of breast cancer cell lines treated with PBS, scrambled siRNA (Sc; 20nMol) or MCL-1 siRNA (20nMol) for 72hrs compared to non-treated cells with associated genomic info.

<p>Apoptosis induction by ABBV-075 across cancer cell lines</p>

Supplemental Figure 5: Flavopiridol synergizes with navitoclax in breast cancer cell lines in vitro.

Supplemental Figure 1: Validation of MCL-1 siRNA.

1. Measured pKa values of ABBV-167 (Table S1. Ionization constants of ABBV-167) 2. Preparation of ABBV-167 on large scale (Scheme S1: Synopsis of GMP synthesis of ABBV-167) 3. Preparation of clinical ABBV-167 drug supply 4. Animal pharmacokinetics (Table S2. Single dose pharmacokinetics of prodrug 3 and parent venetoclax in mouse and dog after IV dosing of prodrug 3. Table S3. Single dose pharmacokinetics of 3 after oral dosing in mouse and dog. Table S4. Single dose pharmacokinetics of parent venetoclax in mouse and dog after oral dosing of 3.) 5. Clinical study (Study design, Pharmacokinetic and statistical analysis, Safety and tolerability assessments, Clinical pharmacokinetic results, Table S5. Effect of prodrug and food on venetoclax bioavailability, Safety results) 6. Crystallography methods (Protein preparation. BCL-2 ABBV-167 complex crystallization. X-ray structure determination. Table S6. Diffraction data collection and refinement statistics for BCL-2 in complex with ABBV-167 (PDB code 7LHB))

Supplementary Table 1 from A Small-Molecule Inhibitor of Bcl-X_L Potentiates the Activity of Cytotoxic Drugs <i>In vitro</i> and <i>In vivo</i>

Abstract Overexpression of the prosurvival Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) is commonly associated with tumor maintenance, progression, and chemoresistance. We previously reported the discovery of ABT-737, a potent, small-molecule Bcl-2 family protein inhibitor. A major limitation of ABT-737 is that it is not orally bioavailable, which would limit chronic single agent therapy and flexibility to dose in combination regimens. Here we report the biological properties of ABT-263, a potent, orally bioavailable Bad-like BH3 mimetic (Ki's of <1 nmol/L for Bcl-2, Bcl-xL, and Bcl-w). The oral bioavailability of ABT-263 in preclinical animal models is 20% to 50%, depending on formulation. ABT-263 disrupts Bcl-2/Bcl-xL interactions with pro-death proteins (e.g., Bim), leading to the initiation of apoptosis within 2 hours posttreatment. In human tumor cells, ABT-263 induces Bax translocation, cytochrome c release, and subsequent apoptosis. Oral administration of ABT-263 alone induces complete tumor regressions in xenograft models of small-cell lung cancer and acute lymphoblastic leukemia. In xenograft models of aggressive B-cell lymphoma and multiple myeloma where ABT-263 exhibits modest or no single agent activity, it significantly enhances the efficacy of clinically relevant therapeutic regimens. These data provide the rationale for clinical trials evaluating ABT-263 in small-cell lung cancer and B-cell malignancies. The oral efficacy of ABT-263 should provide dosing flexibility to maximize clinical utility both as a single agent and in combination regimens. [Cancer Res 2008;68(9):3421–8]