Prediction of venetoclax activity in precursor B-ALL by functional assessment of apoptosis signaling

Abstract

Deregulated cell death pathways contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Intrinsic apoptosis signaling is regulated by different proapoptotic and antiapoptotic molecules: proapoptotic BCL-2 homology domain 3 (BH3) proteins activate prodeath molecules leading to cellular death, while antiapoptotic molecules including B-cell lymphoma 2 (BCL-2) prevent activation of prodeath proteins and counter-regulate apoptosis induction. Inhibition of these antiapoptotic regulators has become a promising strategy for anticancer treatment, but variable anticancer activities in different malignancies indicate the need for upfront identification of responsive patients. Here, we investigated the activity of the BCL-2 inhibitor venetoclax (VEN, ABT-199) in B-cell precursor acute lymphoblastic leukemia and found heterogeneous sensitivities in BCP-ALL cell lines and in a series of patient-derived primografts. To identify parameters of sensitivity and resistance, we evaluated genetic aberrations, gene-expression profiles, expression levels of apoptosis regulators, and functional apoptosis parameters analyzed by mitochondrial profiling using recombinant BH3-like peptides. Importantly, ex vivo VEN sensitivity was most accurately associated with functional BCL-2 dependence detected by BH3 profiling. Modeling clinical application of VEN in a preclinical trial in a set of individual ALL primografts, we identified that leukemia-free survival of VEN treated mice was precisely determined by functional BCL-2 dependence. Moreover, the predictive value of ex vivo measured functional BCL-2 dependence for preclinical in vivo VEN response was confirmed in an independent set of primograft ALL including T- and high risk-ALL. Thus, integrative analysis of the apoptosis signaling indicating mitochondrial addiction to BCL-2 accurately predicts antileukemia activity of VEN, robustly identifies VEN-responsive patients, and provides information for stratification and clinical guidance in future clinical applications of VEN in patients with ALL.

Fig. 1. Heterogeneous sensitivities for VEN in BCP-ALL.

Cell death induction (flow cytometry, forward/side scatter criteria) upon exposure of a BCP-ALL cell lines (N = 6, 72 h; from left [low EC₅₀, sensitive] to right [high EC₅₀, insensitive]: RS4;11, KOPN-8, UoCB6, REH, RCH-ACV, and Nalm-6), b patient-derived BCP-ALL xenograft samples (N = 27, 24 h, from left [low EC₅₀, sensitive] to right [high EC₅₀, insensitive]: PDX1, PDX2, …, PDX27) or peripheral blood mononuclear cells from healthy donors (N = 3, dashed lines) to increasing concentrations of VEN (0.1, 1, 10, 50, 100, 250, 500 nM, 1, 3, 5, and 10 µM) showing heterogeneous half maximal effective concentrations (EC₅₀) indicating variable VEN sensitivities of BCP-ALL. (See also Supplementary Tables 1 and 2)

Fig. 2. Functional BCL-2 dependence indicates ex vivo VEN activity.

Assaying the dependence of mitochondrial apoptosis signaling on different regulating molecules interrogating binding of specific BH3 peptides (BH3 profiling). a Experimental procedure, ALL cells are permeabilized, incubated with the respective peptide followed by detection of cytochrome c release. b Binding table showing interaction of the BH3-peptides (columns) with the respective apoptosis-regulating molecule (rows). Mitochondrial priming by c VEN, e BAD, and g BAD-HRK is significantly associated with ex vivo venetoclax sensitivity (linear regression; R², correlation coefficient; p, significance) and d, f, h predictive for ex vivo response of ALL cells to venetoclax (ROC/receiver operating characteristic curve; AUC, area under the curve; p, significance)

Fig. 3. Functional BCL-2 dependence indicates preclinical antileukemia activity in vivo.

Individual patient-derived BCP-ALL xenograft samples (N = 12) were transplanted onto pairs of recipient mice and treated with either VEN or vehicle for 10 days. After treatment, mice were tightly monitored for onset of leukemia-related morbidity. a Survival times of leukemia bearing mice treated with VEN (gray bars) or vehicle (black bars) (left diagram) and corresponding VEN-induced survival differences (‘delta survival’, right diagram). b Association of direct VEN priming with preclinical VEN sensitivities of BCP-ALL in vivo (linear regression; R², correlation coefficient; p, significance) and c predictive value of direct VEN priming for post-VEN survival of treated mice (ROC/receiver operating characteristic curve; AUC, area under the curve; p, significance). d Strong association of functional BCL-2 dependence (mitochondrial BAD-HRK priming) with in vivo VEN responses, and e high predictive value of BAD-HRK priming for survival of VEN treated mice. Preclinical in vivo VEN responses analyzed in larger treatment groups confirming f low (PDX13; N = 8 mice per group), g intermediate (PDX10; N = 10 mice per group), and h strong (PDX2, five mice per group) VEN sensitivity of the respective patient-derived xenograft leukemia observed in the preclinical trial (Kaplan–Meier analysis; p, significance by log-rank test; VEN, venetoclax; CTRL control)

Fig. 4. Functional BCL-2 dependence indicates in vivo preclinical antileukemia activity in an independent cohort of patient-derived ALL samples.

Individual patient-derived ALL xenograft samples (BCP-ALL N = 5, T-ALL N = 3) were transplanted onto groups of recipient mice and treated with either VEN or vehicle. After treatment, mice were regularly monitored for the appearance of leukemia cells (≥5% or more of mCD45⁻huCD45⁺huCD19⁺ or huCD7⁺) in the peripheral blood. a Survival times of leukemia bearing mice treated with VEN (gray bars) or vehicle (black bars, left diagram) and corresponding VEN-induced survival differences (‘delta survival’, right diagram). b Significant association of functional BCL-2 dependence (mitochondrial BAD-HRK priming) with preclinical VEN sensitivities of ALL in vivo (linear regression; R², correlation coefficient; p, significance) and c high predictive value for post-VEN survival (ROC/receiver operating characteristic curve; AUC, area under the curve; p, significance)