Acquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia

Abstract

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

Graphical abstract

Figure 1.

Venetoclax-based therapies drive selection of novel BAX variants in relapsed AML. (A) Patients with either primary refractory or relapsed AML divided according to whether venetoclax (VEN) was combined with lower intensity (hypomethylating agent or low-dose cytarabine [LDAC]) or high intensity (infusional cytarabine + idarubicin) chemotherapy. The venetoclax dose used is indicated, along with the FLT3 and TP53 mutation status and BM blast percentage at the time of treatment failure. The time spent in remission (months) is shown as a green bar. Treatment failure is shown as a pink-colored bar cap. Patients refractory to therapy spent no time in remission. No BAX variants were identified among patients with primary refractory disease. At relapse, 7 patients (ID number shown) were found to harbor ≥1 BAX variant (red star within pink cap) and the dominant predicted BAX protein change is shown. (B) The distribution of BAX variant allele frequencies (yellow circles) at relapse are shown in the venetoclax-resistant cohort (n = 41), along with a control population of patients with AML relapsing after prior intensive chemotherapy (n = 34). The predicted protein change is indicated for BAX variants with variant allele frequencies (VAFs) ≥5%. (C) Schematic overview of BAX variants. Protein domain structure of BAX and identified variants observed in patients treated with venetoclax or standard chemotherapy. The presence of four BCL-2 homology (BH) domains and the transmembrane domain (TM) are indicated along with the location of each of the nine alpha helices. The red bar indicates the position of the hydrophobic groove. Missense variants, frameshift/nonsense variants, and splice site variants are denoted by pink, green, and blue circles, respectively. (D-G) Fishplot representation of selected patients with BAX variants (also refer to supplemental Figure 1). Clonal architecture presented in fishplot format was inferred from bulk sequencing showing clonal dynamics of leukemic or preleukemic clones in serial samples from 4 venetoclax-treated patients with detected BAX mutations. The dominant BAX subpopulation is shown in red in each case. (D-E) BAX variants emerging with leukemic relapse. (F) A BAX mutation emerges in remission, suggesting presence in an expanded preleukemic population after suppression of the original diagnostic AML clone. (G) BAX mutation is present at diagnosis and persists in remission after venetoclax-based therapy, despite suppression of several AML-related mutations. Duration of therapy is denoted by the gray arrow, clinical status including first complete remission (CR1), CR2, or measurable residual disease (MRD). Available cytogenetic, BM blast information, and time elapsed to remission and relapse are indicated below each fishplot. ∗indicates time points verified by single-cell DNA sequencing. Freq, frequency.

Figure 2.

BAX variants are observed in leukemic cell populations. (A) Proportion of cells in CAL-021 at relapse identified as WT or containing leukemia-associated mutations (top). Clones with leukemia-containing mutations are listed in decreasing order. Schematic of mutation zygosity for each clone (bottom). (B) Two-dimensional UMAP plot of CAL-021 at relapse showing specific phenotypic populations based on antibody tags. CAL-021 cells clustered into four main blood cell compartments including myeloid and monocytic blast populations is shown (top left). The remaining 5 UMAP plots are colored according to the genotype of each individual DNA variant. (C) Proportion of each mutant clone in CAL-021 observed at relapse within specified blood cell lineages. (D) Proportion of cells in CAL-013 after one week of venetoclax treatment identified as WT or containing leukemia-associated mutations (top). Clones with leukemia-containing mutations are listed in decreasing order. Schematic of mutation zygosity for each clone (bottom). (E) Two-dimensional UMAP plot of CAL-013 after one week of venetoclax treatment according to specific phenotypic populations identified by antibody tags. Cells clustered into six major blood cell compartments, including myeloid and monocytic blast populations (top left). The remaining five UMAP plots are colored according to the genotype of each DNA variant detected. (F) Percentages of each mutant clone observed in CAL-013 after 1 week of venetoclax treatment in specified blood cell lineages. HET, heterozygous; HOM, homozygous; NK, natural killer.

Figure 3.

Acquired resistance to BH3-mimetics selects for BAX loss in vitro. (A) VAF of indicated variants in OCI-AML3 cells with acquired resistance to BH3-mimetics compared with dimethyl sulfoxide (DMSO) control. OCI-AML3 cells, which harbor a naturally occurring BAX E41Gfs∗33 abnormality were continuously passaged with progressively higher concentrations (maximum 3 μM) of BCL-2i resistant (BCL-2i-R), MCL1i resistant (MCL1i-R), or combined BCL-2i-R and MCL1i-R (combo R). Cells were then treated in the absence of drug(s) for 6 weeks and BAX targeted amplicon sequencing was performed and compared with DMSO control. (B) Immunoblot profiling of BCL-2 family proteins in OCI-AML3 cultures tolerant to 3 μM of drug (DMSO, BCL-2i-R, MCL1i-R, or combo R). (C-E) Sensitivity of BH3-mimetic resistant (tolerant to 3 μM) OCI-AML3 cells to various drugs and combinations. OCI-AML3 DMSO control, BCL-2i-R, MCL1i-R, or combo R cells were treated with the indicated drugs as single agents or in equimolar combination (0.001-10 μM). Sensitivity was expressed as the 50% lethal concentration (LC₅₀ μM) determined by flow cytometry after 48 hour-exposure. Error bars are standard deviation (SD) of 3 independent experiments. ∗P < .05, ∗∗P <.01, and ∗∗∗P < .001.

Figure 4.

BAX deficiency confers resistance to combined BCL-2 and MCL1 targeting in vitro. (A) BAX expression in CRISPR-Cas9–edited OCI-AML3 cells. Immunoblot demonstrating CRISPR-Cas9–induced BAX depletion in OCI-AML3 cells using guide RNAs (gRNAs) targeting BAX (gRNA-1.1 or gRNA-2.1) or a nontargeted (empty vector [EV]) control. Cells were treated with 5 μg/mL doxycycline for 72 hours to induce BAX loss. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Drug sensitivity of CRISPR-Cas9–edited OCI-AML3 cells. OCI-AML3 cells transduced with gRNA targeting BAX or a nontargeted control were treated with indicated drugs (0.001-10 μM for BH3-mimetics, 0.01-100 μM for cytarabine, and 0.001 to 1μM for idarubicin) and the LC₅₀ determined by flow cytometry after 48-hour exposure. Error bars are SD of 2 independent experiments. ∗P < .05, ∗∗P <.01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. (C) Survival of mice engrafted with BAX knockout cells in response to BH3-mimetic therapy. Irradiated NSG mice were transplanted with 10⁵ OCI-AML3 nontargeted EV control or OCI-AML3 BAX knockout cells (gRNA 2.1). Dosing commenced on day 4 posttransplant and mice were randomly divided into cohorts of 6 mice and treated with vehicle or a combination of venetoclax (75 mg/kg, weekdays by gavage) and S63845 (25 mg/kg, IV weekly) for 4 weeks. Kaplan-Meier survival analysis (ethical end point) shows that combined treatment with venetoclax/S63845 significantly prolongs survival in OCI-AML3 EV but not OCI-AML3 BAX knockout cohorts (black bar indicates duration of treatment). (D) Survival of mice engrafted with BAK knockout cells in response to BH3-mimetic therapy. Irradiated NSG mice were transplanted with 10⁵ OCI-AML3 nontargeted EV control or OCI-AML3 BAK knockout cells (gRNA 2.1). Dosing commenced on day 4 posttransplant and mice were randomly divided into cohorts of 6 mice and treated with vehicle or a combination of venetoclax (75 mg/kg, weekdays by gavage) and S63845 (25 mg/kg, IV weekly) for 4 weeks. Kaplan-Meier survival analysis (ethical end point) shows that combined treatment with venetoclax/S63845 significantly prolongs survival in both the OCI-AML3 EV and OCI-AML3 BAK knockout cohorts (black bar indicates duration of treatment). ns, not significant.

Figure 5.

The BAX P168A variant impairs BAX translocation and confers resistance to venetoclax in vitro and in vivo. (A) Expression of BAX P168A in BAX/BAK DKO MOLM-13 cells. Western blot showing BAX expression levels in MOLM-13 WT, BAX/BAK DKO (−) cells, or BAX/BAK DKO engineered to express BAX WT or BAX P168A upon transduction with retroviral expression constructs. (B) Sensitivity of BAX/BAK DKO plus BAX P168A cells to venetoclax. The indicated MOLM-13 cell lines (BAX/BAX DKO, BAX/BAX DKO + BAX WT, BAX/BAX DKO + BAX P168A, and parental MOLM13 WT) were treated with increasing concentrations of venetoclax (0.05-5 μM) for 24 hours and cell viability determined by annexin V/4′,6-diamidino-2-phenylindole staining and flow cytometry. Data are means ± standard error of the mean of 3 independent experiments. (C) BAX P168A does not have dominant-negative activity. BAX WT or BAX P168A was retrovirally expressed in MOLM-13 WT cells and cells treated with 500 nM venetoclax for 24 hours. Cell viability was determined by annexin V/4′,6-diamidino-2-phenylindole staining and flow cytometry. Data are means ± standard error of the mean of 2 independent experiments. (D) The BAX P168A mutation reduces MOM translocation and integration of BAX. MOLM-13 DKO cells expressing BAX WT or BAX P168A were pre-incubated with 25 μM Q-VD-OPh for 1 hour and then treated with 500 nM venetoclax. After 5 hours, cells were subjected to carbonate extraction and fractions run on sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for BAX. The results are representative of 3 independent experiments. (E-F) MOLM-13 BAX P168A cells are resistant to venetoclax in vivo. Irradiated NSG mice were transplanted with 5 × 10⁵ MOLM13 WT or MOLM13 BAX/BAK DKO cells expressing BAX P168A. On day 4 posttransplant, cohorts of 3 mice were treated with vehicle or venetoclax 75 mg/kg gavage weekdays for 2 weeks. Cohorts were euthanized and human CD45+ cells in BM and peripheral blood enumerated by flow cytometry. Error bars represent SD of 3 mice per treatment arm.