Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53

Abstract

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

Keywords: BCL-2; MCL-1; MYC; apoptosis; cancer; p53.

Figure 1.

Homozygous loss of Bcl-x has only a minor impact on the growth of Eμ-Myc lymphomas. (A) Survival curves of C57BL/6-Ly5.1⁺ recipient mice transplanted with Eμ-Myc;CreERT2;Bcl-x^fl/fl (green line) or control (Eμ-Myc;CreERT2; black line) lymphoma cells and treated with tamoxifen to inactivate Bcl-x where applicable. (n) Total number of recipient mice analyzed; (N) number of independent lymphomas tested. Homozygous deletion of Bcl-x resulted in a small but significant delay in tumor growth. (*) P = 0.0367. For the mice transplanted with the control Eμ-Myc;CreERT2 lymphomas, 3% regressed, and the overall median survival was 19 d. For the mice transplanted with the Eμ-Myc;CreERT2;Bcl-x^fl/fl lymphomas, 4% regressed, and the overall median survival was 25 d. (B) Bioluminescence imaging of the tumor burden in C57BL/6-albino recipient mice injected with primary Eμ-Myc;CreERT2;Bcl-x^fl/fl lymphoma cells that had been transduced with a lentiviral vector coexpressing GFP and luciferase. At 7 d post-transplant, a cohort of these mice was treated with tamoxifen. Mice were subsequently imaged for bioluminescence to monitor lymphoma burden every 3–4 d by measuring the total photon flux per second emitted from a region of interest (ROI) drawn around the whole mouse. See also Supplemental Figure 3.

Figure 2.

Loss of Mcl-1, even loss of a single allele, greatly impairs the sustained growth of Eμ-Myc lymphomas within the whole animal. (A) Survival curves of C57BL/6-Ly5.1⁺ recipient mice transplanted with Eμ-Myc;CreERT2;Mcl-1^fl/+ (red line), Eμ-Myc;CreERT2;Mcl-1^fl/fl (blue line), or control (Eμ-Myc;CreERT2; black line) lymphoma cells and treated with tamoxifen to inactivate Mcl-1 where applicable. (n) Total number of recipient mice analyzed; (N) number of independent lymphomas tested. Heterozygous and homozygous deletion of Mcl-1 significantly delayed lymphoma growth. (****) P < 0.0001. For the mice transplanted with the control Eμ-Myc;CreERT2 lymphomas, 3% regressed, and the overall median survival was 19 d. For the mice transplanted with the Eμ-Myc;CreERT2;Mcl-1^fl/+ lymphomas, 20% regressed, and the overall median survival was 23 d. For the mice transplanted with the Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas, 30% regressed, and the overall median survival was 35 d. (B) Bioluminescence imaging of the tumor burden in C57BL/6-albino recipient mice injected with primary Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphoma cells. At 10 d post-transplant, a cohort of these mice was treated with tamoxifen. These mice were subsequently imaged for bioluminescence.

Figure 3.

Most Eμ-Myc lymphomas that relapse following tamoxifen treatment have escaped Mcl-1^fl allele recombination. (A) FACS analysis to detect expression of huCD4 (reporter for Mcl-1^fl recombination) on the surface of two representative Eμ-Myc;CreERT2;Mcl-1^fl/+ and Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas that had relapsed in recipient mice after tamoxifen treatment (transplanted lymphoma cells stained positive for Ly5.2). Approximately 60% of the relapsed Eμ-Myc;CreERT2;Mcl-1^fl/+ and 40% of the relapsed Eμ-Myc;CreERT2;Mcl-1^fl/f lymphoma cells had escaped Mcl-1^fl allele recombination and, as such, were huCD4-negative (like the sample at the left of each pair), whereas ∼40% of the relapsed Eμ-Myc;CreERT2;Mcl-1^fl/+ and 60% of the relapsed Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas had efficiently recombined at least one Mcl-1^fl allele, as reflected by staining positive for huCD4 (like the sample at the right of each pair). (B) Immunoblotting to detect MCL-1 and CreERT2 protein expression (probing for Actin was used as a loading control) in extracts from the spleens, lymph nodes, or thymuses of sick mice that had been transplanted with Eμ-Myc;CreERT2;Mcl-1^fl/+ or Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas. Three independent, paired control, and tamoxifen-treated tumors of each genotype were analyzed (note that one of the Eμ-Myc;CreERT2;Mcl-1^fl/fl tumors [#3] examined never relapsed after tamoxifen treatment). Mcl-1 knockout (KO) mouse embryonic fibroblasts (MEFs) were used as a control to confirm the specificity of the MCL-1 antibody (neg control). (C) DNA PCR analysis of recombined Mcl-1^fl and unrecombined (intact) floxed alleles in Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas that relapsed following tamoxifen treatment. (D) Immunoblotting to detect MCL-1, CreERT2, p53, p19/ARF and Actin (loading control) protein expression in extracts from the spleens, lymph nodes, or thymuses of sick mice transplanted with Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas that relapsed as huCD4-positive following tamoxifen treatment. Three independent, paired control, and tamoxifen-treated tumors were analyzed. Mcl-1 knockout (KO) MEFs were used as a control (neg control) to confirm the specificity of the MCL-1 antibody. (E) Survival curves of C57BL/6-Ly5.1⁺ recipient mice transplanted with Eμ-Myc;CreERT2;Mcl-1^fl/+ (red line) or Eμ-Myc;CreERT2;Mcl-1^fl/fl (blue line) lymphoma cells that had wild-type p53 genes following tamoxifen treatment and excluding those lymphomas that escaped Cre-mediated deletion of one or both Mcl-1^fl alleles. C57BL/6-Ly5.1⁺ recipient mice transplanted with control (Eμ-Myc;CreERT2; black line) lymphoma cells and treated with tamoxifen are shown for comparison. (n) Total number of recipient mice analyzed; (N) number of independent lymphomas tested. Efficient heterozygous and homozygous deletion of Mcl-1 significantly delayed lymphoma growth. (****) P < 0.0001. For the mice transplanted with the control Eμ-Myc;CreERT2 lymphomas, 3% regressed, and the overall median survival was 19 d. For the mice transplanted with the Eμ-Myc;CreERT2;Mcl-1^fl/+ lymphomas, 94% regressed, and the overall median survival was >180 d. For the mice transplanted with the Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas, 100% regressed, and the overall median survival was >180 d.

Figure 4.

MCL-1 blockade kills human Burkitt lymphoma cells, but some also show minor dependency on BCL-X_L. (A) Immunoblotting to detect MCL-1, BCL-X_L, BCL-2, and β-Actin (loading control) in extracts from Burkitt lymphoma cell lines and, as controls for BCL-2 expression, the X50-7 and Awia lymphoblastoid cell lines. (B) Schematic showing the binding specificities of the BIM_S variants to the BCL-2 prosurvival proteins. (C) Immunoblotting to detect the inducible expression of the BIM_S variants before and after 24 h of dox treatment in the Rael-BL cell lines carrying the different lentiviral-inducible BIM_S expression constructs. Cells were maintained in medium supplemented with the pan-caspase inhibitor qVD-OPh to prevent protein degradation due to apoptosis. Note that the endogenous BIM_EL, BIM_L, and BIM_S proteins can also be detected but at a lower level. Probing for β-Actin served as a loading control. (D) Viability of BL cell lines stably infected with lentiviral constructs carrying vectors for dox-inducible expression of BIM_S variants was determined 72 h after the addition of dox to the medium by staining the cells with propidium iodide (PI) followed by FACS analysis for PI and GFP fluorescence (GFP is expressed from the lentivirus encoding the BIM_s variants). The PI-negative/GFP-positive viable cells were recorded. The percentage of viable/GFP-positive untreated cells was assigned an arbitrary value of 1, and the percentage of viable/GFP-positive dox-treated cells was expressed as a proportion of this. Each line was assayed in triplicate, and data are presented as the mean and standard error of the mean of three independent experiments. Statistical analysis using a paired two-tailed t-test showed that BIMs2A induced significantly more death in all of the Burkitt lymphoma cells examined compared with the negative control BIMs4E (BL31, [*] P = 0.0179; Ramos-BL, [**] P = 0.0025; Rael-BL, [**] P = 0.0017; Sav-BL, [**] P = 0.0044) and that BIMs2A induced significantly more death than BIMsBAD in BL31 ([**] P = 0.0056), Ramos-BL ([*] P = 0.0421), and Rael-BL ([**] P = 0.0023) cells but not in the Sav-BL cells ([ns] P = 0.1348). See also Supplemental Figure 4.

Figure 5.

Mutations in p53 reduce but do not ablate the dependency of c-MYC-driven mouse and human lymphomas on MCL-1. (A) Immunoblotting to detect stabilized p53 proteins, indicative of a mutant p53 protein, in extracts from 17 Eμ-Myc;CreERT2;Mcl-1^fl/+ and six Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas. All but two of the samples were extracted from transplanted and tamoxifen-treated lymphomas. The two exceptions were Eμ-Myc;CreERT2;Mcl-1^fl/+ #9, which was a primary lymphoma, and Eμ-Myc;CreERT2;Mcl-1^fl/fl #3, which was a transplanted but not tamoxifen-treated lymphoma (this particular lymphoma has the deletion of two bases that introduces a premature stop codon in the p53 gene). Probing for HSP70 served as a loading control. (B) Summary of the p53 mutations detected in Eμ-Myc;CreERT2;Mcl-1^fl/+ and Eμ-Myc;CreERT2;Mcl-1^fl/fl lymphomas as detected by DNA sequence analysis. The mutations are listed as wild-type (wt) amino acid (aa) affected, amino acid position, and mutant amino acid. The frequencies at which the mutations were detected in the lymphomas and the human equivalents of the mutations are listed. The sequence alignment for murine p53 was performed using Ensembl transcript number ENSMUST00000108658 as the reference transcript. (C) Immunoblotting to detect the expression of HDM2, p53, and β-Actin (loading control) in human Burkitt lymphoma cell lines. (D) Summary of the p53 pathway aberrations detected in the human Burkitt lymphoma cell lines. The p53 mutations present in each cell line is listed as wild-type (wt) amino acid (aa) affected, amino acid position, and mutant amino acid, and the number of alleles affected is also detailed.

Figure 6.

A proposed model in which c-MYC-driven lymphoma cells that are highly dependent on MCL-1 for their sustained expansion can no longer survive once MCL-1 expression is reduced and the balance between this prosurvival protein and the p53 proapoptotic targets PUMA/NOXA is disturbed. The c-MYC-driven lymphoma cells that have acquired p53 mutations display an accelerated lymphoma progression. These lymphomas express less PUMA/NOXA, and, consequently, less MCL-1 expression is required to maintain the lymphoma progression. Despite this, we showed that targeting of MCL-1 in lymphomas with p53 mutations was sufficient to result in tumor regression. Note that the mouse protein nomenclature is used.