To the Editor:

The gene encoding the anti-apoptotic BCL-2 family member MCL-1 is frequently amplified (~10%) in diverse human malignancies [1] and the finding that its inducible deletion impairs the growth of several types of tumours in vivo makes it an attractive target for anti-cancer therapy [2]. Lymphomas driven by c-MYC are particularly dependent on MCL-1, with loss of only one Mcl-1 allele preventing expansion of such murine lymphomas in vivo [2]. Accordingly, MYC-driven lymphomas are highly sensitive to MCL-1 inhibitors [3] and deletion of one Mcl-1 allele greatly delays c-MYC-driven lymphomagenesis in mice [4]. However, some c-MYC-driven lymphomas can tolerate the loss of one Mcl-1 allele, which reduces MCL-1 protein levels by ~40% [5]. These lymphomas uniformly display mutations of the tumour suppressor Trp53 [2]. Wild-type (wt) TRP53 function is required for optimal responses to BH3-mimetic drugs [6], and loss of Trp53 overcomes the delayed lymphomagenesis seen in Eμ-Myc;Mcl1+/− mice [4]. These studies suggest that loss or mutation of Trp53 diminishes the dependence of c-MYC-driven lymphomas on MCL-1. To explore this further, we conditionally deleted Mcl-1 in TRP53 deficient Eμ-Myc mouse lymphomas in vivo, to determine whether the loss of TRP53 allows them to tolerate the complete absence of MCL-1. This is an important question given that several BH3-mimetic drugs targeting MCL-1 have entered clinical trials in haematological cancers, where defects in TRP53 function frequently underlie resistance to standard therapies.

We utilised Eμ-Myc transgenic mice carrying conditional loxP flanked (floxed) Mcl-1 alleles [2] and a constitutively expressed but conditional tamoxifen-inducible Cre-recombinase (Rosa-CreERT2) (Supplementary Fig. S1). We engineered Eμ-Myc mice with germline loss of one Trp53 allele and one or two floxed Mcl-1 alleles plus the CreERT2 transgene (Supplementary Fig. S1). Eμ-Myc;Trp53+/−;Mcl1fl/+;RosaCreERT2 and Eμ-Myc;Trp53+/−;Mcl1fl/fl;RosaCreERT2 mice rapidly developed pre-B/B lymphomas (median survival ~30 days), and all selected for loss of the wt Trp53 allele, rendering them TRP53 deficient (Supplementary Fig. S2d). Ly5.2+ Eμ-Myc;Trp53−/−;Mcl1fl/+;RosaCreERT2 (N = 3; n = 29), Eμ-Myc;Trp53−/−;Mcl1fl/fl;RosaCreERT2 (N = 6; n = 18) and control Eμ-Myc;Trp53−/−;Mcl1+/+ RosaCreERT2 (N = 1; n = 3) lymphomas were transplanted into immune-competent C57BL/6-Ly5.1 recipients that were treated with either vehicle or tamoxifen on days 5 and 6 post-transplantation to induce Mcl-1fl deletion in the malignant cells in vivo (Supplementary Fig. S1b). Recipients bearing Eμ-Myc;Trp53−/−;Mcl1+/+;CreERT2 control lymphomas exhibited a marginal survival advantage (3 days) following tamoxifen treatment (Supplementary Fig. S2e). In the absence of TRP53, removal of one Mcl-1 allele in Eμ-Myc;Trp53−/−;Mcl1fl/+;RosaCreERT2 lymphomas did not impact lymphoma growth or survival of recipient mice (Fig. 1a). Deletion of the floxed Mcl-1 gene and encoded protein were confirmed in these lymphoma cells following treatment of mice with tamoxifen (Supplementary Fig. S2a-c). Remarkably, biallelic Mcl-1 deletion in Eμ-Myc;Trp53−/−;Mcl1fl/fl;RosaCreERT2 (N = 6; n = 18) lymphomas, resulting in complete removal of MCL-1, significantly prolonged tumour-free survival, with 14/18 (77.8%) of recipients cured (Fig. 1a). The four relapsed lymphomas may have selected against Mcl-1fl recombination and hence loss of MCL-1 protein or acquired changes that compensated for MCL-1 loss.

Fig. 1: Eμ-Myc lymphoma cells with loss of TRP53 remain dependent on anti-apoptotic MCL-1.
figure 1

a Kaplan–Meier survival curve for mice transplanted with Eμ-MycT/+;CreERT2KI/+;Trp53−/− lymphomas bearing either wt Mcl-1, a single floxed Mcl-1 allele or two floxed Mcl-1 alleles and treated with tamoxifen on days 5 and 6 to delete the floxed Mcl-1 alelle(s) in the lymphomas. p value determined by Log-rank (Mantel–Cox) test. N indicates number of independent lymphomas of a given genotype tested; n indicates the number of recipient mice transplanted with the indicated lymphomas examined. b Response of Eμ-Myc lymphoma-derived cell lines to the MCL-1 inhibitor, S63845, comparing a panel of cell lines with deficient vs wild-type TRP53 function; N = 9 of each. Data represent non-linear regression of the means with IC50 and 95% confidence interval (CI) indicated. c Schematic depicting the intersecting and opposing roles of TRP53 and MCL-1 in the regulation of apoptosis.

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We further assessed the impact of Trp53 loss on MCL-1 dependency in Eμ-Myc lymphomas by using the MCL-1 inhibitor S63845. Eμ-Myc lymphoma-derived cell lines with spontaneous Trp53 loss (N = 5) or mutation (N = 4) were compared with control Eμ-Myc lymphoma lines with intact TRP53 function (N = 9). Loss of TRP53 function conferred only minor resistance to MCL-1 inhibition (increase in IC50 value), with higher doses of MCL-1 inhibitor effectively killing Trp53-deficient lymphoma cells (Fig. 1b). In the human diffuse large B cell lymphoma (DLBCL) line DoHH2, CRISPR-mediated loss of TP53, validated by resistance to the MDM2 inhibitor Nutlin-3a (Supplementary Fig. S3a), did not impact the response to S63845 in short-term killing assays (Supplementary Fig. S3b). However, the TP53 deficient DoHH2 cells had a competitive advantage over their wt TP53 counterparts when cultured together in sub-optimal (~IC25) doses of S63845 for 14 days (Supplementary Fig. S3c), consistent with previous observations [6].

Considering the potential underlying mechanism, there is a direct relationship between TRP53 and MCL-1 in their opposing functions in regulating apoptosis (Fig. 1c). TRP53 can induce apoptosis by increasing the expression of the pro-apoptotic BH3-only proteins, PUMA/Bbc3, NOXA/Pmaip1 and BIM/Bcl2l11 [7].

In conclusion, we demonstrate that loss/mutation of TRP53/TP53 renders mouse c-MYC-driven lymphomas and a human DLBCL cell line less dependent on MCL-1 for sustained survival and expansion. Importantly though, lymphomas with loss of TRP53/TP53 can still be effectively killed using either higher concentrations of the MCL-1 inhibitor or by homozygous deletion of Mcl-1. This is important for the clinical translation of MCL-1 inhibitors because it suggests that patients bearing cancers with loss/mutation of TP53 may require higher doses of MCL-1 inhibitors or combination therapies to augment the levels of pro-apoptotic BH3-only proteins [6].