Abstract
The importance of c-MYC in regulating lymphopoiesis and promoting lymphomagenesis is well-established. Far less appreciated is the vital supporting role of MYC’s relative MNT. Using Rag1Cre-mediated Mnt deletion in lymphoid progenitor cells, we show here that, during normal T cell development, MNT loss enhances apoptosis, at least in part by elevating expression of the pro-apoptotic BH3-only protein BIM. Moreover, using T lymphoma-prone VavP-MYC transgenic mice, we show that Mnt deletion reduces the pool of pre-malignant MYC-driven T lymphoid cells and abrogates thymic T lymphomagenesis. In addition, we establish that Mnt deletion prevents T lymphoma development in γ-irradiated mice, most likely by enhancing apoptosis of T lymphoid cells repopulating the depleted thymus. Taken together with our recent demonstration that MNT is vital for the survival of MYC-driven pre-malignant and malignant B lymphoid cells, these results suggest that MNT represents an important new drug target for both T and B lymphoid malignancies.
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Introduction
The transcription factor c-MYC (hereafter MYC) controls expression of numerous genes involved in the proliferation, growth, metabolism and DNA damage responses of normal cells in adult tissues [1, 2] and its deregulated over-expression is a major driver of human cancer [3, 4]. However, stressed cells expressing elevated MYC are prone to apoptosis [5,6,7], which serves as a critical restraint on neoplastic transformation. Hence, genetic defects that impede apoptosis boost MYC’s oncogenic potential, as first revealed by the seminal demonstration that anti-apoptotic BCL-2 synergises with MYC in lymphomagenesis [8, 9].
To activate transcription, MYC heterodimerises with MAX, another ubiquitously expressed basic Helix-Loop-Helix Leucine Zipper (bHLHLZ) protein, and together they bind E-box motifs (CACGTG) in target genes [1]. However, MAX also binds several MYC-related transcriptional repressors containing bHLHLZ domains [1, 10]. MNT, an important member of this MXD (MAX Dimerization) family of c-MYC antagonists [11,12,13] is essential for embryonic development [14] and widely expressed in mammalian tissues.
We recently established that MNT is critical for lymphomagenesis in Eμ-Myc transgenic mice [15], which model human Burkitt’s lymphoma [16], and showed that MNT aids MYC by suppressing apoptosis in both pre-malignant and fully malignant B lymphoid cells.
Many human T cell neoplasms are associated with poor prognosis and new therapeutic approaches are sorely needed [17, 18]. Here, building on earlier studies [19,20,21], we investigate how MNT loss, mediated by a Rag1Cre transgene in lymphoid progenitor cells [22], impacts normal T cell development and T lymphomagenesis. We confirm that MNT loss promotes T cell apoptosis and provide new genetic and biochemical evidence regarding the underlying mechanism. We establish, for the first time, that MNT loss reduces the competitive fitness of T (and B) lymphopoiesis. We show that MNT loss enhances apoptosis of developing T lymphoid cells and abrogates T lymphoma development in vavP-MYC transgenic mice and γ-irradiated C57BL/6 mice. Our studies encourage investigation of MNT as an important new therapeutic target.
Materials and methods
Mice
Mice used were Mntfl/fl [23], Rag1Cre [22], MYC10hom [24] and Bim−/−del339 [25], all on a C57BL/6 background. Note that homozygous Mnt deletion, initially reported as perinatal lethal [14], is fatal at ~E10 in C57BL/6 mice bred in our facility [15]. Details of breeding, genotyping, immunophenotype analysis, OP9-DL1 co-culture, CFSE labelling and CRISPR/Cas9 genome editing are provided in Supplementary Materials and Methods and figure legends.
Competitive bone marrow reconstitution assays
C57BL/6 mice (Ly5.1+) (3 per test) were lethally irradiated (2 × 5.5 Gy) and reconstituted with 106 bone marrow cells from Ly5.1+ C57BL/6 mice (competitor cells) and 106 Ly5.2+ bone marrow cells harvested from either Mnt+/+ Rag1Cre (3 F and 1 M) or Mntfl/fl Rag1Cre (2 F, 3 M) mice (test cells). Ly5.1+ competitor and Ly5.2+ test cells of the same sex were injected into Ly5.1+ recipient mice of the same sex. Twelve weeks after transplantation, the relative proportions of test cell-derived lymphoid and myeloid cells (Ly5.2+) and competitor cell derived lymphoid and myeloid cells (Ly5.1+) of the indicated cell subsets in the thymus, spleen and bone marrow were determined by flow cytometry, using Ly5.1 and Ly5.2 antibodies to distinguish the two types of competing cells (see Supplementary Fig. 1).
Radiation induction of T lymphomas [26]
33 day-old C57BL/6 mice (WT, Mnt+/+ Rag1Cre and Mntfl/fl Rag1Cre) were exposed weekly, 4 times, to 1.5 Gy γ-irradiation from a 60Co source (Theratron Phoenix, Theratronics) and monitored until 350 days old. Sick mice (evidenced by breathing difficulties, enlarged thymus and/or enlarged spleen) were euthanised, autopsied and tumours characterised by immunophenotyping and immunoblot expression analysis.
Statistical analysis
Statistical comparisons were made using unpaired two-tailed Student’s t-test with Prism v8.0 software (GraphPad, San Diego, CA, USA). Data are shown as means ± SEM with P ≤ 0.05 considered statistically significant. Mouse survival analysis was carried out using GraphPad Prism (Version 8.0) and significance determined using log-rank (Mantel–Cox) test.
Results
MNT loss in lymphoid cells reduces competitive fitness
To avoid embryonic lethality conferred by MNT loss [15], we deployed the Rag1Cre transgene, which is expressed only in early lymphoid progenitors [22]. This strategy enabled us to compare MNT-deficient vs normal lymphopoiesis in adult mice, using competitive bone marrow reconstitution. Lethally irradiated C57BL/6-Ly5.1+ mice were injected with a 50:50 mixture of Ly5.1+ WT cells and test Ly5.2+ Mntfl/flRag1Cre or Mnt+/+Rag1Cre bone marrow cells (Fig. 1A). Analysis by flow cytometry after 12 weeks (Supplementary Fig. S1) revealed that the bone marrow cells from Mntfl/flRag1Cre mice had competed poorly against WT cells in regenerating lymphoid populations compared to those from Mnt+/+Rag1Cre mice. Thymi displayed a significantly lower proportion of Ly5.2+ Mntfl/flRag1Cre cells (gold bars) than Ly5.2+ Mnt+/+Rag1Cre cells (brown bars) in all major thymic sub-populations (Fig. 1B). Similarly, the spleen of reconstituted mice contained significantly fewer Mntfl/flRag1Cre than Mnt+/+Rag1Cre CD4+ or CD8+ T cells (Fig. 1C). Comparable outcomes were noted for B lineage cells (CD19+) in the spleen and bone marrow (Fig. 1C, D). In contrast, as anticipated, Ly5.2+ Mnt+/+Rag1Cre and Ly5.2+ Mntfl/flRag1Cre myeloid cells (Mac1+), were present in comparable numbers in the spleen and bone marrow in competitively reconstituted mice. We conclude that MNT loss puts both T and B lymphopoiesis at a significant competitive disadvantage.
Impact of MNT loss on T lymphopoiesis
We have previously investigated the impact of MNT loss in B lymphopoiesis [15]. To investigate how MNT loss affects T lymphopoiesis, we analysed the thymus and spleen in 6–7 wk-old Mntfl/flRag1Cre, Mnt+/+Rag1Cre and WT mice. Mnt deletion mediated via the Rag1Cre transgene was very efficient, as shown by PCR and Western blot analysis (Fig. 2A).
Thymic weight and cellularity were reduced to ~65% of normal in Mntfl/flRag1Cre mice, primarily due to fewer DP T cells (P ≤ 0.001), although DN and SP CD4+ populations were also significantly reduced (Fig. 2B). DN4 (CD25-CD44-) cells were more affected than DN2 (CD25+CD44+) or DN3 (CD25+CD44-) cells (Fig. 2C and Supplementary Fig. S2A). This deficit was not due to a failure of Τcrβ gene rearrangement because intracellular TCRβ protein was readily detectable in Mntfl/flRag1Cre DN4 cells (Supplementary Fig. S2B).
Spleen cellularity was also reduced in young Mntfl/flRag1Cre mice (Fig. 2D), primarily due to decreased B lymphoid cells (~37%; p < 0.0001), as reported previously [15]. In addition, T cells were reduced, particularly CD4+ T cells (~60%; p < 0.001), but the relative proportions of naïve, memory and effector T cells were equivalent between WT and Mnt-deficient T cells (Supplementary Fig. S2C, D). Myeloid (Mac1+) cellularity was unaffected (Fig. 2D), as expected from the lack of Rag1Cre expression in myeloid cells [22].
MNT loss increases T cell apoptosis
The T cell deficit in Mntfl/flRag1Cre mice seemed likely to reflect increased apoptosis and/or reduced MYC levels. All four major thymic sub-populations in Mntfl/flRag1Cre mice displayed a significantly increased proportion of annexin V-positive cells compared to their WT or Mnt+/+Rag1Cre control counterparts (Fig. 3A, Supplementary Fig. S3) and there was a similar trend for CD4+ and CD8+ T cells in the spleen (Fig. 3E). However, MNT loss did not alter endogenous MYC protein levels in any of these cell populations, as shown by flow cytometric and immunoblot analysis (Fig. 3B–D).
Enhanced apoptosis probably also explains the reduced DN4 population in Mntfl/flRag1Cre mice (Fig. 2C). When sorted DN3 and DN4 cells were cultured on OP9-DL1 stromal cells with IL-7, conditions which are permissive for T lymphoid cell proliferation and differentiation (Fig. 4A), MYC levels and proliferation were unaffected (Supplementary Fig. S4A, B). However, the Mnt KO DN4 cells produced considerably fewer viable cells, of all differentiation stages, than Mnt WT DN4 cells (Fig. 4B, C), while the DN3 cell cultures showed no major differences. Thus, MNT loss apparently renders DN4 cells, but not DN3 cells, more vulnerable to apoptosis during culture.
MNT loss increased apoptosis of splenic CD4+ T cells activated in vitro by PMA and ionomycin. The proportion of annexin-V-positive cells was ~2-fold higher in the Mnt KO than the Mnt+/+ CD4+ T cell population and there were fewer viable cells (Fig. 4D, Supplementary Fig. S4C). In contrast, MNT loss had little consequence for CD8+ T cells under these conditions.
Taken together, these observations suggest that increased apoptosis is the major determinant of the T cell deficit in Mntfl/flRag1Cre mice.
BIM is a critical mediator of apoptosis in MNT-null T cells
Cellular stress causes cell death via the mitochondrial apoptosis pathway, which is regulated by opposing factions of the BCL-2 family [27, 28] and extensive genetic studies have identified BIM (BCL2L11), a pro-apoptotic BH3-only protein, as a key trigger of lymphocyte death [29,30,31,32]. We therefore hypothesised that BIM contributed to the enhanced apoptosis of MNT-deficient T cells.
Notably, western blot analysis and intracellular flow cytometry revealed increased BIM protein in Mnt KO DP thymocytes compared to WT DP thymocytes but no significant change in anti-apoptotic MCL-1, an important regulator of T cell survival [33] (Fig. 5A, B). A modest increase in Bim transcription in MNT-deficient T cells (Fig. 5C) may partly account for the increased BIM protein.
BIM protein was also notably higher in mitogen-activated Mnt KO CD4+ splenic T cells, but not in activated CD8+ T cells (see Fig. 4E, F), paralleling their apoptosis susceptibility under these conditions (Fig. 4D). These results suggest that MNT suppresses Bim expression in T lymphoid cells, as we previously proposed for B lymphoid cells [15].
To directly test the importance of BIM in the apoptosis of MNT-deficient T cells, we bred Bim+/− Mntfl/flRag1Cre mice (Bim is functionally haplo-insufficient [34]). Indeed, apoptosis in thymocyte populations from Bim+/− Mntfl/flRag1Cre mice (rust bars) was significantly less than in those from Mntfl/fl Rag1Cre mice (gold bars), and comparable to that in WT mice (black bars) (Fig. 5D). Notably, the cellularity of the major thymic sub-populations was restored (Fig. 5E), as was that of the DN4 sub-population (Fig. 5F). Splenic T cell cellularity was also restored to normal in the Bim+/− Mntfl/flRag1Cre mice (Fig. 5G). Furthermore, loss of one Bim allele prevented the enhanced apoptosis of Mntfl/flRag1Cre CD4+ splenic T cells stimulated in vitro by PMA + ionomycin (Fig. 5H). In summary, MNT loss upregulates BIM, thereby enhancing the vulnerability of T cells to apoptosis during normal T lymphopoiesis.
MNT also constrains BIM levels in other cell types. Thus, using CRISPR/Cas9, we found that human MNT KO HEK 293 T and HeLa cells, and Mnt KO Bax−/−Bak−/− mouse embryonic fibroblasts (MEFs) express more BIM protein than their parental cells (Supplementary Fig. S5). Since apoptosis is blocked in Bax−/−Bak−/− MEFs, MNT regulation of BIM levels must occur upstream of mitochondrial permeability changes. Of note, the mechanism is reversible, because when the MEFs (Mnt+/+ or Mnt−/−) cells were infected with MntERT2 retrovirus and treated with 4-OHT to activate the exogenous MNTERT2 protein [35], BIM levels were again reduced (Supplementary Fig. S5I).
Mnt deletion prevents T lymphoma development in MYC10 hom transgenic mice
To examine the impact of MNT loss on MYC-driven T lymphomagenesis, we utilised our MYC10hom mice [24, 36], which are homozygous for a transgene expressing human MYC cDNA via the pan-haemopoietic VavP transgenic vector [37]. In these mice, expression of transgenic MYC protein in T lymphoid cells is significantly higher than in B lymphoid and myeloid cells, and thymic T lymphoma is the major cause of morbidity, although these mice can also develop disseminated histiocytic myeloid (monocyte/macrophage) (Mac1+F4/80+Gr1-) tumours affecting the spleen and other organs [36].
Mntfl/flMYC10hom/Rag1Cre mice survived significantly longer than the control Mnt+/+MYC10hom and Mnt+/+MYC10hom/Rag1Cre mice (median of 158 d compared to 136 d and 148 d; p ≤ 0.001, p ≤ 0.01, respectively) (Fig. 6A) and autopsy of euthanised sick mice revealed a major difference in pathology. Whereas the control mice presented with massively enlarged thymi and/or splenomegaly, Mntfl/flMYC10hom/Rag1Cre mice presented with splenomegaly but not thymic enlargement (Fig. 6B).
Importantly, Rag1Cre-mediated Mnt deletion specifically prevented T lymphoma development in MYC10hom mice (Fig. 6C). None of the 26 mice in the Mntfl/fl MYC10hom/Rag1Cre cohort developed thymic T lymphomas (Supplementary Table S3) and, where analysed, their thymic T cells had polyclonal rather than monoclonal TCR gene rearrangement, consistent with not being transformed (eg # 1313 and #1316 in Fig. 6D). In contrast, 12/26 Mnt+/+MYC10hom and 7/24 Mnt+/+MYC10hom/Rag1Cre control mice developed massive thymi (up to 1440 mg) and 14/15 of those immunophenotyped were CD4+CD8+ T lymphomas (the other being a CD19+ B lymphoma) (Supplementary Fig. S6A–C and Tables S1, S2). Seven of these thymic T lymphomas analysed by PCR showed 1 or 2 dominant Tcrβ gene rearrangements, indicative of mono- or bi- clonality (Fig. 6D, E). Curiously, the T lymphomas were also Mac-1-positive (Supplementary Fig. S6A, B), which may be due to high MYC expression, because activated CD8+ T cells express Mac1 [38].
The grossly enlarged spleens arising in either Mnt+/+ or Mnt KO MYC10hom mice contained a high proportion of transplantable Mac-1+ myeloid cells (Fig. 6F, Supplementary Table S4). Histological review revealed invasion of many other tissues by these tumour cells, as described previously [36]. Although the splenic CD4+ T cells in these mice were clearly activated (CD44+CD62L-) (Supplementary Fig. S6D), they were not transplantable (Supplementary Table S4).
In summary, lymphoid cell-specific Mnt deletion prevented the development of MYC-driven thymic T lymphomas in MYC10hom transgenic mice but not their myelomonocytic tumours. Whether the MYC-driven myeloid tumorigenesis requires MNT is not addressed by these studies as Rag1Cre is expressed only in lymphoid progenitors [39].
MNT loss impairs T cell development in MYC10 hom transgenic mice
To clarify why T lymphomagenesis was abrogated in Mntfl/flMYC10hom/Rag1Cre mice, we analysed healthy young ie premalignant (8 wk-old) mice. PCR and western blot analysis of DP thymocytes confirmed efficient Mnt deletion (not shown). Of note, thymic cellularity was reduced ~50% in Mntfl/flMYC10hom/Rag1Cre mice (green) compared to control Mnt+/+MYC10hom mice (blue) (p ≤ 0.001), and all thymocyte sub-populations were reduced around two-fold (Fig. 7A).
As reported previously [36], the level of MYC protein in thymocytes of MYC10hom transgenic mice greatly exceeds endogenous MYC levels (compare first 2 tracks in Fig. 7D). Concomitantly, MNT levels are also elevated 3-fold (Fig. 7D, Supplementary Fig. S7A).
MNT loss did not affect MYC protein level or cell size in pre-malignant MYC10hom T cells (Supplementary Fig. S7B–D). However, the proportion of annexin-V-positive cells was significantly higher in Mnt KO MYC10hom than Mnt+/+ MYC10hom thymocyte sub-populations (compare green to blue bars), which in turn tended to be higher than comparable WT sub-populations (compare blue to black bars; Fig. 7B). Furthermore, when cultured in vitro, Mnt KO MYC10hom DP thymocytes died faster than their Mnt+/+MYC10hom or WT counterparts (Fig. 7C). Thus, an overt consequence of MNT loss was increased apoptosis.
The increased apoptosis paralleled elevated BIM protein levels (Fig. 7D, E) and increased Bim transcription (Fig. 7F) in thymic DP T cells. Anti-apoptotic MCL-1 protein levels were higher in MYC10hom than WT DP thymocytes, but not affected by MNT loss (Fig. 7D). BCL-XL levels were comparable in cells from all three genotypes (Supplementary Fig. S7A). The tumour suppressor p53, which can activate apoptosis via transcriptional induction of pro-apoptotic BH3-only proteins PUMA and NOXA [40], was not detectable, by either western blot or qRT-PCR analyses (not shown).
MNT loss also resulted in a deficit of CD4+ and CD8+ T cells in the spleen of pre-malignant MYC10hom mice (Fig. 7G, green bars), and annexin V staining (Fig. 7H) indicated greater predisposition to apoptosis. Pertinently, the high MYC levels did not further increase in the absence of MNT (Supplementary Fig. S8A, B). Staining for CD44 and CD62L indicated that, as expected, the MYC transgenic splenic T cells were enlarged and highly activated (Supplementary Fig. S8C, D).
MNT loss also greatly reduced CD19+ B lymphoid cells in the spleen and bone marrow of the young MYC10hom mice, as reported previously for Mntfl/fl Eμ-Myc/Rag1Cre mice [15], but myeloid cells (Mac1+, Gr1+, Mac1+Gr1+) were unaffected (Fig. 7G, Supplementary Fig. S9C). Indeed, myeloid cell numbers were still normal at this age in both MYC10hom genotypes, despite the disseminated myeloid disease that inevitably develops as these mice age.
We conclude that abrogation of thymic T lymphoma development in Mnt KO MYC10hom mice is largely due to the increased apoptosis of the highly proliferative pre-malignant thymic T cells, driven by elevated BIM levels.
MNT loss prevents γ-radiation-induced T lymphoma development
To investigate the MNT-dependency of T lymphomagenesis in the absence of a Myc transgene, we performed serial total body γ-irradiation of C57BL/6 mice. In this well-studied model [26, 41], γ-irradiation decimates leukocytes and the thymus is repeatedly regenerated from bone marrow-derived haemopoietic stem/progenitor cells, some of which have sustained γ-irradiation-induced oncogenic mutation.
As expected, almost all γ-irradiated WT mice and Mnt+/+/Rag1Cre controls developed thymic T lymphomas (median survival 172 and 204 d respectively). These usually presented in the thymus and often also in the spleen, and had either a CD4+CD8+, CD8+ or mixed surface marker expression profile (Fig. 8A–C, Supplementary Table S5). Western blot analysis (Fig. 8D) showed that, with one exception, MYC protein was lower in γ-irradiation-induced T lymphomas than in MYC10hom transgenic T lymphomas, but nevertheless still far higher than in a normal thymus. NOTCH1 and p53/p19Arf pathway mutations were frequent, as reported previously [42], and all lymphomas expressed MNT and BIM.
Remarkably, however, none of the γ-irradiated Mntfl/fl/Rag1Cre mice developed lymphomas (Fig. 8A). To understand why, we analysed thymi recovering from the first γ-irradiation dose (Fig. 8E–G). All major T cell populations were greatly reduced on d2 compared to unirradiated controls but had largely recovered by d21, irrespective of genotype (Fig. 8E). However, the Mnt KO T cells exhibited greater levels of apoptosis compared to controls (Fig. 8F) and intracellular staining revealed significantly elevated BIM (Fig. 8G). We infer that MNT loss ‘enhances BIM-induced apoptosis of cells repopulating the thymus, including any clones expanding from stem cells carrying irradiation-induced oncogenic mutations. Consequently, lymphoma development is prevented.
Discussion
Using Rag1Cre-mediated deletion of Mnt in immature lymphoid progenitor cells in otherwise normal young mice, and competitive bone marrow reconstitution of lethally irradiated mice, we have shown that MNT-deficient lymphoid cells are more vulnerable to apoptosis during their development than those expressing MNT ([15], this paper). MNT loss elevated apoptosis and reduced cellularity in the thymus, bone marrow and spleen. In the T lineage, the phenotype affected all major sub-populations and was apparent as early as the DN4 pro-T cell stage, during which pre-TCR and Notch-1 signalling elevates c-MYC expression [43, 44]. In the B lineage, pro-B, pre-B and B cells were all affected [15].
The T cell phenotype of Mntfl/fl Rag1Cre mice was milder than that reported previously for Mntfl/fl LckCre mice [19], which had severe T cell loss, progressive inflammatory disease and late onset T lymphoma. The phenotypic differences may be due to continuous (Lck promoter-driven) vs transient (Rag1 promoter-driven) CRE expression. Continuous CRE exposure greatly perturbs T cell development [45], presumably due to accumulated DNA breaks, and this phenotype may be exacerbated by concomitant loss of MNT.
We showed that MNT loss in T cells provoked upregulation of BIM protein, a major initiator of apoptosis [29,30,31,32], and that the enhanced apoptosis and T cell deficit were largely prevented by loss of a single Bim allele. Both BIM protein and Bim mRNA levels were elevated in T cells of Mntfl/fl Rag1Cre mice. MNT may directly suppress Bim transcription, as MNT binding sites in the Bim locus have been identified in mouse B cells (see CUT&RUN data GSE132967 reported by Mathsyaraja et al. [46]). Indirect mechanisms may also be involved, but these do not appear to involve either ERK phosphorylation [47] or down-regulation of microR17-92 [48, 49] (data not shown).
Our data analysing MNT KO HEK 293 T, HeLa and MEF cell lines (Figure S5) suggest that MNT can also dampen BIM expression in non-lymphoid cell types. Importantly, since expression of exogenous MNT in Mnt KO cells reduced the elevated BIM levels, the mechanism is reversible. Furthermore, since the MEFs used in this experiment lacked the apoptosis effectors BAX and BAK, MNT-mediated modulation of BIM levels must occur upstream of mitochondrial outer membrane permeability (MOMP) changes.
To gauge how MNT affects T lymphomagenesis, we first used VavP-MYC10hom transgenic mice [24, 36], which are prone to both thymic T lymphomas and disseminated myeloid tumours. Strikingly, lymphoid-specific Mnt loss abrogated T lymphoma development in the MYC10hom transgenic mice. None of the Mntfl/fl MYC10hom/Rag1Cre cohort (n = 26) developed thymic lymphomas and morbidity was solely due to myeloid tumours.
To explore why T lymphomas failed to develop, we compared phenotypes of young mice, prior to any sign of emerging malignancy. MNT loss halved T cell numbers in both the thymus and spleen, and the Mnt KO MYC10hom T cells were significantly more susceptible to apoptosis than Mnt+/+MYC10hom T cells at all major stages of development. Indeed, their apoptosis is likely greater than suggested by annexin-V labelling, as apoptotic cells are rapidly engulfed by phagocytes in vivo [50]. These results showing the MNT-dependence of VavP-MYC transgene-driven T lymphomagenesis confirm and extend those reported by Hurlin’s group for transgenic mice engineered for T cell-specific (Lck-Cre-dependent) expression of a stable mutant MYCT58A protein from within the ROSA 26 locus [20, 51].
Importantly, we also showed that the MNT-dependency of T lymphomagenesis extends beyond a Myc transgenic setting. Irradiation-induction of T lymphomas, where NOTCH 1 and p53 mutations are principal oncogenic drivers [42], was totally prevented by Rag1Cre-mediated Mnt deletion.
Quantification of major apoptosis regulators revealed that pro-apoptotic BIM protein was elevated in Mnt KO as compared to Mnt+/+ DP thymocytes in healthy young MYC10hom transgenic mice. Similarly, apoptosis and BIM protein levels were elevated in DP thymocytes of regenerating thymi from irradiated Mntfl/fl Rag1Cre versus irradiated Mnt+/+ Rag1Cre or WT C57BL/6 mice. We infer that BIM upregulation after Mnt deletion is a major factor triggering apoptosis of proliferating T cell populations. Our data suggest that the mechanism is reversible and takes place upstream of BAX/BAK activation and MOMP. Further investigations of mechanism are planned, using RNA-Seq and CUT&TAG.
As a MYC antagonist, MNT was originally considered a tumour suppressor and this was supported by an early study showing that mice with mammary-specific Mnt deletion developed mammary adenocarcinoma [23]. The tumour suppressor role received further support when deletions involving the MNT gene locus (usually monoallelic) were noted in ~10% of cancers in The Human Cancer Genome Atlas [4], including certain cases of chronic lymphocytic leukaemia [52], a B cell malignancy, and Sezary syndrome, a cutaneous T-cell lymphoma/leukaemia [53]. However, MNT is localised on human chromosome 17p13.3 [12], near the potent tumour suppressor gene TP53 (17p13.1), making it difficult to ascribe any impact of large deletions solely, if at all, to MNT deletion.
While MNT might act as a tumour suppressor in certain settings, genetic studies from ourselves and others ([15, 20, 21] and this paper), using four independent mouse models, demonstrate unequivocally that MNT facilitates MYC-driven lymphomagenesis, rather than acting as a tumour suppressor, and that it does so by limiting apoptosis. Importantly, we have shown that MNT suppresses apoptosis by dampening expression of BIM, one of the most important apoptosis triggers for B and T lymphoid cells [54]. Like MYC, MNT binds to E boxes near many genes [1, 46]. Therefore, in addition to suppressing apoptosis, MNT may have other roles in facilitating MYC-driven oncogenesis.
MYC is a major driver for many (perhaps most) lymphoid and myeloid tumours and indeed a variety of solid tumours [3, 4]. However, the protracted search for a clinically effective MYC inhibitor has not yet succeeded [55, 56]. The realisation that MNT suppresses MYC-driven apoptosis opens an entirely new therapeutic approach: inhibition of MNT to amplify MYC’s capacity to drive apoptosis.
References
Diolaiti D, McFerrin L, Carroll PA, Eisenman RN. Functional interactions among members of the MAX and MLX transcriptional network during oncogenesis. Biochim Biophys Acta. 2015;1849:484–500.
Kress TR, Sabo A, Amati B. MYC: connecting selective transcriptional control to global RNA production. Nat Rev Cancer. 2015;15:593–607.
Kalkat M, De Melo J, Hickman KA, Lourenco C, Redel C, Resetca D, et al. MYC deregulation in primary human cancers. Genes (Basel). 2017;8:151.
Schaub FX, Dhankani V, Berger AC, Trivedi M, Richardson AB, Shaw R, et al. Pan-cancer alterations of the MYC oncogene and its proximal network across the cancer genome atlas. Cell Syst. 2018;6:282–300.
Askew DS, Ashmun RA, Simmons BC, Cleveland JL. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991;6:1915–22.
Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992;69:119–28.
Murphy DJ, Junttila MR, Pouyet L, Karnezis A, Shchors K, Bui DA, et al. Distinct thresholds govern Myc’s biological output in vivo. Cancer Cell. 2008;14:447–57.
Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988;335:440–2.
Strasser A, Harris AW, Bath ML, Cory S. Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2. Nature. 1990;348:331–3.
Grandori C, Cowley SM, James LP, Eisenman RN. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu Rev Cell Dev Biol. 2000;16:653–99.
Hurlin PJ, Queva C, Eisenman RN. Mnt, a novel Max-interacting protein is coexpressed with Myc in proliferating cells and mediates repression at Myc binding sites. Genes Dev. 1997;11:44–58.
Meroni G, Reymond A, Alcalay M, Borsani G, Tanigami A, Tonlorenzi R, et al. Rox, a novel bHLHZip protein expressed in quiescent cells that heterodimerizes with Max, binds a non-canonical E box and acts as a transcriptional repressor. EMBO J. 1997;16:2892–906.
Liano-Pons J, Arsenian-Henriksson M, Leon J. The multiple faces of MNT and its role as a MYC modulator. Cancers (Basel). 2021;13:4682.
Toyo-oka K, Hirotsune S, Gambello MJ, Zhou ZQ, Olson L, Rosenfeld MG, et al. Loss of the Max-interacting protein Mnt in mice results in decreased viability, defective embryonic growth and craniofacial defects: relevance to Miller-Dieker syndrome. Hum Mol Genet. 2004;13:1057–67.
Nguyen HV, Vandenberg CJ, Ng AP, Robati MR, Anstee NS, Rimes J, et al. Development and survival of MYC-driven lymphomas require the MYC antagonist MNT to curb MYC-induced apoptosis. Blood. 2020;135:1019–31.
Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S, et al. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature. 1985;318:533–8.
Vose J, Armitage J, Weisenburger D. International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes. J Clin Oncol. 2008;26:4124–30.
Horwitz S, O’Connor OA, Pro B, Illidge T, Fanale M, Advani R, et al. Brentuximab vedotin with chemotherapy for CD30-positive peripheral T-cell lymphoma (ECHELON-2): a global, double-blind, randomised, phase 3 trial. Lancet. 2019;393:229–40.
Dezfouli S, Bakke A, Huang J, Wynshaw-Boris A, Hurlin PJ. Inflammatory disease and lymphomagenesis caused by deletion of the Myc antagonist Mnt in T cells. Mol Cell Biol. 2006;26:2080–92.
Link JM, Ota S, Zhou ZQ, Daniel CJ, Sears RC, Hurlin PJ. A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis. Proc Natl Acad Sci USA. 2012;109:19685–90.
Campbell KJ, Vandenberg CJ, Anstee NS, Hurlin PJ, Cory S. Mnt modulates Myc-driven lymphomagenesis. Cell Death Differ. 2017;24:2117–26.
McCormack MP, Forster A, Drynan L, Pannell R, Rabbitts TH. The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development. Mol Cell Biol. 2003;23:9003–13.
Toyo-oka K, Bowen TJ, Hirotsune S, Li Z, Jain S, Ota S, et al. Mnt-deficient mammary glands exhibit impaired involution and tumors with characteristics of myc overexpression. Cancer Res. 2006;66:5565–73.
Smith DP, Bath ML, Harris AW, Cory S. T-cell lymphomas mask slower developing B-lymphoid and myeloid tumors in transgenic mice with broad hematopoietic expression of MYC. Oncogene. 2005;24:3544–53.
Bouillet P, Cory S, Zhang L-C, Strasser A, Adams JM. Degenerative disorders caused by Bcl-2 deficiency are prevented by loss of its BH3-only antagonist Bim. Dev Cell. 2001;1:645–53.
Kaplan HS, Brown MB. A quantitative dose-response study of lymphoid-tumor development in irradiated C57 black mice. J Natl Cancer Inst. 1952;13:185–208.
Strasser A, Cory S, Adams JM. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J. 2011;30:3667–83.
Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49–63.
Bouillet P, Metcalf D, Huang DCS, Tarlinton DM, Kay TWH, Köntgen F, et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science. 1999;286:1735–8.
Bouillet P, Purton JF, Godfrey DI, Zhang L-C, Coultas L, Puthalakath H, et al. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Nature. 2002;415:922–6.
Hildeman DA, Zhu Y, Mitchell TC, Bouillet P, Strasser A, Kappler J, et al. Activated T cell death in vivo mediated by pro-apoptotic Bcl-2 family member, Bim. Immunity. 2002;16:759–67.
Pellegrini M, Bouillet P, Robati M, Belz GT, Davey GM, Strasser A. Loss of Bim increases T cell production and function in interleukin 7 receptor-deficient mice. J Exp Med. 2004;200:1189–95.
Opferman JT, Letai A, Beard C, Sorcinelli MD, Ong CC, Korsmeyer SJ. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature. 2003;426:671–6.
Egle A, Harris AW, Bouillet P, Cory S. Bim is a suppressor of Myc-induced mouse B cell leukemia. Proc Natl Acad Sci USA. 2004;101:6164–9.
Littlewood TD, Hancock DC, Danielian PS, Parker MG, Evan GI. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res. 1995;23:1686–90.
Smith DP, Bath ML, Metcalf D, Harris AW, Cory S. MYC levels govern hematopoietic tumor type and latency in transgenic mice. Blood. 2006;108:653–61.
Ogilvy S, Metcalf D, Gibson L, Bath ML, Harris AW, Adams JM. Promoter elements of vav drive transgene expression in vivo throughout the hematopoietic compartment. Blood. 1999;94:1855–63.
Christensen JE, Andreasen SO, Christensen JP, Thomsen AR. CD11b expression as a marker to distinguish between recently activated effector CD8(+) T cells and memory cells. Int Immunol. 2001;13:593–600.
Kuo TC, Schlissel MS. Mechanisms controlling expression of the RAG locus during lymphocyte development. Curr Opin Immunol. 2009;21:173–8.
Villunger A, Michalak EM, Coultas L, Mullauer F, Bock G, Ausserlechner MJ, et al. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science. 2003;302:1036–8.
Kaplan HS. The role of radiation on experimental leukemogenesis. Natl Cancer Inst Monogr. 1964;14:207–20.
Lee CL, Brock KD, Hasapis S, Zhang D, Sibley AB, Qin X, et al. Whole-exome sequencing of radiation-induced thymic lymphoma in mouse models identifies Notch1 activation as a driver of p53 wild-type lymphoma. Cancer Res. 2021;81:3777–90.
Douglas NC, Jacobs H, Bothwell AL, Hayday AC. Defining the specific physiological requirements for c-Myc in T cell development. Nat Immunol. 2001;2:307–15.
Dose M, Khan I, Guo Z, Kovalovsky D, Krueger A, von Boehmer H, et al. c-Myc mediates pre-TCR-induced proliferation but not developmental progression. Blood. 2006;108:2669–77.
Carow B, Gao Y, Coquet J, Reilly M, Rottenberg ME. lck-driven cre expression alters T cell development in the thymus and the frequencies and functions of peripheral T cell subsets. J Immunol. 2016;197:2261–8.
Mathsyaraja H, Freie B, Cheng PF, Babaeva E, Catchpole JT, Janssens D, et al. Max deletion destabilizes MYC protein and abrogates Emicro-Myc lymphomagenesis. Genes Dev. 2019;33:1252–64.
Luciano F, Jacquel A, Colosetti P, Herrant M, Cagnol S, Pages G, et al. Phosphorylation of Bim-EL by Erk1/2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. Oncogene. 2003;22:6785–93.
Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J, et al. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol. 2008;9:404–14.
Li Y, Choi PS, Casey SC, Dill DL, Felsher DW. MYC through miR-17-92 suppresses specific target genes to maintain survival, autonomous proliferation, and a neoplastic state. Cancer Cell. 2014;26:262–72.
Dzhagalov IL, Chen KG, Herzmark P, Robey EA. Elimination of self-reactive T cells in the thymus: a timeline for negative selection. PLoS Biol. 2013;11:e1001566.
Wang X, Cunningham M, Zhang X, Tokarz S, Laraway B, Troxell M, et al. Phosphorylation regulates c-Myc’s oncogenic activity in the mammary gland. Cancer Res. 2011;71:925–36.
Edelmann J, Holzmann K, Miller F, Winkler D, Buhler A, Zenz T, et al. High-resolution genomic profiling of chronic lymphocytic leukemia reveals new recurrent genomic alterations. Blood. 2012;120:4783–94.
Vermeer MH, van Doorn R, Dijkman R, Mao X, Whittaker S, van Voorst Vader PC, et al. Novel and highly recurrent chromosomal alterations in Sezary syndrome. Cancer Res. 2008;68:2689–98.
Strasser A, Bouillet P. The control of apoptosis in lymphocyte selection. Immunol Rev. 2003;193:82–92.
Dang CV, Reddy EP, Shokat KM, Soucek L. Drugging the ‘undruggable’ cancer targets. Nat Rev Cancer. 2017;17:502–8.
Chen H, Liu H, Qing G. Targeting oncogenic Myc as a strategy for cancer treatment. Signal Transduct Target Ther. 2018;3:5.
Mingueneau M, Kreslavsky T, Gray D, Heng T, Cruse R, Ericson J, et al. The transcriptional landscape of alphabeta T cell differentiation. Nat Immunol. 2013;14:619–32.
Eischen CM, Weber JD, Roussel MF, Sherr CJ, Cleveland JL. Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis. Genes Dev. 1999;13:2658–69.
Acknowledgements
The authors thank Peter Hurlin (Shriner’s Hospital for Children, Portland OR USA) for his kind gift of Mnt floxed mice; our colleagues Andreas Strasser, Gemma Kelly, Jerry Adams, Steve Nutt and Jacob Jackson for comments on the manuscript and useful discussions; Andreas Strasser, Lorraine O’Reilly and Steve Nutt for fluorescent antibodies; David Huang for Bax−/−Bak−/− MEFs; Krystal Hughes, Crystal Stivala, Michael Watters, Sam Kelly, Dan Fayle and Giovanni Siciliano for mouse husbandry; Simon Monard for assistance with flow cytometry; and Peter Maltezos for skilful preparation of Figures. This work was supported by grants from the National Health and Medical Research Council (NHMRC) (program grant 1016701), the Leukemia & Lymphoma Society (SCOR grant 7001-13) and a Grant-in-Aid from the Cancer Council Victoria App 118768 (to SC); NHMRC project grants GNT1060179 and GNT1122783 (to APN), philanthropic support to the Walter and Eliza Hall Institute; and operational infrastructure grants through the Australian Government IRISS and the Victorian Government OIS.
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HVN designed and performed experiments, with assistance from MRR; CJV set up the mouse breeding and performed early analyses; haematologist APN performed histological analysis; SC conceived the project, wrote the manuscript with input from the authors, and secured funding.
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Mice were bred, housed and monitored in WEHI’s Bioservices Facility under the supervision of trained veterinarians and in accordance with the WEHI animal ethics committee regulations and the Australian Code for the Care and Use of Animals for Scientific purposes.
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Nguyen, H.V., Vandenberg, C.J., Robati, M.R. et al. MNT suppresses T cell apoptosis via BIM and is critical for T lymphomagenesis. Cell Death Differ 30, 1018–1032 (2023). https://doi.org/10.1038/s41418-023-01119-y
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DOI: https://doi.org/10.1038/s41418-023-01119-y
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