MNT suppresses T cell apoptosis via BIM and is vital for MYC-driven T lymphomagenesis

doi:10.21203/rs.3.rs-1555936/v1

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Research Article

MNT suppresses T cell apoptosis via BIM and is vital for MYC-driven T lymphomagenesis

https://doi.org/10.21203/rs.3.rs-1555936/v1

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The importance of c-MYC in regulating normal lymphopoiesis and promoting lymphomagenesis is well-established, but far less appreciated is the vital supporting role of MYC’s relative MNT. We show here that, during normal T cell development, MNT loss enhances apoptosis and establish the mechanism as elevated expression of the pro-apoptotic BH3-only protein BIM. Using a MYC transgenic mouse model, we also show that MNT loss reduces the pool of premalignant MYC-driven T cells and totally abrogates thymic T lymphomagenesis. Taken together with our recent demonstration that MNT is vital for the survival of MYC-driven premalignant and malignant B lymphoid cells, these results suggest that MNT represents an important new drug target for both T and B lymphoid malignancies.

Immunology

Cell Survival and Cell Death

Cancer Biology

Molecular Biology

MYC

MNT

BIM

apoptosis

T lymphomagenesis

T lymphopoiesis

transgenic mice

The transcription factor c-MYC (hereafter MYC) controls expression of many genes involved in the proliferation, growth, metabolism and DNA damage responses of normal cells in adult tissues ^{1, 2} and its deregulated overexpression is widely accepted as a major driver of human cancer ^{3, 4}. Importantly, under conditions of stress (e.g. cytokine or nutrient deprivation), cells expressing elevated MYC undergo apoptosis ^{5, 6, 7}, and this 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 showed that MNT is vital for B lymphomagenesis in Eµ-Myc transgenic mice ¹⁵, which model human Burkitt’s lymphoma ¹⁶. We found that MNT aids MYC by suppressing apoptosis in both premalignant MYC-driven pre-B cells and fully malignant B lymphoid tumour cells.

Human T cell malignancies are associated with a very poor prognosis and new therapeutic approaches are sorely needed ^{17, 18}. Here, building on earlier studies ^{19, 20, 21}, we investigate how MNT loss 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 also establish, for the first time, that MNT loss reduces the competitive fitness of both T and B lymphopoiesis. We show that MNT loss prevents T lymphoma development, and elucidate the cause by dissecting T cell development in young MNT-null MYC transgenic mice. Taken together, these studies indicate that MNT can facilitate MYC-driven tumorigenesis rather than acting as a tumour suppressor as previously suggested ^{4, 22, 23, 24}. The results have important therapeutic implications for MYC-driven T and B lymphoid tumours and perhaps many other MYC-driven tumour types.

Mice

Mice used were Mnt^{fl/fl 22}, Rag1Cre ²⁵, MYC10^{hom 26} and Bim^−/−del339 ²⁷, 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. Breeding, genotyping and phenotype analyses are detailed in Supplementary Materials and Methods.

Competitive bone marrow repopulation

C57BL/6 mice (Ly5.1⁺) were lethally irradiated (2 x 550 Rad) and reconstituted with 10⁶ bone marrow cells from C57BL/6 mice (Ly5.1⁺ competitor cells) and 10⁶ bone marrow cells from either Mnt^+/+Rag1Cre or Mnt^fl/flRag1Cre mice (Ly5.2⁺ test cells). Twelve weeks after transplantation, the relative proportions of test- and competitor-derived cells in the thymus, spleen and bone marrow of recipients was determined by flow cytometry, using CD45.1 and CD45.2 antibodies to distinguish Ly5.1⁺ and Ly5.2⁺ cells respectively.

OP9-DL1 co-culture

DN3 and DN4 T progenitor cells sorted from thymi of Mnt^+/+Rag1Cre and Mnt^fl/fl Rag1Cre mice using a-CD4, a-CD8, a-CD25 and a-CD44 antibodies (see Supplementary Fig. S1) were cultured at 37^oC in 6-well plates containing a confluent monolayer of (unirradiated) OP9 cells expressing Notch ligand Delta-like 1 (OP9-DL1) ²⁸ in the presence of 5ng/mL recombinant murine IL-7 for 3–4 d, after which lymphoid cells were recovered via a 40 micron cell strainer.

Statistical analysis

Statistical comparisons were made using unpaired two-tailed Student’s t-test or ANOVA with Prism v8.0 software (GraphPad, San Diego, CA, USA). Data are shown as means ± SEM with P-values ≤ 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.

Impact of MNT loss on T lymphopoiesis.

In adult mice, c-MYC is essential for normal T cell development and immune responses, regulating proliferative bursts in both the thymus and spleen ^{29, 30, 31, Mingueneau, 2013 #21545}. To ascertain MNT’s role in T cell development, we crossed Mnt^fl/fl mice ²² with Rag1Cre mice ²⁵ and analysed the cellular composition of the thymus and spleen in 6–7 week-old offspring of each genotype. Mnt deletion mediated via the Rag1Cre transgene, which is expressed only in early lymphoid progenitors ²⁵, was very efficient, as shown by PCR and western blot analysis of the major thymic sub-populations (Fig. 1A).

Thymic weight and cellularity were reduced to ~ 65% of normal in Mnt^fl/flRag1Cre mice, primarily due to fewer DP T cells (P ≤ 0.001), although the DN and SP CD4⁺ populations were also significantly reduced compared to their counterparts in Mnt^+/+Rag1Cre controls (Fig. 1B). When the DN population was further dissected by staining with CD44 and CD25 (Supplementary Fig. S1A), it became apparent that DN4 (CD25⁻CD44⁻) cells were more affected than DN2 (CD25⁺CD44⁺) or DN3 (CD25⁺CD44⁻) cells (Fig. 1C). This deficit was not due to a failure of TCR b gene rearrangement because intracellular TCRb protein was readily detectable in the Mnt^fl/flRag1Cre DN4 cells (Supplementary Fig. S1B).

Spleen cellularity was also reduced in young Mnt^fl/flRag1Cre mice (p < 0.0001) (Fig. 1D), primarily due to the decrease in the B lymphoid population (~ 37%; p < 0.0001), as reported previously ¹⁵. In addition, T cells were reduced, particularly CD4⁺ T cells (~ 60%; p < 0.001). Cell surface expression analysis of CD44 and CD62L showed that the proportions of naïve, memory and effector T cells were equivalent between WT and Mnt-null T cells (Supplementary Fig. S1C, D). Myeloid (Mac1⁺) cellularity was unaffected (Fig. 1D), as expected from the lack of Rag1Cre expression in this cell lineage.

MNT loss reduces competitive fitness.

To compare MNT-deficient versus normal lymphopoiesis, we performed competitive bone marrow reconstitution experiments. Lethally irradiated Ly5.1⁺ mice were injected with a 50:50 mixture of Ly5.1⁺ WT cells and test Ly5.2⁺ Mnt^fl/flRag1Cre bone marrow cells, or a 50:50 mixture of Ly5.1⁺ WT and test Ly5.2⁺ Mnt^+/+Rag1Cre bone marrow cells (Fig. 2A). Analysis by flow cytometry after 12 weeks (Supplementary Fig. S2) revealed that the bone marrow cells from Mnt^fl/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⁺ Mnt^fl/flRag1Cre (gold bars) than Ly5.2⁺ Mnt^+/+Rag1Cre (brown bars) cells in all major thymic subpopulations (Fig. 2B). Similarly, the spleen of reconstituted mice contained significantly fewer Mnt^fl/flRag1Cre than Mnt^+/+Rag1Cre CD4⁺ or CD8⁺ T cells (Fig. 2C). Comparable outcomes were noted for B lineage cells in the spleen and bone marrow (Fig. 2C, D). In contrast, as anticipated, Ly5.2⁺ Mnt^+/+Rag1Cre and Ly5.2⁺ Mnt^fl/flRag1Cre myeloid cells, were present in comparable numbers in the spleen and bone marrow. We conclude that MNT loss puts both T and B lymphopoiesis at a significant competitive disadvantage.

MNT loss increases T cell apoptosis.

The T cell deficit in Mnt^fl/flRag1Cre mice seemed likely to reflect increased apoptosis and/or reduced MYC levels. All four major thymic sub-populations in Mnt^fl/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 the levels of endogenous MYC protein in any of these populations, as shown by flow cytometric and immunoblot analysis (Fig. 3B-D). Thus, MNT loss promoted apoptosis but did not affect MYC levels.

Enhanced apoptosis probably also explains the reduced DN4 population in Mnt^fl/flRag1Cre mice. When sorted DN3 and DN4 cells were cultured on OP9-DL1 cells in IL-7, conditions which are permissive for 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). In contrast, the DN3 cultures showed no major differences. These observations suggest that MNT loss 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 ~ two-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 the major determinant of the T cell deficit in Mnt^fl/flRag1Cre mice is increased apoptosis.

BIM is a critical mediator of apoptosis in MNT-null T cells.

Cellular stress provokes apoptosis via the mitochondrial cell death pathway, which is regulated by opposing factions of the BCL-2 family ^{32, 33}) and genetic studies have identified pro-apoptotic BIM (BCL2L11), a BH3-only protein, as a key trigger of lymphocyte death^{34, 35, 36, 37}. 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 pro-survival MCL-1, an important regulator of T cell survival ³⁸ (Fig. 5A, B). Increased Bim transcription in MNT-deficient T cells (Fig. 5C) may at least 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 mitogen-activated CD8⁺ T cells (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 have previously proposed for B lymphoid cells ¹⁵.

To directly test BIM involvement in the apoptosis of MNT-deficient T cells, we bred Bim^+/− Mnt^fl/flRag1Cre mice (Bim is functionally haplo-insufficient ³⁹). Indeed, apoptosis in thymocyte populations from Bim^+/− Mnt^fl/flRag1Cre mice (rust bars) was significantly less than in those from Mnt^fl/f Rag1Cre mice (gold bars), and comparable to that in WT mice (black bars) (Fig. 5D). Furthermore, 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^+/− Mnt^fl/flRag1Cre mice (Fig. 5G). Furthermore, loss of one Bim allele prevented the enhanced apoptosis of Mnt^fl/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. Importantly, MNT may also constrain BIM levels in other cell types. We found that MNT KO HEK 293T cells and HeLa cells have more BIM than their parental cells, and BIM was reduced when MNT was reintroduced into MNT KO cells (Supplementary Fig. S5A-F).

Mnt deletion prevents T lymphoma development in MYC10^hom transgenic mice.

To ascertain the impact of MNT loss on MYC-driven T lymphomagenesis, we utilised our MYC10^hom mice ^{26, 40}, which are homozygous for a transgene expressing a human MYC cDNA via the pan-haemopoietic VavP transgenic vector ⁴¹. 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 they also develop disseminated histiocytic myeloid (monocyte/macrophage) (Mac1⁺F4/80⁺Gr1⁻) tumours affecting the spleen and other organs ⁴⁰.

Mnt ^fl/fl MYC10 ^hom/Rag1Cre mice survived significantly longer than the control Mnt^+/+MYC10^hom and Mnt^+/+MYC10^hom/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 euthanased sick mice revealed a major difference in pathology. Whereas the control mice presented with massively enlarged thymi and/or splenomegaly, Mnt^fl/flMYC10^hom/Rag1Cre mice presented only with splenomegaly (Fig. 6B).

Importantly, Rag1Cre-mediated Mnt deletion had specifically prevented T lymphoma development in MYC10^hom mice (Fig. 6C). None of the 26 mice in the Mnt^fl/f MYC10^hom/Rag1Cre cohort developed thymic T lymphomas (Supplementary Table S3) and, where analysed, their thymic T cells were polyclonal (e.g. # 1313 and #1316 in Fig. 6D). In contrast, 12/26 Mnt^+/+MYC10^hom and 7/24 Mnt^+/+MYC10^hom/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 TCRb gene rearrangements, indicative of clonality/biclonality (Fig. 6D, E). Curiously, the T lymphomas were also Mac-1-positive (Supplementary Fig. S6A, B) (see Discussion).

Grossly enlarged spleens arising in either Mnt^+/+ or Mnt KO MYC10^hom mice contained a high proportion of Mac-1⁺ myeloid cells (Fig. 6F), which were transplantable (Supplementary Table S4), and histological review revealed invasion of many other tissues, as described previously ⁴⁰. However, although the splenic CD4⁺ T cells were clearly activated (CD44⁺CD62L⁻) (Supplementary Fig. S6D), they were not transplantable (Supplementary Table S4).

In summary, our data establish that lymphoid-specific Mnt deletion prevented MYC-driven T lymphomagenesis in MYC10^hom transgenic mice but not the development of fatal MYC-driven myeloid tumours. Whether MYC-driven myeloid tumorigenesis in vavP-MYC transgenic mice requires MNT is not addressed by these studies as Rag1Cre is only expressed in lymphoid progenitor cells ⁴².

MNT loss impairs T cell development in MYC10^hom transgenic mice.

To clarify why T lymphomagenesis was abrogated in Mnt^fl/flMYC10^hom/Rag1Cre mice, we analysed healthy young (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 Mnt^fl/flMYC10^hom/Rag1Cre mice (green) compared to control Mnt^+/+MYC10^hom mice (blue) (p ≤ 0.001), and all thymocyte sub-populations were reduced around two-fold (Fig. 7A).

As reported previously ⁴⁰, the level of MYC protein in thymocytes of MYC10^hom 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 premalignant MYC10^hom T cells (Supplementary Fig. S7B to D). However, as shown in Fig. 7B, the proportions of annexin-V-positive cells were significantly higher in Mnt KO MYC10^hom than Mnt^+/+ MYC10^hom 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). Furthermore, when cultured in vitro, Mnt KO MYC10^hom DP thymocytes died faster than their Mnt^+/+MYC10^hom or WT counterparts (Fig. 7C). Thus, the overt consequence of MNT loss was increased apoptosis.

Elevated BIM protein levels paralleled the increased apoptosis, as shown by immunoblot and intracellular FACS analysis of DP thymocytes (Fig. 7D, E), and RT PCR analysis suggested the rise at least in part reflected increased Bim transcription (Fig. 7F). Anti-apoptotic MCL-1 protein levels were higher in the MYC10^hom than WT DP thymocytes, but they were not affected by MNT loss (Fig. 7D). BCL-X_L levels were comparable in cells from all three genotypes (Supplementary Fig. S7A) and expression of pro-apoptotic p53 was not detectable in T cells from these healthy young Mnt^fl/flMYC10^hom mice, 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 premalignant MYC10^hom mice (green bars in Fig. 7G). This might have been due partly to reduced migration from the thymus, but annexin V staining (Fig. 7H) indicated that they were also more susceptible 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 the MYC trangenic splenic T cells were enlarged and highly activated, particularly the CD4⁺ cells lacking MNT (Supplementary Fig. S8C, D).

MNT loss also greatly reduced CD19⁺ B lymphoid cells in the spleen and bone marrow of the young MYC10^hom mice, as reported previously for pre-leukaemic Mnt^fl/fl Em-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 MYC10^hom genotypes, despite the disseminated myeloid disease that inevitably develops as the mice age.

We conclude that the abrogation of thymic lymphoma development in Mnt KO MYC10^hom mice is due at least in part to the increased apoptosis of their pre-malignant T lymphoid populations, driven by elevated BIM levels.

Impact of MNT loss on normal T cell development.

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 (ref 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 ^{29, 30}. In the B lineage, pre-pro-B, pro-B, pre-B and B cells were all affected ¹⁵.

The mild T cell phenotype of our Mnt^fl/fl Rag1Cre mice differed markedly from that reported earlier for Mnt^fl/fl LckCre mice ¹⁹, which had a more severe T cell loss, progressive inflammatory disease and a high predisposition to late T lymphoma. No tumours have developed in our Mnt^fl/fl Rag1Cre colony, although we have not aged a large cohort. The phenotypic differences may be due to continuous (Lck promoter-driven) versus transient (Rag1 promoter-driven) CRE expression. Continuous CRE exposure greatly perturbs T cell development ⁴³, perhaps due to accumulated DNA breaks, and this phenotype may be exacerbated by concomitant loss of MNT. However, the reduced B lymphoid population may have contributed to the milder T phenotype in Mnt^fl/fl Rag1Cre mice.

We found that MNT loss in T cells provoked upregulation of BIM, a major apoptosis trigger (e.g. ^{34, 35, 36, 37}), as we reported previously for B lymphoid cells ¹⁵. Both BIM protein and Bim mRNA levels were elevated in T cells of Mnt^fl/fl Rag1Cre mice. Importantly, the enhanced apoptosis and T cell deficit provoked by lymphoid-specific Mnt KO was reversed by loss of a single Bim allele. 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 ⁴⁴). Importantly, our data with human HEK 293T and HeLa cells (Supplementary Fig. S5) suggest that MNT can also dampen BIM expression in non-lymphoid cell types. Indirect as well as direct mechanisms may be involved.

Impact of MNT loss on T lymphomagenesis.

To gauge how MNT affects MYC-driven T lymphomagenesis, we utilised VavP-MYC10^hom mice, in which transgene expression is pan-haemopoietic but highest in T cells ^{26, 40}. These mice are prone to both thymic T lymphomas and disseminated myeloid tumours. Strikingly, lymphoid-specific Mnt loss totally ablated T lymphoma development in the MYC10^hom mice. None of the mice in the Mnt^fl/flMYC10^hom/Rag1Cre cohort (n = 26) developed thymic lymphomas. Instead, their morbidity was exclusively due to highly invasive myeloid tumour cells.

In contrast, approximately 40% of the 50 control Mnt WT MYC transgenic mice (Mnt^+/+MYC10^hom plus Mnt^+/+MYC10^hom/Rag1Cre) developed thymic lymphomas. Apart from one CD19⁺ lymphoma, all 15 thymic tumours immunophenotyped in these mice were unusual CD4⁺CD8⁺ T lymphomas expressing Mac-1. Activated CD8⁺ T cells express Mac-1⁴⁵, so its expression on the DP T lymphomas may reflect activation induced by the MYC10^hom transgene. Alternatively, these tumours may arise from a T/myeloid progenitor cell, as do rare mixed-phenotype human acute leukaemias (e.g. ref ^{46, 47}).

Our results showing that MNT loss abrogates 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 MYC^T58A protein from within the ROSA 26 locus ^{20, 48}.

To explore why T lymphomas failed to develop in Mnt KO MYC10^hom mice, 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 MYC10^hom T cells were significantly more susceptible to apoptosis than Mnt ^+/+MYC10^hom T cells, at all major stages of development. Indeed, their apoptosis is likely to be much greater than suggested by annexin-V labelling, as apoptotic cells are very rapidly engulfed by phagocytes in vivo ⁴⁹.

Quantification of the expression of major apoptosis regulators revealed that pro-apoptotic BIM was elevated in Mnt KO as compared to Mnt^+/+ DP thymocytes in MYC10^hom transgenic mice. In contrast, the transcription factor TP53, which triggers apoptosis by transcriptionally activating expression of two other pro-apoptotic BH3-only proteins, PUMA and NOXA, was undetectable. Moreover, the level of MCL-1, an important regulator of T cell survival ³⁸, was unchanged. We infer that BIM upregulation is a major factor responsible for reducing T cell populations in healthy young Mnt KO MYC10^hom mice.

Tumour development requires acquisition of oncogenic mutations and overcoming the protective apoptosis threshold. BIM-directed apoptosis has been shown to be one of the major factors limiting MYC-driven cellular proliferation, as loss of even one Bim allele greatly accelerated lymphomagenesis in Em-Myc mice ³⁹. By suppressing BIM, MNT reduces the risk of apoptosis in proliferating cells driven by MYC, thereby boosting the population at risk of acquiring oncogenic mutations. Conversely, MNT loss elevates apoptosis risk and reduces lymphoma risk.

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 ²². The tumour suppressor role received further support when focal deletions involving the MNT locus (usually monoallelic) were noted in ~ 10% of cancers in The Human Cancer Genome Atlas ⁴, including certain cases of chronic lymphocytic leukaemia ²⁴, a B cell malignancy, and Sezary syndrome, a cutaneous T-cell lymphoma/leukaemia ²³. 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 (ref^{15, 20, 21} and this paper), using three independent transgenic 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 ⁵⁰. Like MYC, MNT binds to E boxes near many genes ^{1, 44}. Therefore, in addition to suppressing apoptosis, MNT may prove to 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 ^{51, 52}. 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.

ACKNOWLEDGEMENTS

The authors thank Peter Hurlin (Shriner’s Hospital for Children, Portland OR USA) for his kind gift of Mnt floxed mice; our colleagues Jerry Adams, Andreas Strasser, Gemma Kelly, Steve Nutt and Jacob Jackson for comments on the manuscript and useful discussions; Steve Nutt and Andreas Strasser and Lorraine O’Reilly for fluorescent antibodies; 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.

AUTHOR CONTRIBUTIONS

H.V.N, C.J.V and M.R.R. performed the experiments; A.P.N performed histological analysis; S.C. conceived the project, wrote the manuscript with input from the authors and secured funding.

FUNDING

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.

COMPETING INTERESTS

The authors declare no competing financial interests.

ADDITIONAL INFORMATION

Supplementary information The online version contains supplementary material available at

Correspondence: Suzanne Cory, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, VIC 3052, Australia; e-mail: [email protected]

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posted 15 Apr, 2022

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MNT suppresses T cell apoptosis via BIM and is vital for MYC-driven T lymphomagenesis

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Abstract

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Introduction

Materials And Methods

Mice

Competitive bone marrow repopulation

OP9-DL1 co-culture

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