T-cell Dysfunction upon Expression of MYC with Altered Phosphorylation at Threonine 58 and Serine 62

This article is featured in Highlights of This Issue, p. 1011

The c-MYC oncoprotein (MYC) plays an important role as a transcription factor in balancing cellular growth, proliferation, differentiation, and programmed cell death through the regulation of many target genes. The ability to promote cell growth and proliferation is modulated by MYC's ability to also promote apoptosis, which feasibly occurs in normal cells as a part of a feedback loop in response to high levels of MYC to keep proliferation in balance. With such important biological functions, it is not surprising that Myc knock-out mice are embryonically lethal and MYC null cells have severe growth and proliferation defects, while sustained overexpression of MYC in mice leads to cancer (1–3). Increased levels of MYC protein have been reported in about 70% of human cancers, but only approximately 20% of these show MYC gene amplification suggesting a role of posttranslational deregulation (4, 5). Studies have also shown that mutations in MYC, primarily at or around the conserved Threonine 58 (T58) and Serine 62 (S62) phosphorylation sites, are found in viral isolates, B-cell Burkitt lymphomas, and AIDS associated B- and T-cell lymphomas (6–9). Together, these observations support the importance of posttranslational regulation of MYC.

Normal cycling cells regulate the levels of MYC by tightly controlling its transcription, translation, and protein stability. One mechanism regulating MYC protein stability includes the sequential phosphorylation of two highly conserved phosphorylation sites at residues T58 and S62 in the transactivation domain (TAD) region (10–12). In response to growth signals, MYC is phosphorylated at S62 by one of several proline-directed kinases including ERK or CDKs, which transiently increases MYC stability. Subsequently, the phosphorylation at T58 is mediated by the processive kinase GSK3β (10) or BRD4 (13), initiating the dephosphorylation of S62 by Protein Phosphatase 2A (PP2A) and the ubiquitination of MYC by the E3 ligase complex SCF-FBW7, directing proteasomal degradation. The dephosphorylation at S62 is facilitated by the peptidyl prolyl isomerase, PIN1, which catalyzes a cis to trans isomerization at proline 63 (11, 14). This tightly regulated MYC degradation pathway is coordinated by the scaffold protein AXIN1 (15, 16), which is a known tumor suppressor (17, 18). The genes involved in this MYC degradation pathway such as FBW7, AXIN1, and the PP2A subunits, as well as proteins upregulating ERK and CDK kinase activity have been reported mutated in cancer cells including hematologic cancers and play a role in the wide-spread deregulated MYC phosphorylation and increased protein stability in tumors (15, 16, 19–23).

In addition to controlling MYC protein degradation, functional studies with the MYC T58A and S62A mutants have reported altered MYC activity indicating that aberrant phosphorylation at these sites can impact MYC function. The T58A mutation leads to increased levels of S62 phosphorylation and increased protein stability whereas the S62A mutant cannot be phosphorylated at either residue, which also generally leads to increased stability as it cannot be regulated through the S62/T58-mediated degradation pathway (11). Functionally, in vitro studies with fibroblasts and epithelial cells indicate that the T58A mutant (MYC^T58A) has increased transforming and DNA binding activity and the S62A mutant (MYC^S62A) has decreased transforming and DNA binding activity relative to wild-type MYC ((MYC^WT; refs. 24–27). This increased oncogenic potential of MYC^T58A may be in part due to a decreased capacity of MYC^T58A to induce apoptosis, which has been shown in tissue culture and mouse models (27–30). Consistently, human tumors and cancer cell lines display an altered ratio of decreased T58 to increased S62 phosphorylation, which correlates with impairment in the degradation pathway and increased MYC protein stability, has been broadly observed (16, 20, 29).

Mutations in c-MYC are rare and only appreciably observed in Burkitt and AIDS-associated lymphomas. These mutations occur at hot spots around the T58 and S62 residues, with previous studies reporting the predominant mutations to be T58I and T58A (7, 24, 31). Phospho-peptide mapping analysis of the T58I mutant has shown that unlike the T58A mutant, which has higher than wildtype S62 phosphorylation, MYC^T58I lacks phosphorylation at both the T58 and S62 sites suggesting that MYC lacking S62 phosphorylation can be oncogenic in B-cell lymphomagenesis (24). Thus, studying the effects of phosphorylation mutants at both T58 and S62 on MYC function and involvement in tumorigenesis in vivo remains an important area of research (6, 7), particularly for the MYC^S62A mutant which is not phosphorylated at either residue, shows low oncogenic activity in vitro, and has been understudied in vivo. This mutant has been previously observed using a mammary expressing Cre recombinase (Cre) with leaky expression in the brain to drive choroid plexus and pituitary tumors in mouse brain (29), however further examination of MYC^S62Ain vivo function is lacking.

High levels of MYC have been broadly observed in human T-ALLs, due to a variety of mechanisms including a rare MYC gene translocation (32), frequent aberrant NOTCH1 signaling, and FBW7 mutations (19, 33–35), which are associated with increased MYC protein stabilization (33, 36–38). Given these observations of increased MYC stabilization in T-ALL (19, 20), in this study, we investigated the oncogenic potential of the two phosphorylation mutants of MYC utilizing our conditional knock-in mouse model expressing Myc^WT, Myc^T58A, or Myc^S62A from the endogenous ROSA26 locus in response to Cre-mediated excision of a transcription stop site and the T-cell–specific Lck-Cre to mediate recombination (29, 30, 39–41). This model allows for the study of the MYC phosphorylation mutants in cells that pass through distinct phases of growth, proliferation, and apoptosis, allowing elucidation of the function of MYC without phosphorylation at T58 and/or S62 in T-cell development and lymphomagenesis.

ROSA26-floxed stop (RFS)--c-Myc^WT, -c-Myc^T58A, and -c-Myc ^S62A mice have been previously described (29). RFS-c-Myc^WT, -c-Myc^T58A, and -c-Myc ^S62A mice were crossed with Lck-Cre mice for T-cell–specific expression (30). To conditionally ablate endogenous Myc, RFS-Myc^WTMyc^fl/flLck-Cre, RFS-Myc^T58AMyc^fl/flLck-Cre, and RFS-Myc^S62AMyc^fl/flLck-Cre mice (MYC^WTMYC^fl/fl, MYC^T58AMYC^fl/fl, & MYC^S62AMYC^fl/fl) were generated by breeding with C57Bl/6 conditional Myc^fl/fl mice (42, 43). Simultaneously, cohorts of RFS-c-Myc^WT, -c-Myc^T58A, and -c-Myc ^S62A mice were generated by breeding out the Myc^fl/fl allele to control for strain background in lymphomagenesis. Male or female control mice were homozygous for either RFS-Myc^WT, RFS-Myc^WTMyc^fl/fl, RFS-Myc^T58A, RFS-Myc^T58AMyc^fl/fl, RFS-Myc^S62A, RFS-Myc^S62AMyc^fl/fl or Myc^fl/fl mice, but had no Lck-Cre allele. All mice were handled and maintained in accordance with the Oregon Health and Science University (OHSU; Portland, OR) Institutional Animal Care and Use Committee.

Lck-Cre mediated recombination in RFS-c-Myc^WT, -c-Myc^T58A, and -c-Myc ^S62A mice were determined by PCR analysis and specific primer sets. Genotyping for the presence of RFS, Myc^fl/fl and Lck-Cre was done using tail DNA and specific primer sets (see Supplementary Table S1 for primer sequences).

All cohorts of male or female experimental mice and control mice with no Cre expression were monitored for development of thymic lymphoma, splenomegaly, or both. Mice were subjected to euthanasia and necropsied when presented with accelerated breathing, a distended abdomen, cachexia, were moribund, and/or reached about 730 days of age. Kaplan–Meier survival plot was done using Prism (Graphpad Software Inc., RRID:SCR_002798).

RNA was extracted from male and female mice with thymic lymphomas or normal thymi from control mice of any genotype with no Cre expression with Trizol (Invitrogen) following the manufacturer's protocol. The resulting RNA was treated with DNaseI (Roche) and further purified with a RNeasy mini kit (Qiagen). cDNA was synthesized using random primers with the High Capacity Reverse Transcription kit (Applied Biosystems). qRT-PCR for total, ectopic, and endogenous c-Myc mRNA was carried out on a StepOne qRT-PCR machine (Applied Biosystems) with SYBR Green (Invitrogen) and normalized to Tbp message (see Supplementary Table S2 for primer sequences).

Thymi and spleens were harvested from male or female 6-week–old experimental mice. Littermate controls were male or female mice homozygous for either RFS-Myc^WT, RFS-Myc^T58A, RFS-Myc^T58AMyc^fl/fl, RFS-Myc^S62A, or RFS-Myc^S62AMyc^fl/fl with no Cre expression. Tissue was disrupted into a single cell suspension in 1X PBS with 5% FBS, 0.5% BSA, and 2 mmol/L EDTA and filtered through a 70-μm mesh filter. Cells were spun down at 1,200 rpm for 5 minutes. Red blood cells were lysed using RBC lysis buffer (155 mmol/L NH₄Cl, 12 mmol/L NaHCO₃, 0.1 mmol/L EDTA) Cells were respun and refiltered through a 70-μm mesh filter and counted. Two million splenocytes were plated per well (DMEM with 10% FBS, 4.5 g/L glucose, 2 mmol/L L-glut, NEAA, and 50 μmol/L β-mercaptoethanol) labeled with BrdU and stimulated with 1 μg Concanavalin A (Con A; Sigma-Aldrich) for 24 hours. FITC or APC BrdU Flow Kit (BD Pharmigen) and antibodies for CD4 (GK1.5, Thermo Fisher Scientific, catalog no. 553051, RRID:AB_464902) and CD8 (53–6.7, Thermo Fisher Scientific, catalog no. 553033, RRID:AB_467086) were used to assess CD4 and CD8 labeled splenic T-cell proliferation. Apoptosis of CD4- and CD8-positive (CD4⁺, CD8⁺) thymic T cells were assayed using a FITC Annexin V Apoptosis Detection Kit (BD Pharmigen) 24 hours postculture. Cells were analyzed with a BD FACS Canto II (BD Biosciences) and 100,000 live cell events were collected. Data files were analyzed using FlowJo X (Tree Star, Inc., RRID:SCR_008520).

Tissues were fixed in 10% formalin overnight at 4°C, washed in 70% EtOH the following day, and embedded in paraffin. A microtome (Leica) was used to cut 5-μm–thick sections, which were stained with Mayer hematoxylin and eosin (H&E). For immunofluorescence (IF) staining, antigen retrieval was achieved by pressure-cooking in citrate buffer pH 6 for 10 minutes (Sigma-Aldrich). Slides were allowed to cool for 1 hour. The following antibodies were used for staining: CD3 1:50 (pAb, Dako, catalog no. A045229–2, RRID:AB_2335677), CD45R 1:50 (RA3–6B2, Abcam, catalog no. ab64100, RRID:AB_1140036), pS62 MYC rat monoclonal (4B12, conjugated to AF647) 1:25 (44). Secondary antibodies include Alexa Fluor 488 and Alexa Fluor 750 (Invitrogen) and mounting media with antifade containing DAPI (Thermo Fisher Scientific). H&E and IF stains were imaged using a Zeiss Axio Scan.Z1 slide scanner at 20x and 40x. Analysis was done with Zen software (Zeiss Microscopy) and FIJI (RRID:SCR_002285).

Tumor and control thymic tissue were disrupted into a single-cell suspension in 1X PBS with 5% FBS, 0.5% BSA, and 2 mmol/L EDTA and filtered through a 70-μm mesh filter. Cells were spun down at 1,200 rpm for 5 minutes, resuspended in 70% DMEM, 10% DMSO, 20% FBS, and frozen and stored at −80°C. Cells were stained in 1X PBS with 2% FBS and 0.1% NaN₃ for 20 minutes, on ice in the dark. Antibodies used are as follows: CD4-BUV737 (GK1.5, BD Biosciences, catalog no. 612761, RRID:AB_2870092), CD8-BUV395 (53–6.7, BD Biosciences, catalog no. 563786, RRID:AB_2732919), CD24-PE (M1/69, BD Biosciences, catalog no. 553262, RRID:AB_394741), CD25-BV650 (PC61, BD Biosciences, catalog no. 564021, RRID:AB_2738547), CD45-BUV496 (30F-11, BD Biosciences, catalog no. 749889, RRID:AB_2874129), CD138-PE (281–2, BD Biosciences, catalog no. 553714, RRID:AB_395000), Ly6G-BUV395 (1A8, BD Biosciences, catalog no. 565964, RRID:AB_2739417), and TCRβ-BUV737 (H57–597, BD Biosciences, catalog no. 612821, RRID:AB_2870145), CD4-PE/Cy7 (RM4–5, BioLegend, catalog no. 100528, RRID:AB_312729), CD8-PE/Cy7 (53–6.7, BioLegend, catalog no. 100722, RRID:AB_312761), CD19-BV650 (6D5, BioLegend, catalog no. 115541, RRID:AB_11204087), CD19-PerCP/Cy5.5 (6D5, BioLegend, catalog no. 115534, RRID:AB_2072925), CD21-APC/Cy7 (7E9, BioLegend, catalog no. 123417, RRID:AB_1953274), CD44-FITC (IM7, BioLegend, catalog no. 103021, RRID:AB_493684), CD49β-APC/Cy7 (DX5, BioLegend, catalog no. 108920, RRID:AB_2561458), B220-BUV395 (RA3–6B2, BD Biosciences, catalog no. 563793, RRID:AB_2738427), IgD-A488 (11–26c.2a, BioLegend, catalog no. 405718, RRID:AB_10730619), Sca-I-BV711 (D7, BioLegend, catalog no. 108131, RRID:AB_2562241), MHC II-BV421 (M5/114, BioLegend, catalog no. 107631, RRID:AB_10900075), Thy1.2-BV785 (30-H12, BioLegend, catalog no. 105331, RRID:AB_2562900), DAPI (BioLegend, catalog no. 422801), CD11b-FITC (M1/70, Thermo Fisher Scientific, catalog no. 11–0112–82, RRID:AB_464935), CD11c-PE (N418, Thermo Fisher Scientific, catalog no. 12–0114–81, RRID:AB_465551), CD23-PE/Cy7 (B3B4, Thermo Fisher Scientific, catalog no. 25–0232–82, RRID:AB_469604), B220-A647 (RA3–6B2, BioLegend, catalog no. 103226, RRID:AB_389330), Ly-6C-PerCP/Cy5.5 (HK1.4, Thermo Fisher Scientific, catalog no. 45–5932–82, RRID:AB_2723343), TACI-APC (ebio8F10–3, Thermo Fisher Scientific, catalog no. 17–5942–81, RRID:AB_842759), and TCRγδ-A647 (eBioGL3, Thermo Fisher Scientific, catalog no. 17–5711–82, RRID:AB_842756), IgM-BV421 (pAb, Jackson ImmunoResearch Labs, catalog no. 115–675–075, RRID:AB_2651085). Cells were analyzed with a BD FACS Symphony A5 (BD Biosciences). Data files were analyzed using FlowJo X (Tree Star, Inc., RRID:SCR_008520).

Tumor cell lines were generated as previously described in Jinadasa and colleagues (45). Briefly, tumors were mechanically disassociated into single cells, suspensions were diluted, and grown at different densities in culture media (DMEM with 10% FBS, 4.5 g/L glucose, 2 mmol/L L-glut, NEAA, and 50 μmol/L β-mercaptoethanol) until clones emerged.

For baseline MYC protein levels, total thymi from male and female 6-week–old MYC^WT, MYC^T58A, and MYC^S62A mice or control mice of any genotype with no Cre expression were homogenized in Ab lysis buffer (20 mmol/L Tris, pH 7.5, 50 mmol/L Nacl, 0.5% Triton X-100, 0.5% DOC, 0.5% SDS, 1 mmol/L EDTA) with protease and phosphatase inhibitors (Roche) and sonicated (Branson). Protein levels were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) and equivalent protein levels were used to immunoprecipitate with 2 μg total MYC antibody (N262, Santa Cruz Biotechnology, catalog no. SC-764, RRID:AB_631276) or 2 μg anti-HA antibody (G036, Applied Biological Materials, RRID:AB_2801472) overnight at 4°C and then pulled down with either protein-A IPA300 beads (Repligen), or protein-G plus agarose beads (Santa Cruz Biotechnology), respectively. For detection of phosphorylated S62 MYC, tumor lysates were prepared as described above and immunoprecipitated with 2 μg anti-MYC (N262, Santa Cruz Biotechnology, catalog no. SC-764, RRID:AB_631276) overnight at 4°C and then pulled down with protein-A IPA300 beads (Repligen; refs. 44, 46). Immunoprecipitated protein was resolved via SDS-PAGE and was transferred to an Immobilon-FL PDVF membrane (Millipore). AquaBlock (EastCoast Bio) was used to block membranes. The membranes were probed with the following primary antibodies as indicated: total c-MYC antibody (Y-69, Abcam, catalog no. ab32072, RRID:AB_1191623), phosphorylated S62 MYC (33A12E10, Abcam, catalog no. ab78318, RRID:AB_10678479), and GAPDH (6C5, Thermo Fisher Scientific, catalog no. AM4300, RRID:AB_627679). Species-specific secondary antibodies conjugated to either a 680 or 800 fluorophore (LI-COR, Molecular Probes) were applied and detected with the LI-COR Odyssey Infrared Imager (LI-COR).

Genomic DNA was extracted from tumor tissue or single cell suspensions using the DNAeasy Blood and Tissue Kit (Qiagen). Deep sequencing of the CDR3 V/J region of the T-cell receptor β (TCRβ) was performed using methods previously described (47).

RNA was extracted from previously frozen tumor tissue (n = 3 from each group and n = 2 as control) using Trizol (Invitrogen) and libraries were generated using a NEB Next Ultra Directional RNA Library Prep Kit for Illumina (NEB) per manufacturer instructions. Libraries were submitted to the OHSU Massively Parallel Sequencing Shared Resource for short read sequencing assays. Differentially expressed genes were selected with DESeq2 using an adjusted P value threshold of 0.05 and a fold change (FC) threshold of ±1.5. Genes were further filtered using a coefficient of variation threshold of 1.0. For unsupervised hierarchical clustering, Voom normalization was performed on tumor samples using all genes and reduced to unique gene symbols, then Ward clustering was used to identify subgroups. Row scaled, hierarchically clustered heatmaps were generated using log scaled and transcripts per million (TPM) normalized values for the selected genes. Gene set enrichment analysis (GSEA) using MSigDB and ImmuneSigDB was performed to determine terms related to predominating hallmark and immune gene sets or pathways (48–50).

The raw RNA sequencing (RNA-seq) files generated in this study can be found in the National Center for Biotechnology Information's Gene Expression Omnibus repository under the accession: GSE193995 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193995), with token ID: ifcfccouftqvten.

To examine the functional roles of stabilized MYC lacking T58 phosphorylation with or without S62 phosphorylation in T-cell development and tumorigenesis, we utilized knock-in mice that conditionally express either a C-terminal HA-tagged murine MYC^WT, MYC with T58 mutated to Alanine (MYC^T58A) that has elevated S62 phosphorylation, or MYC with S62 mutated to Alanine (MYC^S62A) that lacks both T58 phosphorylation and S62 phosphorylation (refs. 29, 30; Fig. 1A). The ROSA-Myc knock-in mice were bred to gain one copy of Lck-Cre for T-cell–specific ectopic expression of homozygous RFS-c-Myc^WT, -c-Myc^T58A, or -c-Myc ^S62A specifically in T cells by the double negative III (DNIII) stage of T-cell development. These mice are denoted here as MYC^WT, MYC^T58A, and MYC^S62A mice. PCR analysis of genomic DNA taken from various tissues at 6 weeks of age confirmed recombination of the allele primarily in thymic tissue (Supplementary Fig. S1A). Consistent with previous analysis of the RFS-Myc model (29, 51, 52), we found equivalent mRNA levels of ROSA promoter driven ectopic Myc^WT, Myc^T58A, and Myc^S62A mRNA expression in the three strains indicative of similar levels of recombination. This results in approximately two- to three-fold higher total cMyc mRNA expression in thymus tissues from the MYC^WT, MYC^T58A, and MYC^S62A mice as compared with no Cre controls (Supplementary Fig. S1B). As expected, based on increased MYC protein stability (Supplementary Fig. S1C), the levels of both ectopic MYC^T58A and MYC^S62A protein were about five-fold higher than the level of ectopic MYC^WT, despite similar mRNA levels (Supplementary Fig. S1B and S1D).

In our mice, both ectopic and endogenous MYC can heterodimerize with MAX to promote transcription of MYC target genes; moreover, MYC/MAX heterotetramers may include both ectopic and endogenous MYC (53). To determine the effects of ectopic MYC expression in the absence of endogenous MYC, C57Bl/6 mice with the LoxP/Cre-driven conditional deletion of endogenous Myc (designated MYC^fl/fl; ref. 42) were bred with the MYC^WT, MYC^T58A, and MYC^S62A mice. This generated new cohorts of RFS-Myc^WT;Myc^fl/fl;Lck-Cre, RFS-Myc^T58A;Myc^fl/fl;Lck-Cre, and RFS-Myc^S62A;Myc^fl/fl;Lck-Cre mice, designated MYC^WTMYC^fl/fl, MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl, respectively (Fig. 1A). All experimental mice contained only one copy of Lck-Cre to minimize Cre effects on T-cell development (54).

With these new cohorts, we first compared thymic weights from male and female 6-week–old mice expressing ectopic MYC with and without endogenous Myc to no Cre control mice. In mice with endogenous Myc, we found a significantly higher thymic mass in MYC^T58A mice as compared with control and MYC^S62A mice (Fig. 1B). Ablation of endogenous MYC significantly reduced thymic weights for all strains. MYC^T58AMYC^fl/fl was capable of maintaining normal thymic weight relative to control while MYC^S62AMYC^fl/fl was not. MYC^T58AMYC^fl/fl thymic weights were significantly higher than MYC^S62AMYC^fl/fl, and both were significantly higher when compared with thymi lacking both ectopic and endogenous Myc expression (designated MYC^fl/fl). Overall total thymocyte numbers, composed of mainly CD4 and CD8 double-positive (DP) T cells, from all strains except MYC^T58A were significantly lower than control (Supplementary Fig. S2A). Further reduction in thymocyte number was seen with loss of endogenous Myc, with the lowest numbers of thymocytes in MYC^S62AMYC^fl/fl and MYC^fl/fl (Fig. 1C). Ectopic expression of MYC^T58A increased, and ablation of endogenous MYC decreased, spleen weight in 6-week–old mice, but ectopic MYC^WT and MYC^S62A did not have significant effects (Supplementary Fig. S2B). Total splenocyte number was also significantly higher in MYC^T58A mice when compared with control and MYC^WT mice (Supplementary Fig. S2C). This increase may be due to an early expansion of CD3-positive MYC^T58A T cells and loss of follicular architecture with disruption of marginal zones in the spleen (Supplementary Fig. S2D).

In addition to contributing to mitogenic cell division, MYC contributes to cell growth and can help drive apoptosis (55, 56). We did observe a significant increase in MYC^T58A and MYC^S62A DP thymocyte size (either with or without endogenous MYC) when normalized to control littermates from each strain, suggesting both unphosphorylated or phosphorylated MYC can drive increased thymocyte size in DP T cells in the thymus at 6 weeks (Fig. 1D; Supplementary Fig. S2A). Expression of MYC^T58A or MYC^S62A in a MYC null background did not show a loss in mature T-cell survival ex vivo suggesting that both can support T-cell survival programs (Fig. 1E; refs. 30, 43). The larger cell sizes without a loss in survival may in part support the ability of MYC^T58A and MYC^S62A to maintain thymic weight, despite having lower total thymocytes in vivo.

Mitogen stimulation of splenic T cells triggers cell growth through nutrient uptake and macromolecular synthesis, and the transition from quiescence to proliferation (30, 35, 57). Ectopic expression of Myc enhances ex vivo T-cell proliferation during mitogen stimulation despite a concomitant increase in apoptosis (30). To look at ex vivo proliferative potential of MYC^T58A and MYC^S62A, and their ability to compensate in a MYC-null background we cultured splenic T cells isolated from 6-week–old mice with the lectin Con A for 24 hours. Total BrdU incorporation of CD4⁺ and CD8⁺ T cells from MYC^WT, MYC^T58A, MYC^T58AMYC^fl/fl, MYC^S62A, or MYC^S62AMYC^fl/fl mice were normalized to stimulated T cells from control littermates. Splenic T cells from MYC^S62AMYC^fl/fl mice showed the least ex vivo proliferative potential compared with all other strains (Fig. 1F; Supplementary Fig. S2E). MYC^T58A T cells proliferated significantly more than MYC^WT T cells but this potential was lost when endogenous MYC was ablated. In general, the ablation of endogenous MYC in MYC^T58A and MYC^S62A T cells significantly diminished their ex vivo proliferative potential when compared with their counterparts, suggesting a cooperativity of endogenous MYC in this assay. However, unlike MYC^S62AMYC^fl/fl, MYC^T58AMYC^fl/fl T cells maintained control levels of proliferation. Together, these data indicate that while MYC^T58A and MYC^S62A are both stabilized and expressed at similar higher levels compared with MYC^WT, only MYC^T58A mice showed significant difference in thymic and splenic mass, cell count, cell size, and ex vivo proliferative potential when compared with control mice with no Lck-Cre expression, and with endogenous Myc loss, MYC^T58A was mostly able to maintain control levels of these metrics while MYC^S62A was not.

Following our 6-week studies on the different MYC strains we conducted survival studies. Overall survival of MYC^T58A mice with or without endogenous MYC was significantly shortened when compared with all other strains including control mice. Interestingly, survival of MYC^T58AMYC^fl/fl mice was significantly shorter than syngeneic MYC^T58A mice (136 days and 193 days, respectively), suggesting that the expression of endogenous Myc dampens the tumorigenic activity of Myc^T58A in vivo. Median overall survival of MYC^S62A and MYC^S62AMYC^fl/fl mice was significantly longer than the MYC^T58A cohort (364 and 324 days, respectively), but significantly shorter than MYC^WT mice (Fig. 2A). Median survival of both MYC^WTMYC^fl/fl and MYC^WT mice were 675 days and 686 days, respectively. The survival curve data suggests that expression of MYC^WT in the presence or absence of endogenous MYC has a very low tumorigenic penetrance in our model where total Myc expression is only approximately two- to three-fold above endogenous levels (Supplementary Fig. S1B; ref. 29). This cohort did not show any significant difference in survival when compared with control mice not expressing Lck-Cre.

Necropsies of moribund mice revealed thymic lymphomas developed in MYC^T58A and MYC^T58AMYC^fl/fl mice with 100% penetrance. A similar presentation of thymic lymphomas occurred in some MYC^S62A and MYC^S62AMYC^fl/fl mice, but necropsy also revealed a high prevalence of splenomegaly in the MYC^S62A and MYC^S62AMYC^fl/fl mice that in some mice cooccurred with an enlarged thymus (hereafter, thymic mass) with unconfirmed etiology due to the presence of splenomegaly and these mice showed similar survival ages compared with the same strain of mice with only splenomegaly (395 and 450 days, respectively) (Supplementary Fig. S3A; Fig. 2B; and Table 1). In contrast, MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice that developed thymic lymphomas without evidence of splenomegaly had significantly shorter but similar survival ages (144 and 168 days, respectively) indicating the lethality of a rapidly expanding lymphoma. Thus, despite the decrease in ex vivo proliferative potential in MYC^S62AMYC^fl/fl T cells, reduction in thymic weight and thymocyte number, MYC^S62AMYC^fl/fl mice can develop thymic lymphomas, but at a substantially lower penetrance (∼36%) than MYC^T58A and MYC^T58AMYC^fl/fl mice (Fig. 2C; Table 1). Together, this indicates that MYC^S62AMYC^fl/fl mice can spontaneously develop thymic lymphomas, however if they escape early thymic lymphoma development, they either succumb to splenomegaly with or without a thymic mass, or persist tumor free (Fig. 2B–C; Table 1).

Tumor tissue from MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice with only thymic lymphomas were histologically similar. These thymic lymphomas were relatively homogenous lymphocytic infiltrates in the thymic compartments with disrupted architectures and were categorized as T-cell lymphomas by cytopathology analysis performed by a hematopathologist (Fig. 3A; Supplementary Table S3). In contrast, the predominant cells in thymic masses that cooccurred with splenomegaly in MYC^S62AMYC^fl/fl mice had greater cytoplasm:nucleus ratios, irregular nuclei, and showed B-cell plasmacytic infiltrates which were characterized as malignant B-cell lymphomas (Fig. 3B; Supplementary Fig. S3B; and Supplementary Table S3). These thymic masses were further characterized by IF to show heterogeneous populations of CD45R (B220)– and CD3-positive cells (CD3⁺) unlike cells in thymic lymphomas from MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice, which were almost entirely CD3⁺ T cells (Fig. 3C). We developed a flow cytometry panel capable of identifying T cells at varying stages of development (Table 2; Supplementary Table S4; and Supplementary Fig. S4A). All ten MYC^T58AMYC^fl/fl and two MYC^S62AMYC^fl/fl thymic lymphomas analyzed were 80% to 100% Thy1.2⁺, confirming they were T-cell lymphomas. Eight tumors contained primarily CD4⁺ CD8⁺ DP thymocytes (Table 2; Supplementary Fig. S4B). MYC^T58AMYC^fl/fl tumors H128 and H520 each contained approximately 45% DP thymocytes and 40% to 50% CD4⁺ single-positive (SP) thymocytes, suggesting about half of the tumor cells had progressed past the DP stage. Tumor H204 was CD4⁺ SP, the most differentiated phenotype among MYC^T58AMYC^fl/fl tumors. MYC^T58AMYC^fl/fl tumor H144 was at the earliest stage of development, as 26% of cells were CD4⁻ and CD8⁻ double-negative (DN), primarily DN4 (Table 2; Supplementary Fig. S4C), while the remaining cells were primarily CD8⁺, most likely CD8⁺ immature SP (ISP) thymocytes (58). Altogether, these data indicate that MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl thymic lymphomas represent T-ALL tumors at various stages of T-cell development (Table 2; Supplementary Table S4).

As described above, mice expressing the MYC^S62A mutation sometimes developed T-cell lymphomas, but other times developed splenomegaly (Table 1), with thymic masses being primarily B220-positive (Fig. 3C), suggesting a B-cell lineage. We developed a flow cytometry panel that could distinguish B1 B cells, B2 marginal zone B cells, B2 follicular B cells, and plasma cells (59, 60), and a panel to identify other immune cell types and applied them to thymic masses cooccurring with splenomegaly in MYC^S62AMYC^fl/fl mice (Table 2; Supplementary Table S4; and Supplementary Figs. S5A and S6). MYC^S62AMYC^fl/fl tumor I442 expressed IgD and CD23, consistent with B2 follicular B cells (Table 2; Supplementary Fig. S5B). MYC^S62AMYC^fl/fl tumors I259 and I411 lacked surface immunoglobulin (not shown) but were CD138⁺ and thus identified as plasma cells (Table 2; Supplementary Fig. S5C). Interestingly, MYC^S62AMYC^fl/fl tumors I86 and I440 expressed B220 and/or CD19, consistent with tumors of a B-cell lineage, but lacked any other B-cell subset markers (Table 2). While a significant fraction of I86 and I440 cells were T cells, these were likely normal mature CD4 and CD8 T cells trafficking through the spleen, and relatively few cells in any sample were identified as myeloid or natural killer (NK) cells (Supplementary Table S4; Supplementary Fig. S6). Histology indicated a starry sky appearance for I440, a hallmark of Burkitt lymphoma (Supplementary Fig. S3B), and consistent with the lack of B1, B2, and plasma cell markers.

To further validate the ability of MYC^S62A to drive T-cell lymphoma in a MYC^fl/fl background, we first tested if deletion of endogenous Myc was complete. Lck-Cre–mediated recombination can be variable and incomplete in T cells (61) and the concern was that tumor forming T cells may be from a subset population that have escaped Cre-mediated recombination of endogenous Myc. Thus, we measured Myc mRNA and protein levels from several T-cell lymphomas from both MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice (n = 5 from each strain; Fig. 4A and B). The mRNA levels of endogenous Myc in the T-cell lymphomas from both MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice were significantly lower when normalized to control thymi from any genotype with no Cre expression, consistent with deletion of endogenous MYC in the models, while expression of ectopic message was equivalently high in both knock-in strains (Fig. 4A). In addition, pS62 MYC was only detected in MYC^T58AMYC^fl/fl and not MYC^S62AMYC^fl/fl T-cell lymphomas and generated lymphoma cell lines (Fig. 4B and C). Likewise, CD3⁺ cells in MYC^T58AMYC^fl/fl T-cell lymphomas stained robustly for pS62 MYC, unlike MYC^S62AMYC^fl/fl, which showed little pS62 MYC staining in CD3⁺ cells; although pS62 MYC was strongly detected in the CD45R-positive (CD45R⁺) cells in thymic masses from MYC^S62AMYC^fl/fl mice with splenomegaly (Fig. 4D). Taken together, these data indicate that the T cells in MYC^S62AMYC^fl/fl tumors do not express endogenous Myc and that this mutant form of MYC lacking phosphorylation at T58 and S62 is capable of driving T-cell lymphoma in our model, albeit at a lower penetrance.

MYCN is essential in early DN stages of T-cell development but decreases in the late DN stage as MYC expression increases (62). However, MYCN can substitute for MYC during embryogenesis (63), and can drive indolent B-cell lymphomas (64). We hypothesized that MYCN was playing a role in MYC^S62AMYC^fl/fl T-cell lymphomagenesis however, analysis of mRNA levels of Mycn in these T-cell lymphomas revealed low levels of expression compared with control thymi, decreasing the likelihood of an oncogenic compensatory role by MYCN in T-cell lymphomagenesis in the MYC^S62AMYC^fl/fl model (Supplementary Fig. S7A). Although Mycn mRNA expression was variable among MYC^S62AMYC^fl/fl tumors, it was not significantly higher than in MYC^T58AMYC^fl/fl tumors.

B1 cells, a subclass of B cells have been reported to express Lck (65–67), which could lead to leaky ectopic MYC expression in the spleen in our model (Supplementary Fig. S1A). To address whether this was accounting for the B-cell expansion in MYC^S62AMYC^fl/fl mice that develop splenomegaly and B-cell lymphomas, we looked at the number of copies of HA-tag Myc mRNA from RNA sequence data of thymic masses from MYC^S62AMYC^fl/fl mice accompanied with splenomegaly compared with MYC^S62AMYC^fl/fl and MYC^T58AAMYC^fl/fl mice that had T-cell lymphoma (Fig. 4E). Ectopic Myc-HA-tag mRNA levels in thymic masses from MYC^S62AMYC^fl/fl mice with splenomegaly was not significantly higher than control thymi without the Lck-Cre transgene, confirming that these malignant B cells infiltrate the thymi without expressing ectopic Myc^S62A and consistent with their robust pS62 MYC staining (Fig. 4D).

Sequencing of the CDR3 V/J region of the TCR (n = 3 for each tumor phenotype) from thymic tumor tissue were predominantly clonal in T-cell lymphomas from both MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice compared with a more diverse population of T- and B-cells in MYC^S62AMYC^fl/fl thymic masses with accompanying splenomegaly (Fig. 4F; ref. 47). When comparing clonality indexes, the T-cell lymphomas in MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice, as well as cell lines derived from representative T-cell lymphomas showed clonality while MYC^S62AMYC^fl/fl thymic masses with splenomegaly did not (Fig. 4G). Together, these results indicate that malignant B cells infiltrating into the thymic cavity underlie the appearance of thymic lymphomas in the MYC^S62AMYC^fl/fl mice with splenomegaly; and this infiltration is not originating from cells with MYC^S62A transgene expression. This is consistent with the longer life span of mice in which transgene-driven T-cell lymphoma is not the cause of mouse study endpoint. Taking this into consideration reveals that the difference in penetrance of T-cell lymphoma between the MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice is stark (Fig. 2C).

Clonal T-cell expansion is a hallmark of T-cell lymphomas in MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice without splenomegaly. To determine if similar gene expression profiles are present in the T-cell lymphomas in the MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice, we used bulk RNA-sequencing data of tissue from T-cell lymphomas from MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice, as well as from thymic masses in MYC^S62AMYC^fl/fl mice with accompanying splenomegaly. Unsupervised hierarchical clustering of the RNA-seq data (n = 9) resulted in three distinct groups, with T-cell lymphomas from MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice clustering separately, but closer together than thymic masses with splenomegaly from MYC^S62AMYC^fl/fl mice (Fig. 5A). Distinct clustering between MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl T-cell lymphomas was also seen with immunologic gene sets when performing analysis using the ImmuneSigDB gene set (Supplementary Fig. S7B; ref. 50), suggesting the different forms of mutant MYC drove transcriptionally distinct tumors. Visualization of differentially regulated genes between MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl T-cell lymphomas with an adjusted P value cut-off point of 0.05 and FC cut-off point of ± 1.5 revealed 486 genes (Fig. 5B; Supplementary Table S5). Performing GSEA with the Hallmarks of Cancer gene sets revealed the T-cell lymphomas from MYC^T58AMYC^fl/fl mice were enriched for genes involved in KRAS signaling, angiogenesis, and IFNα response (Fig. 5C; Supplementary Table S6). Interestingly, MYC^S62AMYC^fl/fl T-cell lymphomas were enriched for gene sets involved in the G₂–M checkpoint, MYC, and mitotic spindle, consistent with a potential role of genomic instability in tumor progression for the MYC^S62A mutant as was previously observed (ref. 29; Fig.5D; Supplementary Table S6). Taken together, these data suggest that these two mutant forms of MYC may drive T-cell lymphomas through mechanistically different pathways.

We found 1,747 differentially regulated genes between thymic masses from MYC^S62AMYC^fl/fl mice with splenomegaly versus T-cell lymphomas from MYC^S62AMYC^fl/fl (Fig. 5E; Supplementary Table S7) with an adjusted P value cut-off point of 0.05 and FC cut-off point of ±1.5. The thymic masses with splenomegaly showed enrichment of IFNα and γ gene sets (Fig. 5F; Supplementary Table S7), which may be due to the infiltration of malignant B cells.

MYC protein levels are found to be constitutively high in human tumors and play a role in tumorigenic activity. High MYC levels can activate both apoptosis and proliferation pathways and are also associated with genomic instability. MYC protein levels are affected by stability, which is regulated by phosphorylation at T58 and S62, and dysregulation of phosphorylation at these sites is often associated with cancer (29, 68). By using the constitutive but relatively weak ROSA promoter, we were able to express c-Myc phosphorylation mutant mRNA at near physiologic levels. This allowed us to investigate the effects of phosphorylation at these sites in vivo, particularly the MYC^S62A mutant, the activity of which has been poorly understood with conflicting reports including reduced transforming activity in vitro, contribution to genomic instability in vivo, and the presence of MYC^T58I mutations in Burkitt lymphoma that similarly lacks T58 and S62 phosphorylation (7, 24, 26, 29). We used the Lck-Cre model of recombination, since this occurs as earlier as the DN2 stage of T-cell differentiation (41, 69) and therefore allowed us to perturb an in vivo developmental process involving stages of proliferation, differentiation, and apoptosis.

Consistent with a reduced function of the MYC^S62A mutant, we found that thymic weight and number of thymocytes in MYC^S62AMYC^fl/fl mice was significantly reduced, although higher than the MYC^fl/fl mice, indicating some activity of this mutant on its own. Further, the proliferative potential of MYC^S62AMYC^fl/fl splenic T cells ex vivo showed a substantial decrease relative to littermate control levels when exposed to the mitogen Con A indicating that MYC^S62A has a poor capacity to drive acute proliferation in response to these mitogenic signals in the absence of endogenous MYC. However, despite these deficiencies, MYC^S62AMYC^fl/fl mice developed T-cell lymphomas histologically similar to MYC^T58AMYC^fl/fl mice, although at a substantially lower penetrance. We found that these T-cell lymphomas resembled human T-ALL tumors by flow cytometry. Most lymphomas expressed CD4 and CD8, indicating that DP thymocytes were the most likely cell of origin. These tumors were clonal and did not show escape from endogenous Myc ablation or strong compensation by Mycn. While 36% of MYC^S62AMYC^fl/fl mice developed these T-cell lymphomas and 14% remained tumor free to study endpoint, approximately 50% of necropsied mice succumbed to splenomegaly with or without an accompanying thymic mass. Thymic masses that accompanied splenomegaly lacked a clonal T-cell population, and were found to be predominantly from the B-cell lineage, with Burkitt lymphoma, follicular B-cell lymphoma, and multiple myeloma (i.e., plasma-cell tumors) all identified. Importantly, these B cells did not show Cre-mediated loss of endogenous MYC, and showed high pS62 MYC staining presumably from endogenous MYC, indicating that they were not directly driven by expression of the MYC^S62A transgene. No MYC^WT and only one MYC^WTMYC^fl/fl mouse developed a thymic lymphoma in our model indicating that normal regulation of the ectopically expressed MYC^WT protein is nontumorigenic at levels driven by the ROSA promoter. This underscores the functional relevance of regulated phosphorylation at S62 and T58.

In contrast to MYC^S62AMYC^fl/fl mice in which loss of endogenous Myc resulted in reduced total thymocytes and a lower level of T-cell lymphomagenesis, we saw a more rapid onset of T-cell lymphoma and decreased survival in MYC^T58AMYC^fl/fl mice lacking endogenous Myc. This finding indicates that endogenous Myc dampens the tumorigenic effects of MYC^T58A, suggesting that the possibility of MYC-MAX heterodimers (53) forming with either endogenous MYC or MYC^T58A in the transcriptional machinery suppresses tumor onset in our model. The differential effect of the presence of endogenous Myc on lymphomagenesis in MYC^S62A versus MYC^T58A mice suggests altered transcriptional regulatory activity between these phosphorylation site mutants. This is consistent with the role of S62 phosphorylation in directing MYC promoter binding and activated gene expression (14, 70). In addition, our previous studies indicated that regulated sequential phosphorylation of S62 and T58 is important for both temporal cycling of MYC on and off target genes and the spatial regulation of MYC to associate with a subset of target genes located near the nuclear pore complexes (14, 70). In these studies, the MYC^T58A and MYC^S62A mutants had different temporal and spatial target gene binding, suggesting that they may differentially regulate gene transcription. Indeed, RNA-seq data revealed distinct pathway enrichment between the T-cell lymphomas from MYC^T58AMYC^fl/fl and MYC^S62AMYC^fl/fl mice, with enrichment of KRAS, angiogenesis, and IFNα response pathways in the MYC^T58AMYC^fl/fl tumors, and G₂–M checkpoint, MYC targets, and mitotic spindle pathways in the MYC^S62AMYC^fl/fl tumors. Thus, MYC^S62A appears to drive T-cell lymphomas in our model through the dysregulation of cell-cycle checkpoint, upregulation of MYC targets, and mitotic spindle genes.

A diverging pattern of gene expression was also seen when comparing thymic masses in MYC^S62AMYC^fl/fl mice presenting with splenomegaly, showing an enrichment of IFNα and γ gene sets consistent with the etiologic differences between these thymic masses that were predominantly malignant B cells. The mechanism for B-cell lymphomagenesis and infiltration of B cells into the thymic cavity in MYC^S62AMYC^fl/fl mice still remains to be elucidated. However, based on our observations in early thymic development in MYC^S62AMYC^fl/fl mice of reduced total thymocytes and ex vivo proliferation, we would hypothesize that the expression of MYC^S62A contributes to an initial defect in T-cell proliferation that allows for the mis-regulation and expansion of the B-cell population which, in some cases resulted in terminally differentiated plasma B cells. This B-cell lymphomagenesis appears to be independent of leaky transgene expression and is probably due to the interdependencies of T- and B-cell development in vivo.

Multiple studies have shown the oncogenic potential of MYC^T58A and the importance of S62 phosphorylation in the regulation, transcriptional activity, and degradation of MYC. In this study, we demonstrate for the first time, the ability of the MYC^S62A mutant to directly drive T-cell lymphomas in a mouse model. Despite a significantly lower penetrance, a portion of MYC^S62AMYC^fl/fl mice developed rapid clonal T-cell lymphomas. Conversely, we also observed defects in T-cell proliferation and expansion ex vivo and in vivo in the MYC^S62AMYC^fl/fl mice and that this defect in proper T-cell development was indirectly associated with progressive B-cell lymphomas originating from the spleen. These mechanisms remain to be elucidated, however, these results reaffirm the importance of MYC in T- and B-cell biology and human disease, and demonstrate the protumorigenic properties of posttranslationally deregulated MYC (8, 9, 20, 29, 32, 68).

S.A. Byers reports grants from NIH; and grants from Collins Medical Trust during the conduct of the study. E. Juarez reports grants from NIEHS during the conduct of the study. L.M. Coussens reports personal fees and nonfinancial support from Cell Signaling Technologies; nonfinancial support and other support from ZellBio, Inc., Syndax Pharmaceuticals Inc.; personal fees, nonfinancial support, and other support from Hibercell, Inc.; other support from Prospect Creek Foundation, Lustgarten Foundation for Pancreatic Cancer Research, Pharmacyclics, Inc. (steering committee for PCYC-1137-CA), AstraZeneca Partner of Choice Network, Cancer Research Institute, The V Foundation for Cancer Research, American Association for Cancer Research (AACR), AACR (Cancer Discovery); personal fees and other support from Susan G. Komen Foundation; personal fees from Carisma Therapeutics Inc., Verseau Therapeutics, Inc., CytomX Therapeutics, Inc., Kineta Inc., Alkermes, Inc., PDX Pharmaceuticals, Inc., Zymeworks, Inc., Genenta Sciences, Pio Therapeutics Pty Ltd., (P30) Koch Institute for Integrated Cancer Research, Massachusetts Institute of Technology, Bloomberg-Kimmel Institute for Cancer Immunotherapy, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins (Baltimore, MD), (P50) Dana-Farber Cancer Center Breast SPORE (Boston, MA), (P30) Dana-Farber/Harvard Cancer Center, (P30) University of California, San Diego Moores Cancer Center (San Diego, CA), (P30) The Jackson Laboratory Cancer Center, (P01) Columbia University Medical Center (New York, NY), (P50) MDACC GI SPORE, Lustgarten Foundation for Pancreatic Cancer Research (Therapeutics Working Group), NIH/NCI-Frederick National Laboratory Advisory Committee, AbbVie Inc., Shasqi, Inc., AACR (Cancer Immunology Research); and other support from Cancer Cell during the conduct of the study. R.C. Sears reports grants from NIH, Leukemia and Lymphoma Society; and grants from Brenden-Colson Center Foundation during the conduct of the study. No disclosures were reported by the other authors.

C.J. Daniel: Conceptualization, data curation, formal analysis, investigation, visualization, methodology, writing–original draft, writing–review and editing. C. Pelz: Formal analysis. X. Wang: Data curation. M.W. Munks: Data curation, writing–review and editing. A. Ko: Data curation, writing–review and editing. D. Murugan: Data curation. S.A. Byers: Conceptualization. E. Juarez: Software. K.L. Taylor: Project administration, writing–review and editing. G. Fan: Formal analysis. L.M. Coussens: Resources, funding acquisition. J.M. Link: Methodology, writing–review and editing. R.C. Sears: Supervision, funding acquisition, investigation, writing–review and editing.

We would like to thank Dr. Peter Hurlin for providing the Myc null mice and members of the Sears lab for helpful suggestions and edits. These studies were supported by funding from the Leukemia and Lymphoma Society and the NIH/NCI (grant nos. R01 CA129040, R01 CA186241, R01 CA196228, U01 CA224012, and U54 CA209988), and the OHSU–Brenden-Colson Center for Pancreatic Care to R.C. Sears, NIH/NCI (grant nos. U01 CA224012, and U2C CA233280), the Knight Cancer Institute, and the OHSU–Brenden-Colson Center for Pancreatic Care to L.M. Coussens, and the Knight NCI Cancer Center Support Grant 5P30CA069533.

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