MAX inactivation deregulates the MYC network and induces neuroendocrine neoplasia in multiple tissues

Abstract

The MYC transcription factor requires MAX for DNA binding and widespread activation of gene expression in both normal and neoplastic cells. Inactivating mutations in MAX are associated with a subset of neuroendocrine cancers including pheochromocytoma, pituitary adenoma, and small cell lung cancer. Neither the extent nor the mechanisms of MAX tumor suppression are well understood. Deleting Max across multiple mouse neuroendocrine tissues, we find that Max inactivation alone produces pituitary adenomas, while Max inactivation cooperates with Rb1/Trp53 loss to accelerate medullary thyroid C cell and pituitary adenoma development. In the thyroid tumor cell lines, MAX loss triggers a marked shift in genomic occupancy by other members of the MYC network (MNT, MLX, MondoA) supporting metabolism, survival, and proliferation of neoplastic neuroendocrine cells. Our work reveals MAX as a broad suppressor of neuroendocrine tumorigenesis through its ability to maintain a balance of genomic occupancies among the diverse transcription factors in the MYC network.

Fig. 1.. Max deletion results in pituitary adenomas that arise with long latency.

(A) Illustration of mouse models used to study roles for Max in neuroendocrine tumorigenesis. (B) Kaplan-Meier curve showing survival with Max inactivation in the Ascl1-Cre-ERT2 model following tamoxifen delivery, with P value from log-rank test shown. (C) Histology showing pituitary lesions in control and Max-deleted mice. (D) Western blot analyses of pituitary tumors arising with Max deletion. As a positive control, pituitary tumors from RP mice with Rb1/Trp53 deletion driven by Ascl1-Cre-ERT2 are also shown. Asterisk indicates position of the N-terminal fragment of the truncated MAX protein in RPMax cells.

Fig. 2.. Max deletion cooperates with Rb1/Trp53 loss to drive medullary thyroid carcinoma and pituitary adenomas.

(A) Illustration of mouse models used to examine synergy between Rb1/Trp53 and Max loss in neuroendocrine tumorigenesis. (B) H&E staining of pituitary and thyroid from RP versus RPMax models at 10 weeks post-tamoxifen, with RP model exhibiting medullary hyperplasia and small pituitary adenomas, and the RPMax model exhibiting bilateral medullary adenomas and large pituitary tumors. Quantification of increase in pituitary tumor size in the RPMax model shown to the right. For pituitary tumor size quantification from H&E-stained sections, the tumor region of interest (ROI) was defined manually in ImageJ using the ROI Manager. Tumor size in pixels was then quantified using the “Measure” function in ImageJ. (C) Kaplan-Meier curve showing accelerated time to morbidity in the RPMax cohort, with P value from log-rank test shown. (D and E) Mice in both RP and RPMax models exhibited medullary thyroid C cell and pituitary adenomas, with representative H&E images of thyroid (D) and pituitary tumors (E) shown. (F and G) Western blot analyses in thyroid tumors (F) and pituitary tumors (G) showing increased abundance of one-carbon pathway proteins SHMT1 and ATIC upon MAX loss. Asterisks indicate position of the N-terminal fragment of the truncated MAX protein in RPMax cells. A separate gel was used for the SHMT1 blot, with second actin blot shown below. (H) Heatmap showing RNA-seq data from pituitary tumors of RP and RPMax models as well as the Max-null only tumors (Rb1/Trp53 WT) that arise with long latency.

Fig. 3.. Increased proliferation in RPMax versus RP cell thyroid carcinoma cell lines.

(A) Western blot analysis showing loss of full-length MAX in thyroid cancer cell lines derived from the RPMax compared to those derived from the RP model. Asterisk indicates position of the N-terminal fragment of the truncated MAX protein in RPMax cells. (B) CellTiter-Glo assays showing cell growth in four RPM and four RP thyroid cancer lines (n = 3 independent experiments). *P < 0.05, unpaired t test. (C) Western blot showing doxycycline (DOX)–inducible return of MAX to the RPMax thyroid cancer cell line RPM-4. WT cell line RPM-3 is MAX+ lane. (D) CellTiter-Glo assays showing suppressed cell growth upon return of MAX to the RPM-4 cell line (n = 3 independent experiments). ANOVA with post hoc testing shows that the pCW MAX + doxycycline group had significantly lower luminescence than the other three groups (**P_adj < 0.02 across the three comparisons), and the other three groups were not significantly different from each other. (E) Venn diagram showing genes with increased expression upon MAX loss (EdgeR analyses, FDR < 0.05) comparing RPMax to RP thyroid tumors, tumor-derived cell lines, and genes with decreased expression upon doxycycline-induced return of MAX to RPM-4 cells. (F and G) Pathway enrichment analyses querying GO Biological Processes (F) and ENCODE/CHEA datasets (G) including the 50 core MAX-regulated genes from (E).

Fig. 4.. MAX genomic occupancy and correlation with gene expression changes in MAX-null thyroid tumors.

(A) Chromatin immunoprecipitation (ChIP) was performed using anti-MAX or control IgG antibody on cell lines established from thyroid tumors with WT Max gene (RP-4 cells, denoted RP, Max WT) or a mutant Max gene (RP-1 cells, denoted RPMax, Max KO). One of three biological replicates are shown for each. Heatmaps were generated centered on the TSS ± 2 kb of flanking regions. (B) CUT&RUN was performed with anti-Myc or IgG control antibody on the same cell lines as in (A). Heatmaps were generated centered on the TSS ± 2 kb of flanking regions for each gene. (C) Volcano plot of RNA-seq data (comparing RPMax versus RP cell lines) showing gene expression change (log₂ fold change, x axis) versus significance (−log₁₀ adjusted P value, y axis) comparing RPMax to RP cell lines. Peak calls on MAX ChIP identified 2408 peaks on 857 gene promoters. Genes were considered occupied if a peak was called within 1 kb of its promoter. Genes occupied by Max in RP cell lines are shown in blue. Genes regulated in all datasets (50 genes, from Fig. 3E) and bound by MAX are shown as red dots. (D) Cumulative distribution plots depicting the ranked order (y axis, Percent of genes) of log₂ fold change (x axis) in RNA-seq (comparing RPMax versus RP cell lines). MAX-bound genes (dark blue) and a random, size-matched set of MAX unbound genes (light blue) are shown. The difference between the distributions was determined using Kolmogorov-Smirnov (KS) statistics. (E) Line plots from ChIP experiments performed on Max WT RP cells (RP-4 cell line) were generated for all significantly changed (blue line), up-regulated (red), and down-regulated (green) genes in the RPMax cells. Each line is centered on the TSS (± 2 kb) of every gene.

Fig. 5.. Alterations in MNT genomic occupancy correlates with gene expression changes in RP and RPMax tumor cells.

(A) Plots of genomic region over gene bodies (x axis) versus normalized coverage (y axis) of MAX/MNT-bound cluster 1 (see fig. S4C), which was a cluster of peaks determined to be differentially occupied by MNT. Coverage of MAX for RP (RP-4 cell line, denoted MAX WT, blue) is compared to that from RPMax (RPM-1 cell line, denoted MAX KO, red) cells. (B) Plots of MNT coverage of MAX/MNT-bound cluster 1 comparing RP (blue) and RPMax (red) cells, similar to (A) using the same cell lines. (C) Genomic tracks showing differential occupation of the Rorc promoter in RP (Max WT) and RPMax (Max KO) cells as determined by ChIP-seq. The antibodies (against MAX, MNT, and MLX) used for ChIP are shown (at left) after the hyphen. (D) Volcano plot of RNA-seq gene expression changes comparing RP versus RPMax thyroid tumors (log₂ fold change on the x axis, −log₁₀ adjusted P value on the y axis). Genes with peaks that map to MAX-bound sites (blue) and in MAX/MNT cluster 1 (red) are shown. (E) Cumulative distribution plots depicting the ranked order (y axis, Percent of genes) versus log₂ fold change (x axis) of RNA-seq data (comparing RPMax versus RP thyroid tumors). MNT cluster 1 genes (dark blue) and a similarly sized set of genes determined to not be MNT occupied (light blue) are shown. The difference between the distributions is statistically determined using KS statistics. (F) Enrichment analysis of genes found to be differentially occupied by MAX and MNT in Max WT RP tumor–derived thyroid cell lines (Mnt cluster 1 from fig. S4C). Enrichment categories are plotted as the −log₁₀ of the adjusted P value (y axis, −log₁₀ P_adj) and odds ratio (x axis).

Fig. 6.. Altered MondoA genomic occupancy in RPMax tumor lines and MAX-addback cell lines.

(A and B) CUT&RUN heatmap analysis of MAX or MondoA in cells derived from Max-mutant thyroid tumor cells (RPM-4) expressing doxycycline-inducible MAX (MAX addback) or control vector (Vector). CUT&RUN was performed on 1 million cells using the Auto CUT&RUN robotic method with anti-Max, anti-MondoA, or control IgG antibody. Duplicate samples were merged, and heatmaps were plotted for Max (left) or MondoA (right) centered on the TSS ± 2 kb of flanking regions for every gene (A) or MondoA centered on MAX peaks as determined by peak calls (B). (C) Genomic tracks showing differential MondoA occupancy of the Npm1 promoter in Max-null cells reconstituted with doxycycline-induced MAX (MAX addback) or vector control as determined by ChIP-seq. The antibodies used for ChIP are shown (left) after the hyphen for each cell type. (D) Volcano plot of RNA-seq gene expression changes comparing RP versus RPMax thyroid tumors (log₂ fold change on the x axis, −log₁₀ adjusted P value on the y axis). Genes with peaks that map to Mlx cluster 1 (blue) and both Mlx cluster 1 and Max (red) are shown. (E) Volcano plot of RNA-seq gene expression changes comparing RP versus RPMax thyroid tumors (log₂ fold change on the x axis, −log₁₀ adjusted P value on the y axis). Genes with peaks that are MondoA bound (blue) or bound by both MondoA and Max (red) are shown.

Fig. 7.. Differential sensitivity and gene expression changes in Max-inactivated thyroid tumor upon inhibition of MNT, MLX, and MondoA.

(A) Thyroid tumor cell lines (three independent RP and three RPMax lines) were transfected with the indicated siRNA and allowed to reconstitute over 4 days. Outgrown spheres were enumerated and normalized to the siCtrl condition. siDeath is a control for transfection efficacy. For (A) to (C), P values were calculated using ANOVA with a Tukey’s adjustment. (B) Thyroid tumor cell lines (three independent RP and three RPMax lines) were seeded at equal density with either vehicle (DMSO) or SBI-477 (Mondo inhibitor, 20 μM). After 4 days, cells were counted and normalized to DMSO. (C) RPMax thyroid tumor cell line RPM-4, reconstituted with either control vector (pCW Vector) or doxycycline-inducible Max construct (pCW MAX), was cultured in the absence or presence of doxycycline and/or SBI-477 for 96 hours. Cells were counted and normalized to untreated cells for each line. P values were calculated using ANOVA with a Tukey’s adjustment. (D) Enrichment analysis of genes that were down-regulated in tumor cells expressing WT Max, but up-regulated in tumor cells with Max deleted upon MondoA silencing (using MondoA inhibitor, SBI-477). Enrichment categories are plotted as the −log₁₀ of the adjusted P value (y axis, −log₁₀ P_adj) and odds ratio (x axis). (E and F) Volcano plots of RNA-seq gene expression changes (log₂ fold change on the x axis, −log₁₀ adjusted P value on the y axis) comparing vehicle control to MondoA inhibitor (SBI-477) for RP cells (D) or RPMax cells (E). Genes involved in the unfolded protein response are highlighted in blue. (G) Plot of expression changes (log₂ fold change) of RP (y axis) versus RPMax (x axis) cells. Genes in the unfolded protein response are highlighted by yellow-red color. Yellow-red shading depicts the adjusted P value (P_adj), and higher red intensity indicates greater significance in gene expression change.

Fig. 8.. Schematic showing putative stages in the development of Max-null neuroendocrine tumors.

Involvement of the MYC network in two general pathways leading to multiple neuroendocrine neoplasia (MEN). WT progenitors increase growth upon MYC amplification, are dependent on MAX, and are further boosted by cooperating mutations in other oncogenes and tumor suppressor genes. By contrast, Max-null progenitors lose MYC-MAX binding and rapidly degrade MYC protein. Max deletion is also predicted to result in loss of DNA binding activity of the growth inhibitory MXD proteins such as MNT, which normally repress a subset of MYC targets. Thus, a subpopulation of Max-mutant cells may transiently maintain accessibility and expression at former MYC-MAX targets, bypassing the requirement for MYC-MAX. Rb1/Trp53 loss would be expected to facilitate survival of these cells. The observed reshuffled genomic occupancies of MNT, MLX, and MondoA may further augment growth and, in addition, lead to an abnormal or discordant response to mitogenic signals, resulting in cell-autonomous growth and eventually neoplasia.