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An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma

Abstract

Translocation events are frequent in cancer and may create chimeric fusions or 'regulatory rearrangements' that drive oncogene overexpression. Here we identify super-enhancer translocations that drive overexpression of the oncogenic transcription factor MYB as a recurrent theme in adenoid cystic carcinoma (ACC). Whole-genome sequencing data and chromatin maps highlight distinct chromosomal rearrangements that juxtapose super-enhancers to the MYB locus. Chromosome conformation capture confirms that the translocated enhancers interact with the MYB promoter. Remarkably, MYB protein binds to the translocated enhancers, creating a positive feedback loop that sustains its expression. MYB also binds enhancers that drive different regulatory programs in alternate cell lineages in ACC, cooperating with TP63 in myoepithelial cells and a Notch program in luminal epithelial cells. Bromodomain inhibitors slow tumor growth in ACC primagraft models in vivo. Thus, our study identifies super-enhancer translocations that drive MYB expression and provides insight into downstream MYB functions in alternate ACC lineages.

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Figure 1: MYB translocations involve alternate partners and frequently retain the MYB 3′ UTR.
Figure 2: Translocation partners contain super-enhancers that loop to the MYB promoter.
Figure 3: MYB protein binds translocated super-enhancers and other active enhancers.
Figure 4: MYB drives alternate cell fates in ACC.
Figure 5: BET bromodomain inhibition slows tumor growth in grade 2 ACC primagrafts.

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Acknowledgements

We thank M. Rivera, N. Riggi, S. Puram, P. van Galen, J. Lohr and J. Kaufman for helpful discussions and critical comments on the manuscript; J. Voisine, R. Isenhart, R. Issner, H. Whitton, A. Spooner, M. Uziel, C. Epstein and N. Shoresh for technical assistance; T. Chan and V. Makarov for help with whole-genome sequencing data access; and the Salivary Gland Tumor Biorepository for providing the primary tumors (National Institute of Dental and Craniofacial Research (NIDCR) award reference HHSN268200900039C 04). This work was supported by the Adenoid Cystic Carcinoma Research Foundation (B.E.B. and B.K.), the Temares Family Foundation and the Howard Hughes Medical Institute. B.E.B. is an American Cancer Society Research Professor.

Author information

Authors and Affiliations

Authors

Contributions

B.K. and Y.D. designed and performed experiments and analyzed the data. B.K. and B.E.B. designed the experimental strategy and supervised the study and analysis. Y.D. carried out computational analyses. Y.D., B.K. and B.E.B. wrote the manuscript. J.C.A., M.J.C., K.E.W., S.M.G., C.D.C., S.J.R., L.M.S. and M.J.W. contributed to experiments and data analysis. A.H.A., R.J.H.R., M.J.K., W.C.F., L.Q., J.Q., J.E.B., C.A.M., A.K.E.-N. and J.E.B. provided reagents, contributed to analysis and gave conceptual advice. All authors discussed the results and implications and reviewed the manuscript.

Corresponding authors

Correspondence to Birgit Knoechel or Bradley E Bernstein.

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Competing interests

J.E.B. is a scientific founder of Tensha Therapeutics, which has licensed drug-like derivatives of the JQ1 bromodomain inhibitor from the Dana-Farber Cancer Institute. The remaining authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PCR validation of rearrangements in four ACC primagrafts.

PCR results across genomic breakpoints (Table 1 and Supplementary Table 1) of high-confidence rearrangements (HC), MYB rearrangements and low-confidence rearrangements (LC) are shown for four ACC primagrafts. Control (C) refers to the same primer used in a different sample—ACC X2 for ACC X9 and ACC X9 for all others.

Supplementary Figure 2 ACC primagrafts and primary ACCs have very similar enhancer profiles.

Spearman correlation between enhancer (H3K27ac) maps of primary ACCs, ACC primagrafts, an HPV-transformed ACC cell line (ACC112), and other normal and malignant tissues and cell lines, based on H3K27ac signal 2 kb outside of the TSS.

Supplementary Figure 3 Several of the translocated enhancers are super-enhancers.

Super-enhancer ‘hockey stick’ plots, showing normalized H3K27ac (top) or BRD4 (bottom) signals across all enhancers genome-wide in ACC primagrafts. Enhancers on the right of the infliction point are considered ‘super-enhancers’. The enhancers translocated near MYB are shown as red, purple or blue dots (color matches the type of MYB rearrangement: MYB-NFIB fusion with 3′ UTR loss in red, MYB-NFIB translocations retaining the 3′ UTR in purple and MYB-TGFBR3 translocation retaining the 3′ UTR in blue).

Supplementary Figure 4 RAD51B locus contains many putative active enhancers.

H3K27ac tracks are shown for the RAD51B locus in five primary tumors and eight primagrafts. H3K27ac signal is shown in fragments per million (fpm).

Supplementary Figure 5 NFIB, TGFBR3 and RAD51B are highly expressed in normal salivary gland.

(a) NFIB and TGFBR3 mRNA are highly expressed in normal salivary gland, as detected by qPCR, normalized to GAPDH expression. OCI-LY-3 (lymphoma cell line) serves as negative control. Error bars, s.e.m. (b) NFIB, TGFBR3 and RAD51B are highly expressed in normal salivary gland, according to RNA-seq data from the Human Protein Atlas20.

Supplementary Figure 6 Translocated MYB-bound enhancers activate transcription.

Putative enhancer sequences of the NFIB enhancers (En3, En6 or En7) or TGFBR3 enhancers (Et3, Et6) or matching controls with the same sequence but scrambled MYB motifs (replacing CNGTT with GTAAG) were cloned upstream of luciferase and a minimal promoter (sequences are listed in Supplementary Table 6). Constructs were delivered by nucleofection into Jurkat cells (Online Methods). Firefly luciferase activity was measured after 36 h and normalized to Renilla luciferase to control for cell number and transfection efficiency. Error bars, s.e.m. for six technical repeats. Four of the five enhancer elements strongly increased activity in the luciferase assay. Two of the NFIB elements (En6 and En7) showed significantly reduced activity with scrambled MYB motifs. Significance was estimated by one-tailed t test. These data support our model that translocated enhancers drive transcription in a MYB-dependent manner.

Supplementary Figure 7 ACCs express the ΔNp63 but not TAp63 isoform of TP63.

(a) H3K27ac, H3K4me3 and MYB tracks are shown for the TP63 locus. MYB binds several intragenic TP63 enhancers in grade 2 primagrafts (X16, X5M1, X2). Only the promoter of the short isoform ΔNp63 is active. Signals are shown in fpm. (b) TP63 is expressed in grade 2 tumors (X2, X5M1) but not in grade 3 tumors (X9, X11). TAp63 is not detected in ACC primagrafts; hence only ΔNp63 is expressed. OCI-LY-3 (lymphoma cell line) serves as a positive control. Error bars, s.e.m. (n = 3).

Supplementary Figure 8 Colocalization of TP63 and MYB in grade 2 ACC.

Heat maps of MYB and TP63 binding in 1-kb windows around TP63 peaks, sorted by overall signal strength. 81% of TP63 peaks are co-bound by MYB. Colors are scaled to show a maximum of 3 fpm per 10-bp bin.

Supplementary Figure 9 TP63, MYB and intercellular NOTCH1 expression in grade 2 and grade 3 ACCs.

Representative IHC staining for TP63, MYB and the intracellular domain of NOTCH1 (ICN1) in grade 2 ACCs (top three rows) and grade 3 ACCs (bottom four rows). 40× magnification is shown for ACCD3 and ACCD4; 40× (upper left corner) and 100× magnification is shown for ACCS1, ACCS4, ACCS5, ACCD1 and ACCD2. Scale bar, 100 µm.

Supplementary Figure 10 Heat map of H3K27ac showing similar patterns across primary tumors and primagrafts and distinct patterns between grade 2 and grade 3 tumors.

Heat map of H3K27ac at the enhancers of five primary tumors and eight primagrafts, showing four clusters of enhancers (unsupervised k-means) in a 5-kb window centered around H3K27ac peaks. Notice that the last cluster is preferentially active in grade 2 primagrafts and primary tumors. This cluster is highly enriched for the TP63 motif, consistent with the loss of TP63-expressing myoepithelial cells in grade 3 tumors. Colors are scaled to show a maximum of 2 fpm per 50-bp bin.

Supplementary Figure 11 Most MYB-binding sites are co-bound by BRD4 and show H3K27 acetylation.

Heat maps of H3K27ac, MYB and BRD4 binding in 5-kb windows around MYB peaks. The large majority of MYB peaks have both BRD4 co-binding and acetylation of H3K27. Colors are scaled to show a maximum of 10 fpm per 50-bp bin.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Table 5 (PDF 2148 kb)

Supplementary Table 1

Rearrangements detected in six ACC primagrafts from whole-genome sequencing. (XLSX 27 kb)

Supplementary Table 2

MYB high-confidence peaks and associated nearby genes. (XLSX 531 kb)

Supplementary Table 3

Enriched annotations of MYB targets. (XLSX 787 kb)

Supplementary Table 4

Transcriptional regulators targeted by MYB ranked by MYB binding, with expression levels in ACC and normal gland. (XLSX 67 kb)

Supplementary Table 6

Primer and reporter sequences used in this study. (XLSX 31 kb)

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Drier, Y., Cotton, M., Williamson, K. et al. An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma. Nat Genet 48, 265–272 (2016). https://doi.org/10.1038/ng.3502

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