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Table S1 has the RNA-Seq sample names and raw counts.

Metastatic disease in pheochromocytomas and paragangliomas (PCC/PGL) is not well-understood. The Cancer Genome Atlas discovered recurrent MAML3 fusion genes in a subset of tumors that lacked known germline or somatic driver mutations and were associated with aggressive disease. Here, we aimed to investigate the role of MAML3 in tumorigenesis. Human PCC/PGLs were used for IHC and genetic analysis. Three neuroendocrine tumor cell lines, SK-N-SH, QGP-1, and BON-1, were transiently transfected with MAML3 (FL) or exon 1 deleted MAML3 (dEx1; mimicking the fusion), and biologic effects of overexpression were examined in vitro. We found 7% (4/55) of human PCC/PGL have UBTF∼MAML3 fusions and all were sporadic cases with metastatic disease. Fusion-positive tumors had intense MAML3 nuclear staining and increased β-catenin by IHC and showed increased WNT4 expression. In vitro, overexpression of FL and dEx1 MAML3 increased invasion in SK-N-SH, QGP-1, and BON-1 (all P < 0.05) and increased soft-agar colony formation in QGP-1 and BON-1 (all P < 0.05). Cotransfection with FL or dEx1 MAML3 and β-catenin increased TCF/LEF promoter activation by luciferase activity and coimmunoprecipitation confirmed interaction between MAML3 and β-catenin. These data suggest MAML3 is involved in WNT signaling pathway activation. In summary, UBTF∼MAML3 fusions are present in a subset of PCC/PGL and associated with metastatic disease without other known drivers. MAML3 overexpression led to increased tumorigenicity in neuroendocrine tumor cells and the mechanism of action may involve WNT signaling pathways.Implications:MAML3 increases tumorigenicity and invasion in neuroendocrine tumor cells and may be a prognostic marker for aggressive disease.

MAML3 overexpression has no effect on proliferation or apoptotic signaling. SK-N-SH, QGP-1 and BON-1 transfected with FL or dEx1 MAML3 and showed A) no increase in proliferation by BrdU assay when grown in full serum media for 72 hours and B) no increase in total or cleaved PARP or Caspase 3 when grown in serum free media for 48 or 72 hours. No cleaved caspase 3 was seen at all. The % of cleaved PARP was calculated after normalizing to beta tubulin and no significant differences were found at either time point for any cell line. Experiment performed N=3 with representative blots shown. T-PARP= total PARP; C-PARP=cleaved PARP; T-Caspase 3=total Caspase 3; C-Caspase 3=cleaved Caspase 3

MAML3 increases cell viability. SK-N-SH, QGP-1 and BON-1 transfected with FL or dEx1 MAML3 showed increased cell viability by Cell Titer Glo assay (* p

MAML3 in vitro cellular localization for QGP-1 and BON-1. Immunofluorescence of cells transfected with either FL or dEx1 MAML3 show nuclear localization of both constructs. FL MAML3 shows more punctate staining and dEx1 MAML3 shows more diffuse staining. No endogenous MAML3 could be found in transfected controls. (100x/1.30 oil Nikon Plan Fluor Objective, AlexaFluor546, Scale bar represents 10 µm)

β-catenin interacts with dEx1 MAML3. Neuroendocrine tumor cell lines were co-transfected with β-catenin and dEx1 MAML3. Immunoprecipitation of MAML3 demonstrated co-precipitation of β-catenin in QGP-1 and BON-1, but not in SK-N-SH (not shown). Blot represents one of three separate experiments.

β-catenin and FL or dEx1 MAML3 colocalize in the nucleus. Cell lines (A. SK-N-SH, B. QGP-1, C. BON-1) were co-transfected with either FL or dEx1 MAML3 and β-catenin or control plasmid. Cells overexpressing FL or dEx1 MAML3 alone showed some faint nuclear accumulation of β-catenin, as denoted by the arrows. When FL or dEx1 MAML3 was co-transfected β-catenin to increase overall levels of β-catenin in the cell, the nuclear accumulation and colocalization with MAML3 was even more apparent. (scale bar represents 10 μm)

C6 Oncogenic pathway analysis. GSEA of MAML3 dEx1 and FL transfected cells compared to control showed dysregulation of WNT and SHH signaling pathways. Filled circles represent an adjusted p value of less than 0.05, whereas empty circles represent an adjusted p value of less than 0.10. (NES- Normalized Enrichment Score)

Western blot for expression of FL and dEx1 MAML3 domain constructs and luciferase assay. Cells for these blots were transfected at the same time as cells used for the luciferase experiment and harvested on the day the luciferase assay was performed. A. Protein expression of FL MAML3 domain constructs: FLâ^†C772 contains amino acids (aa) 1-772, FLâ^†C364 aa 1-364, FLâ^†Q aa 1-364 and 772-1133, FLâ^†S aa 1-195 and 365-1133, FLâ^†Nâ^†S aa 1-155 and 365-1133. B. Protein expression of dEx1 MAML3 domain constructs: dEx1â^†C772 contains amino acids (aa) 156-772, dEx1â^†C364 aa 156-364, dEx1â^†Q aa156-364 and 772-1133, dEx1â^†S aa 156-195 and 365-1133, dEx1â^†Nâ^†S aa 365-1133. C. TCF/LEF promoter activation by MAML3 constructs in QGP-1 and BON-1. Diagrams of each MAML3 construct are shown to the left of their relative promoter activation. All MAML3 constructs were co-transfected with β-catenin. Cells transfected with β-catenin alone, or with neither MAML3 nor β-catenin are shown as controls, in addition to the negative control FOP vector for every combination. Experiments was done three separate times in triplicate.

Metastatic disease in pheochromocytomas and paragangliomas (PCC/PGL) is not well-understood. The Cancer Genome Atlas discovered recurrent MAML3 fusion genes in a subset of tumors that lacked known germline or somatic driver mutations and were associated with aggressive disease. Here, we aimed to investigate the role of MAML3 in tumorigenesis. Human PCC/PGLs were used for IHC and genetic analysis. Three neuroendocrine tumor cell lines, SK-N-SH, QGP-1, and BON-1, were transiently transfected with MAML3 (FL) or exon 1 deleted MAML3 (dEx1; mimicking the fusion), and biologic effects of overexpression were examined in vitro. We found 7% (4/55) of human PCC/PGL have UBTF∼MAML3 fusions and all were sporadic cases with metastatic disease. Fusion-positive tumors had intense MAML3 nuclear staining and increased β-catenin by IHC and showed increased WNT4 expression. In vitro, overexpression of FL and dEx1 MAML3 increased invasion in SK-N-SH, QGP-1, and BON-1 (all P < 0.05) and increased soft-agar colony formation in QGP-1 and BON-1 (all P < 0.05). Cotransfection with FL or dEx1 MAML3 and β-catenin increased TCF/LEF promoter activation by luciferase activity and coimmunoprecipitation confirmed interaction between MAML3 and β-catenin. These data suggest MAML3 is involved in WNT signaling pathway activation. In summary, UBTF∼MAML3 fusions are present in a subset of PCC/PGL and associated with metastatic disease without other known drivers. MAML3 overexpression led to increased tumorigenicity in neuroendocrine tumor cells and the mechanism of action may involve WNT signaling pathways. Implications: MAML3 increases tumorigenicity and invasion in neuroendocrine tumor cells and may be a prognostic marker for aggressive disease.