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Additional file 7: Fig. S2. Heatmap of Differentially Expressed miRNAs between GEP-NETs (G1 and G2) and GEP-NEC plus metastases (G3). The log transformed median for each group is shown.

Additional file 6: Uncropped blots from images in the manuscript.

Additional file 3: Fig. S1. Mutational profile of different NEN classes. (A) Oncoplot representation of the TOP 20 mutated genes in MTC samples. The gray bar in the bottom figure highlights the presence of familiar syndrome MEN2. (B) Oncoplot representation of the TOP 20 mutated genes in GEP-NETs (G1 and G2 NENs). Gray bars at the bottom indicate, the presence of familiar syndrome MEN1 (upper) and metastatic tissues (lower). (C) Oncoplot representation of the TOP 20 mutated genes in GEP-NEC. The bottom bar indicates metastases. In each graph each column represent a sample with its numeric code while each raw represent a gene. Box colors refer to mutation type.

Supplementary Material 4

Additional file 1: Fig. S1. Dot1L and menin expression in breast cancer. Dot1L (A, left panel) and menin (A, right panel) mRNA levels in normal and luminal breast cancer samples from The Cancer Genome Atlas (TCGA), analyzed with UALCAN. B) Dot1L and menin mRNA co-expression from two additional BC patient datasets from TCGA. Only expression values above the first quartile were considered. Fig. S2. Dot1L, menin and ERα binding to MCF-7 cell chromatin. Heatmap (left panel) showing read density within 10 kb regions centred on Dot1L, menin and ERα binding sites in MCF-7 cells. Control (CTRL) was obtained using nonspecific Abs. Overrepresented transcription factor binding motifs within Dot1L+menin+ERα binding sites (green) or Dot1L+menin binding sites (blue) are reported in the word cloud (right panel). Fig. S3. Impact of Dot1L and menin blockade on gene expression and cell proliferation in BC cells. Bar charts from KEGG functional enrichment analysis showingstatistically significant pathways deregulated upon MCF-7 cell treatment with EPZ (A) or MI-2 (B). Combenefit software was used to generate dose-response surface curves in BT474 (C), T47D (D) and Zr75.1 (E) BC cells according to D–R Lowe model, combining increasing dose of EPZ and MI-136 for 9 days. Color scale bar indicate the level of synergy (blue) or antagonism (red) at each combination. Effect of siRNA-mediated Dot1L and menin silencing on AE-sensitive (MCF7; F) and tamoxifen (MCF7 TAM-R; G)- or fulvestrant/ICI (MCF7 ICIR; H)-resistant BC cells assessed by RT-qPCR (top) and western blot (middle) assays. RT-qPCR results are shown as mean ± SD of triplicate determinations from a representative experiment after 72h of silencing. Western blot show Dot1L or menin protein levels following their silencing. β-actin (ACTB) was used as control. Effect of siRNA-mediated Dot1L and menin silencing on AE-sensitive (MCF7; I) and tamoxifen (MCF7 TAM-R; J)- or fulvestrant/ICI (MCF7 ICIR; J)-resistant BC cells assessed on cell proliferation. MTT data are presented as the mean ±SD from six independent replicates after 72 h of silencing. Asterisks indicate statistically significant differences (*p ≤0.05, **p ≤0.01, ***p ≤0.005) to CTRL or to single treatment (black bars). Fig. S4. Technical and functional evaluation of Dot1L and menin interactome analysis by mass spectrometry. A Principal component analysis (PCA) of the mass spectrometry (MS) results obtained from three biological replicates of control (CTRL; IgG: blue), Dot1L (red) and menin (MEN1; green) samples. Numbers between parentheses indicate percentage of total variance. B Box plot showing Dot1L (red) and menin (green) protein levels detected in CTRLs and samples by MS. Asterisks indicate statistically significant differences (*p ≤0.01). C Results of IPA (Ingenuity Pathway Analysis) functional analysis showing statistically significant molecular functions enriched in Dot1L (green) and menin (red) interactomes. Fig. S5. Impact of BAZ1B silencing on gene expression in MCF-7 BC cells. A RT-qPCR analysis of BAZ1B mRNA level after silencing with two different siRNAs compared to scramble (Silencer Select Negative Control: CTRL). RT-qPCR results shown are the mean ± SD of multiple determinations from a representative experiment. Asterisks indicate statistically significant differences (***p ≤0.005). B Heatmap showing top down-regulated (green) and up-regulated (red) transcripts, fold change ≤ and ≥ |1.5|, following BAZ1B silencing with both siRNAs, compared to scramble (Silencer Select Negative Control: CTRL). Fig. S6. Effect of BAZ1B silencing in luminal-like BC cell models. RT-qPCR (upper panel) and western blot analysis of BAZ1B mRNA and protein levels following BAZ1B silencing in BT474 (A), T47D (B) and ZR75.1 (C) BC cells. RT-qPCR results are shown as mean ± SD of triplicate determinations from a representative experiment after 72 h of silencing. Western blot shows BAZ1B protein level following BAZ1B silencing. β-actin (ACTB) was used as control Cell proliferation rate performed following BAZ1B silencing or treatment with fulvestrant (ICI; 100 nM) in BT474 (D), T47D (E) and ZR75.1 (F) BC cells. All data are analyzed respect to the scramble (Silencer Select Negative Control: CTRL). Data are presented as the mean ± SD of determinations from a representative experiment performed in six independent replicates after 72 h of silencing. Asterisks indicate statistically significant differences (*p ≤0.05, **p ≤0.01, ***p ≤0.005). Fig. S7. Effects of BAZ1B silencing on antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Statistically significant functions highlighted by Gene Set Enrichment Analysis (GSEA) in siRNA-mediated BAZ1B kd cell transcriptome of wt (MCF7: A), tamoxifen (MCF7 TAM-R; B)- or fulvestrant/ICI (MCF7 ICI-R; C)-resistant BC cells. NES: Negative Normalized Enrichment Score, FDR: False Discovery Rate. Fig. S8. Analysis of the influence of BAZ1B, Dot1L and menin co-expression in luminal-like BC clinical outcome. Kaplan-Meier curves, generated using the Kaplan-Meier Plotter, showing the probability of overall (A; 205 low and 549 high samples, respectively) and relapse-free (B: 641 low and 261 high samples, respectively) survival of ERα+ BC patients according to BAZ1B, Dot1L and menin concomitant mRNA expression levels.

Supplementary Material 1: Figure S1. Impact of menin silencing on proliferation of OC cells. (A) RT-qPCR, (left panel) and western blot (right panel) of MEN1 mRNA and menin expression levels, respectively, in Caov-3, OVCAR-3, PEO1, PEO14 and PEO4 cells. Error bars represent the mean of replicate values ± SD (* p ≤ 0.05). Changes of MEN1 mRNA relative abundance (B), menin protein expression (C) and cell proliferation (D) determined by RT-qPCR, western blot and MTT assay, respectively, in Caov-3 (left), OVCAR-3 (middle) and PEO14 (right) cells 96 hours post transfection with three siRNAs targeting different regions of the MEN1 mRNA. Scramble siRNA (CTRL) was used as the negative control. Error bars represent the mean of replicate values ± SD (* p ≤ 0.05). Figure S2. Validation of menin silencing effects on OC cells. (A) Correlation coefficient between differentially expressed genes following menin silencing obtained from independent experiments performed with two different sequencing approaches. Graph showing differentially expressed genes belonging to Aryl Hydrocarbon receptor (B), Integrin (C) Cyclins and Cell cycle regulation (D) signaling pathways obtained from independent experiments performed with MGI and Illumina sequencing approaches. The dashed orange line marks the Fold change threshold (|FC| ≥ Q1, padj ≤ 0.05). Figure S3. The effects of menin pharmacological inhibition on OC cells proliferation. Caov-3 (left), OVCAR-3 (middle) and PEO14 (right) relative cell viability assessed in cells treated with increasing concentrations of MI-136. Data represent the mean of six independent replicates ± SD (* p ≤ 0.05). Figure S4. The effects of menin pharmacological inhibition on OC cells proliferation. Caov-3, OVCAR-3, PEO1, PEO14, PEO4 relative cell viability assessed in cells treated with increasing concentrations of MI-136. Data represent the mean of six independent replicates ± SD (* p ≤ 0.05). Figure S5. Menin associates with DOT1L in nucleus of OC cells. Immunoprecipitation-western blot showing the presence of DOT1L among proteins, co-precipitated altogether with menin from PEO4 nuclear extracts. IgG was used as negative control. Figure S6. D-R Lowe graph showing the effects of combinatorial treatment with increasing MI-136 and EPZ5676 concentrations on PEO1 (left) and PEO4 (right) cells proliferation after twelve days of treatment

Additional file 9. Tutorial and use examples of HOME-BIO.