Figure S1. TT and MZ-CRC-1 cell viability and proliferation after Palbociclib treatment. Cell viability assays (A) and cell counting assays (B) were

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1 Figure S1. TT and MZ-CRC-1 cell viability and proliferation after Palbociclib treatment. Cell viability assays (A) and cell counting assays (B) were performed with TT and MZ-CRC-1 cells treated with Palbociclib or vehicle control. WST-8 solution was added at 72h-120h and the optical density was read using a spectrophotometer for viability assays. These data were normalized to the 0µM (vehicle control) wells and represented as percentages. In cell counting assays, the cells were counted for 5 days after treatment, including vehicle control wells at day 0, using the Countess Automated Cell Countess. Each biological replicate was done in triplicate (cell viability assays) or duplicate (cell counting assays), and the error bars represent the standard deviation. Two-way ANOVA tests followed by Holm s procedure were done on both types of assays. In the case of cell counting assays, log2 transformed data was used for statistical analysis. # P < 0.01, + P < 0.05.

2 Figure S2. TT Vandetanib-resistant and age-matched controls viability after Dinaciclib treatment. Cell viability assays for TT Vandetanib-resistant cells and age-matched controls. The cells were treated with Dinaciclib and incubated for h. WST-8 solution was added at each time point and the optical density was read using a spectrophotometer. The data are normalized to the 0nM (vehicle control) wells and represented as percentages. Each biological replicate was done in triplicate, and the error bars represent the standard deviation. Linear mixed models were used to statistically analyze the data after log2 transformation.

3 Figure S3. MZ-CRC-1 Vandetanib-resistant and age-matched controls viability after Dinaciclib treatment. Cell viability assays for MZ-CRC-1 Vandetanib-resistant cells and age-matched controls. The cells were treated with Dinaciclib and incubated for h. WST-8 solution was added at each time point and the optical density was read using a spectrophotometer. The data was normalized to the 0nM (vehicle control) wells and represented as percentages. Each biological replicate was done in triplicate, and the error bars represent the standard deviation. Linear mixed models were used to statistically analyze the data after log2 transformation.

4 Figure S4. Principal component analysis and mrna expression profiles. TT and MZ-CRC-1 cells were treated with vehicle control or Dinaciclib (10nM TT and 20nM MZ-CRC-1) for 12h and 24h; and RNA sequencing was performed. A. Principal component plots showing treatment as the variable that is associated with most of the change. B. mrna expression of treated cells was downregulated compared to that of untreated cells for all time points and replicates. Data represents two biological replicates.

5 Table S1. GSEA results for TT cells after treatment with Dinaciclib. Upregulated in class GeneSet Enrichment Score (ES) Upregulated in class Normalized Enrichment Score (NES) Nominal p- value FDR q- value FWER p- Value 0nM 0nM 0nM 24h_10nM Reactome Generic_Tran scription_path way Fischer_G2_M _Cell_Cycle Dazard_Respon se_to_uv_nhe k_dn Reactome_RNA_ Pol_I_RNA_Pol_ III_and_Mitochon drial_transcriptio n nM 0nM 0nM 24h_10nM < < < < < < Table S2. GSEA results for MZ-CRC-1 cells after treatment with Dinaciclib. Upregulated in class GeneSet Enrichment Score (ES) Normalized Enrichment Score (NES) Nominal p- value FDR q- value FWER p- Value 0nM 0nM 0nM 20nM Reactome_Mit otic_g1_g1_s _Phases Fischer_G2_M _Cell_Cycle Reactome_Dn a_replication Reactome_RNA_P ol_i_rna_pol_iii _and_mitochondri al_transcription < < < < < < 0.001

6 Table S3. Top GSEA pathways downregulated in TT cells treated with 10nM Dinaciclib. NAME SIZE ES NES NOM p- value FDR q- value FWER p-value Burton_Adipogenesis_ < Plasari_Nfic_Targets_Basal_Up Pid_Hedgehog_2pathway Pid_Il3_Pathway < Dazard_Response_To_Uv_Scc_Dn < Kegg_Basal_Cell_Carcinoma < Reactome_G1_S_Specific_Transcription Ly_Aging_Middle_Dn Biocarta_Cellcycle_Pathway Marshall_Viral_Infection_Response_Dn Leonard_Hypoxia < Reactome_Generic_Transcription_Pathway < Dazard_Response_To_Uv_Nhek_Dn < Reactome_Nuclear_Receptor_Transcription_Pathway < Pramoonjago_Sox4_Targets_Up < Nakayama_Fra2_Targets Gentile_Uv_Response_Cluster_D < Figueroa_Aml_Methylation_Cluster_6_Dn < Fischer_G2_M_Cell_Cycle < Gyorffy_Doxorubicin_Resistance <

7 Table S4. Top GSEA pathways upregulated in TT cells treated with 10nM Dinaciclib. NAME SIZE ES NES NOM p-value FDR q- value FWER p-value Reactome_Amyloids < Kegg_Systemic_Lupus_Erythematosus < Reactome_Rna_Pol_I_Promoter_Opening < Reactome_Packaging_Of_Telomere_Ends < Reactome_Meiosis < Reactome_Meiotic_Recombination < Reactome_Rna_Pol_I_Transcription < Reactome_Meiotic_Synapsis < Reactome_Telomere_Maintenance < Reactome_Srp_Dependent_Cotranslational_Protein_ Targeting_To_Membrane < Reactome_Deposition_Of_New_Cenpa_Containing_ Nucleosomes_At_The_Centromere < Reactome_Peptide_Chain_Elongation < Reactome_Respiratory_Electron_Transport < Reactome_Respiratory_Electron_Transport_Atp_ Synthesis_By_Chemiosmotic_Coupling_And_Heat_ Production_By_Uncoupling_Proteins_ < Reactome_Rna_Pol_I_Rna_Pol_III_And_ Mitochondrial_Transcription < Pid_Syndecan_1_Pathway < Naba_Collagens < Kegg_Ribosome <

8 Table S5. Top GSEA pathways downregulated in MZ-CRC-1 cells treated with 20nM Dinaciclib. NAME SIZE ES NES NOM p-value FDR q- value FWER p-value Gary_Cd5_Targets_Dn < Reactome_Mitotic_G1_G1_S_Phases < Reactome_G1_S_Transition < Reactome_M_G1_Transition < Fischer_G2_M_Cell_Cycle < Reactome_S_Phase < Reactome_Dna_Replication < Moreaux_Multiple_Myeloma_By_Taci_Dn < Martinez_Response_To_Trabectedin_Dn < Reactome_Orc1_Removal_From_Chromatin < Chiang_Liver_Cancer_Subclass_Unannotated_Dn < Reactome_Scfskp2_Mediated_Degradation_Of_P27 _P < Berenjeno_Transformed_By_Rhoa_Up < Reactome_Cyclin_E_Associated_Events_During_ G1_S_Transition_ < Reactome_Mitotic_M_M_G1_Phases < Wong_Embryonic_Stem_Cell_Core < Schlosser_Myc_Targets_Repressed_By_Serum < Schlosser_Myc_Targets_And_Serum_Response_Dn < Reactome_Synthesis_Of_Dna < Dacosta_Uv_Response_Via_Ercc3_Xpcs_Up <

9 Table S6. Top GSEA pathways upregulated in MZ-CRC-1 cells treated with 20nM Dinaciclib. NAME SIZE ES NES NOM p-value FDR q- value FWER p-value Reactome_Rna_Pol_I_Promoter_Opening < Reactome_Amyloids < Kegg_Systemic_Lupus_Erythematosus < Reactome_Meiotic_Recombination < Reactome_Rna_Pol_I_Transcription < Reactome_Packaging_Of_Telomere_Ends < Reactome_Meiosis < Reactome_Meiotic_Synapsis < Reactome_Deposition_Of_New_Cenpa_Containing_ Nucleosomes_At_The_Centromere < Reactome_Rna_Pol_I_Rna_Pol_III_And_Mitochondrial _Transcription < Reactome_Telomere_Maintenance < Acevedo_Liver_Cancer_With_H3k27me3_Dn < Reactome_Chromosome_Maintenance < E Reactome_Transcription < E Reactome_Extracellular_Matrix_Organization < E Acevedo_Liver_Cancer_With_H3k9me3_Dn < E Naba_Collagens < E Haslinger_B_Cll_With_6q21_Deletion < E Kegg_Type_I_Diabetes_Mellitus < E Dalessio_Tsa_Response <

10 Table S7. Biological processes downregulated in TT cells treated with 10nM Dinaciclib. #pathway ID pathway description observed gene count false discovery rate GO transcription, DNA-templated E-100 GO RNA biosynthetic process E-97 GO nucleobase-containing compound biosynthetic process E-96 GO regulation of nucleic acid-templated transcription E-93 GO regulation of RNA metabolic process E-93 GO organic cyclic compound biosynthetic process E-92 GO regulation of transcription, DNA-templated E-92 GO nucleic acid metabolic process E-91 GO regulation of cellular macromolecule biosynthetic process E-91 GO cellular macromolecule biosynthetic process E-90 GO regulation of macromolecule biosynthetic process E-90 GO regulation of gene expression E-89 GO macromolecule biosynthetic process E-89 GO cellular nitrogen compound biosynthetic process E-88 GO regulation of nucleobase-containing compound metabolic process E-88 GO nucleobase-containing compound metabolic process E-87 GO negative regulation of transcription from RNA polymerase II promoter E-23 GO cell cycle E-09 GO mitotic cell cycle E-09 GO mitotic cell cycle phase transition E-08

11 Table S8. Biological processes upregulated in TT cells treated with 10nM Dinaciclib. #pathway ID pathway description observed gene count false discovery rate GO DNA methylation on cytosine E-34 GO chromatin silencing at rdna E-31 GO histone H4-K20 demethylation E-17 GO chromatin organization E-17 GO negative regulation of megakaryocyte differentiation E-16 GO nucleosome assembly E-16 GO DNA replication-dependent nucleosome assembly E-15 GO chromatin assembly E-15 GO protein heterotetramerization E-15 GO chromosome organization E-14 GO DNA packaging E-14 GO CENP-A containing nucleosome assembly E-13 GO CENP-A containing chromatin organization E-13 GO centromere complex assembly E-12 GO regulation of gene silencing E-11 GO small GTPase mediated signal transduction E-11 GO telomere maintenance E-11 GO regulation of immune system process E-09 GO positive regulation of defense response to virus by host E-07 GO negative regulation of nitrogen compound metabolic process E-07

12 Table S9. Biological processes downregulated MZ-CRC-1 cells treated with 20nM Dinaciclib. #pathway ID pathway description observed gene count false discovery rate GO mitotic cell cycle E-108 GO mitotic cell cycle process E-95 GO cell cycle process E-84 GO cell cycle E-84 GO G1/S transition of mitotic cell cycle E-77 GO cell cycle phase transition E-73 GO mitotic cell cycle phase transition E-72 GO regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle E-44 GO mitotic cell cycle checkpoint E-44 anaphase-promoting complex-dependent proteasomal ubiquitin-dependent GO protein catabolic process E-43 GO cell cycle checkpoint E-42 positive regulation of ubiquitin-protein ligase activity involved in GO regulation of mitotic cell cycle transition E-42 GO regulation of mitotic cell cycle phase transition E-42 GO regulation of cell cycle E-42 negative regulation of ubiquitin-protein ligase activity involved in mitotic GO cell cycle E-42 GO DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest E-41 GO negative regulation of mitotic cell cycle phase transition E-40 GO negative regulation of cell cycle process E-40 GO DNA integrity checkpoint E-40

13 Table S10. Biological processes upregulated in MZ-CRC-1 cells treated with 20nM Dinaciclib. #pathway ID pathway description observed gene count false discovery rate GO DNA methylation on cytosine E-43 GO chromatin silencing at rdna E-39 GO histone H4-K20 demethylation E-24 GO negative regulation of megakaryocyte differentiation E-23 GO DNA replication-dependent nucleosome assembly E-22 GO protein heterotetramerization E-21 GO chromatin organization E-19 GO histone exchange E-19 GO CENP-A containing nucleosome assembly E-18 GO CENP-A containing chromatin organization E-18 GO centromere complex assembly E-17 GO nucleosome assembly E-16 GO chromosome organization E-16 GO chromatin assembly E-15 GO DNA metabolic process E-15 GO telomere maintenance E-15 GO DNA packaging E-14 GO small GTPase mediated signal transduction E-13 GO regulation of gene silencing E-12 GO regulation of immune system process E-10

14 Figure S5. Effect of Dinaciclib treatment on CDK9 protein stability. TT and MZ-CRC-1 cells were treated with A. Dinaciclib alone, cycloheximide alone, or cotreated with Dinaciclib and cycloheximide; or treated with B. Dinaciclib alone, bortezomib alone, or co-treated with Dinaciclib and bortezomib for 24h or 48h. After treatment, protein extracts were analyzed by Western blot for the level of CDK9, β-actin, and cyclin D1 or Mcl-1. Vehicle controls were collected for all time points in addition to a non-treated sample collected at the time that treatments were added for all experiments. Dinaciclib: 0.1µM, cycloheximide: 5.0µM, bortezomib: 0.1µM.

15 Figure S6. Effect of JQ1 treatment on TT and MZ-CRC-1 cell viability and CDK9 downstream pathway. Cell viability assays (A) were performed with TT and MZ-CRC-1 cells treated with JQ1 or vehicle control. WST-8 solution was added at 72h-120h and the optical density was read using a spectrophotometer. These data were normalized to the 0nM (vehicle control) wells and represented as percentages. Each biological replicate was done in triplicate, and the error bars represent the standard deviation. Two-way ANOVA tests followed by Holm s procedure were done. B. Western blots performed with protein extracts from TT and MZ-CRC-1 cells treated with vehicle control or JQ1 for 120h showed that total CDK9 protein levels were not reduced with treatment, but there was a small downregulation of phosphorylation of RNA pol II at Ser2 on the TT cells. ** P < , * P < 0.001, # P < 0.01, + P < 0.05.

16 Figure S7. Effect of Dinaciclib treatment on CDK9 protein stability. TT and MZ-CRC-1 cells were treated with THZ alone, cycloheximide alone, or co-treated with THZ1 and cycloheximide for 24h or 48h. After treatment, protein extracts were analyzed by Western blot for the level of CDK9, RET, β-actin, and cyclin D1. Vehicle controls were collected for all time points in addition to a non-treated sample collected at the time that treatments were added for all experiments. THZ1: 0.1µM (TT), 0.25µM (MZ-CRC-1), cycloheximide: 5.0µM.

17 Figure S8. Effect of Dinaciclib-Vandetanib combination therapy on MTC cells in vitro. TT and MZ-CRC-1 cells were treated with Dinaciclib for 6h and incubated in 2% FBS- RPMI medium for 72h (Dinaciclib control), or Vandetanib alone for 72h (Vandetanib control), or Dinaciclib for 6h followed by 72h of Vandetanib incubation. Dinaciclib treatment induced RET and ERK activation, which was obliterated by Vandetanib treatment (A). Cell viability assays showed synergistic effect between these drugs in both cell lines (B). Linear mixed models were used to statistically analyze the cell viability. If the difference of the optical density ((combination-dinaciclib)-(vandetanib control)) <0 and the p-value is significant, then we claimed there is significant synergistic interaction at the tested dose. * P < 0.001, # P < 0.01, + P < ^β-actin for RNAP II, pser2 RNAP II, ERK 1/2, and pthr202/tyr204 ERK 1/2 in this figure is the same because they were blotted in the same membrane.

18 Figure S9. Gene Ontology cellular and metabolic processes associated with superenhancers identified in the TT cell line. ChIP sequencing was performed on TT cells looking at H3K27Ac and BRD4 binding sites. The genes associated with super-enhancers were loaded to PANTHER to identify the biological processes associated. The two highest categories within biological processes were cellular (A) and metabolic (B) processes, which in turn contain sub-categories shown here. Among these processes, cell communication and primary metabolic processes are associated with the highest number of genes. Out of the primary metabolic processes, nucleobase-containing compound metabolic processes (C) stand out, with RNA processes accounting for the most genes in this category.

19 Table S11. Array-CGH analysis of CDK9 in human MTCs Sporadic MTC without RET mutation Sporadic MTC with somatic RET mutation Hereditary MTC with germline RET mutation Patient number RET mutation Lymph node metastases present Distant metastases present CDK9 copy number 1 n/a Yes No 3 2 n/a Yes No 2 3 n/a Yes No 3 4 n/a Yes No 2 5 n/a Yes No 2 6 n/a Yes No 2 7 n/a No No 2 8 n/a No No 2 9 n/a No No 3 10 n/a No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes No No No No No Yes Yes Yes Yes No Yes Yes No Yes No Yes No Yes No No No No information No information No information No information 2

20 Figure S10. CDK9 expression in MTC human samples. A. Paraffin-embedded tissue samples from 14 MTC patients were immunostained for CDK9. 10 out of 14 cases had stronger staining in the tumor area vs. the normal thyroid. Two cases are represented. Scale bar indicates 100µm, magnification: 400X. B. An additional 36 cases were immunostained for CDK9 and quantified using the Vectra Automated Quantitative Pathology Imaging System, generating H scores for each field of view (10 per slide). The graph represents the mean H scores per slide. These cases did not have normal thyroid areas. C. A representative image of a separate cohort of 65 MTC tumor microarrays which were immunostained for CDK9. Scale bar indicates 100µm, magnification: 200X.