<p>The most abundant deregulated miRs targeting the main genes essential for thyroid differentiation.</p>

<p>Putative PAX8 binding regions upstream the transcription start site of both human and rat miR-146b.</p>

Supplementary Figure S5. Immunoblotting of Calr-immunoprecipitated proteins and whole cell lysates from Ba/F3-Cas9 and Ba/F3-MPL-Cas9 cells targeted with Calr-directed CRISPR gene editing using a control guide, Calr guide m1, or Calr guide m2 demonstrates increased binding between MPL and mutant Calr (lane 3 - 4 (m1) and lane 5 -6 (m2)) as compared to wild type Calr (lane 2).

Supplementary Figure S4. (A) Comparison between human and mouse wild-type (WT) calreticulin Cterminal tail. Blues indicates non-conserved regions between human and mouse. The wild-type proteins are highly conserved between the two species and contain many negatively-charged glutamic acid (E) amino acids. (B) Comparison between human and mouse amino acid sequences corresponding to the mutant C-terminal part of the human and mouse mutant with red shading indicating non-conserved regions. Despite the large number of mismatches, both mutant C-terminal tails are rich in positively charged amino acids (arginine (R) and lysine (K)).

Supplementary Table S1. Pre-ranked GSEA statistics between mutant and wild-type CALR 24 hours post-IL-3 withdrawal amongst STAT5, STAT3 and JAK2 inhibitor signatures from MSiGDB and literature.

Screening results U251 Val-083

<p>Validation of the most abundantly deregulated miRs.</p>

<p>Selected target genes most extensively regulated by miRs in PTC.</p>

Abstract The presence of differentiated thyroid cells in thyroid cancer is critical for the antitumor response to radioactive iodide treatment, and loss of the differentiated phenotype is a key hallmark of iodide-refractory metastatic disease. The role of microRNAs (miRNA) in fine-tuning gene expression has become a major regulatory mechanism by which developmental and pathologic processes occur. In this study, we performed next-generation sequencing and expression analysis of eight papillary thyroid carcinomas (PTC) to comprehensively characterize miRNAs involved in loss of differentiation. We found that only a small set of abundant miRNAs is differentially expressed between PTC tissue and normal tissue from the same patient. In addition, we integrated computational prediction of potential targets and mRNA sequencing and identified a master miRNA regulatory network involved in essential biologic processes such as thyroid differentiation. Both mature products of mir-146b (miR-146b-5p and -3p) were among the most abundantly expressed miRNAs in tumors. Specifically, we found that miR-146b-3p binds to the 3′-untranslated region of PAX8 and sodium/iodide symporter (NIS), leading to impaired protein translation and a subsequent reduction in iodide uptake. Furthermore, our findings show that miR-146b and PAX8 regulate each other and share common target genes, thus highlighting a novel regulatory circuit that governs the differentiated phenotype of PTC. In conclusion, our study has uncovered the existence of a miR-146b-3p/PAX8/NIS regulatory circuit that may be exploited therapeutically to modulate thyroid cell differentiation and iodide uptake for improved treatment of advanced thyroid cancer. Cancer Res; 75(19); 4119–30. ©2015 AACR.

Over-represented gene sets defined in KEGG (Kyoto Encyclopedia of Genes and Genomes) amongst differentially expressed genes in PTC.

Clinico-histopathological and molecular characteristics of the discovery set.

Supplementary Figure S1. (A) Relative frequency of GFP positive cells in peripheral blood over time in BMT mice receiving empty vector, CALRWT or CALRMUT-expressing c-Kit+ BM cells. (B) Histopathologic H&E sections of BM from representative EV, CALRWT or CALRMUT recipient mice demonstrates megakaryocytic hyperplasia in CALRMUT recipient animals (100X magnification) (C) A N-terminal anti-CALR antibody demonstrated strong, diffuse cytoplasmic reactivity in megakaryocytes in representative normal and CALR-mutant patient bone marrow biopsies. In addition, cytoplasmic reactivity was noted in smaller mononuclear cells, which were morphologically most suggestive of early myeloid forms. Strong reactivity was not observed in mature erythrocytes or in mature granulocytes. (D) Flow cytometric analysis of Lin−Sca-1+c-Kit+ (LSK) and Lin−Sca-1−c-Kit+ (LK) cell numbers in the bone marrow of BMT mice receiving empty vector, CALRWT or CALRMUTexpressing BM cells 16 weeks post bone marrow transplantation demonstrates a significant increase in LSK cells in CALRMUT recipients. All p values were determined by unpaired two-tailed Student's t test. (*0.01 < p < 0.05; (**0.001 < p < 0.01; ns, not significant).

Supplementary Figure S2. Schema of CRISPR/Cas9 gene-editing targeting of exon 9 of Calr.

<p>Main characteristics of the 8 patients including gender, histological variant, Tumor Node Metastasis (TNM) stage, concomitant thyroid disease and total thyroidectomy.</p>

Among the list of target genes of miR146b there are four genes involved in thyroid differentiation including NIS and PAX8.

Screening results T98G Val-083

<p>Deregulated mRNAs in PTC.</p>

Supplementary Figure S3. (A) Pre-ranked GSEA in CALRMUT versus CALRWT expressing Ba/F3-MPL cells 24 hours post-IL-3 withdrawal. Stat5 (left) and Stat3 (right) target genes (Baker et al, 2007) are both enriched in CALRMUT compared to CALRWT-expressing Ba/F3-MPL cells (B) Immunoblotting demonstrates differential phosphorylation of STAT5 in CALRMUT-expressing UT-7-MPL cells. (C) Immunoblotting demonstrates differential phosphorylation of Stat5 and Stat3 in Calr-targeted Ba/F3-MPL-Cas9 cells.

Supplementary Figure Legends from The miR-146b-3p/PAX8/NIS Regulatory Circuit Modulates the Differentiation Phenotype and Function of Thyroid Cells during Carcinogenesis