Kristianne Michelle Oristian

Figure S1. Genetic loss of CDK8 impairs cell growth. Figure S2. Pharmacologic inhibition of CDK8 triggers slowed cell proliferation, minimal apoptosis and cell cycle effects. Figure S3. CDK8 inhibition activates myogenic differentiation-associated gene targets. Figure S4. CDK8 inhibitors induce a muscle differentiation program without altering PAX3::FOXO1. Figure S5. Interruption of the SAGA complex rescues the effects of CDK8 inhibitors. Figure S6. CDK8 inhibition increases SIX4-driven muscle differentiation. Figure S7. The Mediator kinase module is required for maximal CDK8 inhibitor activity

Supplementary Table S1 of gene targets from NGS data

Alveolar rhabdomyosarcoma (aRMS) is a fusion-driven pediatric cancer with poor survival and limited therapeutic options. To uncover novel vulnerabilities, we used complex-Map functional genomic data, identifying CDK8 as a dependency in aRMS. Both CDK8 knockout and pharmacologic inhibition impaired tumor cell growth and induced myogenic differentiation in vitro and in vivo. Compared with genetic loss, CDK8 pharmacologic inhibition induced more dynamic transcriptional changes. With a genome-scale CRISPR–Cas9 drug modifier screen, we determined that the maximal antitumor activity of the CDK8 inhibitor requires the presence of the Mediator kinase module, including CDK8, and transcriptional cooperation with the SAGA complex. We further identified SIX4 as a key transcription factor mediating CDK8 inhibitor–induced transcriptional activation of myogenic differentiation genes and impaired tumor proliferation. These findings suggest a distinct gain-of-function mechanism of the CDK8 inhibitor and establish a strong rationale for CDK8 inhibition as a differentiation-inducing therapeutic strategy in aRMS. Significance: We provide a framework for uncovering therapeutic targets by network-based analysis of functional genomic screens. We identify CDK8 as a druggable target in aRMS and determine that CDK8 inhibition drives myogenic differentiation and impairs tumor progression via a collaborative mechanism involving the Mediator kinase module, SAGA complex, and SIX4. See related commentary by Stevens and Hatley, p. 2210

Background: We recently reported aberrant processing and localization of the precursor PNC (pro‐N‐cadherin) protein in failing heart tissues and detected elevated PNC products in the plasma of patients with heart failure. We hypothesize that PNC mislocalization and subsequent circulation is an early event in the pathogenesis of heart failure, and therefore circulating PNC is an early biomarker of heart failure. Methods and Results: In collaboration with the Duke University Clinical and Translational Science Institute's MURDOCK (Measurement to Understand Reclassification of Disease of Cabarrus and Kannapolis) study, we queried enrolled individuals and sampled 2 matched cohorts: a cohort of individuals with no known heart failure at the time of serum collection and no heart failure development in the following 13 years (n=289, cohort A) and a matching cohort of enrolled individuals who had no known heart failure at the time of serum collection but subsequently developed heart failure within the following 13 years (n=307, cohort B). Serum PNC and NT‐proBNP (N‐terminal pro B‐type natriuretic peptide) concentrations in each population were quantified by ELISA. We detected no significant difference in NT‐proBNP rule‐in or rule‐out statistics between the 2 cohorts at baseline. In participants who developed heart failure, serum PNC is significantly elevated relative to those who did not report development of heart failure ( P 6 ng/mL have a 41% increased risk of all‐cause mortality independent of age, body mass index, sex, NT‐proBNP, blood pressure, previous heart attack, and coronary artery disease ( P =0.044, n=596). Conclusions: These data suggest that PNC is an early marker of heart failure and has the potential to identify patients who would benefit from early therapeutic intervention.

Rhabdomyosarcoma (RMS), a cancer characterized by features of skeletal muscle, is the most common soft‐tissue sarcoma of childhood. With 5‐year survival rates among high‐risk groups at < 30%, new therapeutics are desperately needed. Previously, using a myoblast‐based model of fusion‐negative RMS (FN‐RMS), we found that expression of the Hippo pathway effector transcriptional coactivator YAP1 (YAP1) permitted senescence bypass and subsequent transformation to malignant cells, mimicking FN‐RMS. We also found that YAP1 engages in a positive feedback loop with Notch signaling to promote FN‐RMS tumorigenesis. However, we could not identify an immediate downstream impact of this Hippo‐Notch relationship. Here, we identify a HES1‐YAP1‐CDKN1C functional interaction, and show that knockdown of the Notch effector HES1 (Hes family BHLH transcription factor 1) impairs growth of multiple FN‐RMS cell lines, with knockdown resulting in decreased YAP1 and increased CDKN1C expression. In silico mining of published proteomic and transcriptomic profiles of human RMS patient‐derived xenografts revealed the same pattern of HES1‐YAP1‐CDKN1C expression. Treatment of FN‐RMS cells in vitro with the recently described HES1 small‐molecule inhibitor, JI130, limited FN‐RMS cell growth. Inhibition of HES1 in vivo via conditional expression of a HES1‐directed shRNA or JI130 dosing impaired FN‐RMS tumor xenograft growth. Lastly, targeted transcriptomic profiling of FN‐RMS xenografts in the context of HES1 suppression identified associations between HES1 and RAS‐MAPK signaling. In summary, these in vitro and in vivo preclinical studies support the further investigation of HES1 as a therapeutic target in FN‐RMS.

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for the precursor form of N-cadherin, pro-N-cadherin (PNC), by immunohistochemistry and compared to healthy tissues. Myofibroblast cell lines were analyzed for cell surface pro-N-cadherin by flow cytometry and immunofluorescent microscopy. Soluble PNC products were immunoprecipitated from patient plasmas and an enzyme-linked immunoassay was developed for quantification. All fibrotic tissues examined show aberrant PNC localization. Cell surface PNC is expressed in myofibroblast cell lines isolated from cardiomyopathy and idiopathic pulmonary fibrosis but not on myofibroblasts isolated from healthy tissues. PNC is elevated in the plasma of patients with cardiomyopathy (p ≤ 0.0001), idiopathic pulmonary fibrosis (p ≤ 0.05), and nonalcoholic fatty liver disease with cirrhosis (p ≤ 0.05). Finally, we have humanized a murine antibody and demonstrate that it significantly inhibits migration of PNC expressing myofibroblasts. Collectively, the aberrant localization of PNC is observed in all fibrotic tissues examined in our study and our data suggest a role for cell surface PNC in the pathogenesis of fibrosis.