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Cushing’s syndrome is caused by excessive glucocorticoid production, which may lead to a series of metabolic disorders such as obesity, glu-cose intolerance and hypertension. Adrenal Cushing’s syndrome results from autonomous production of cortisol (ACTH-independent) from adrenocortical tumors (ACTs), which are most common in adult females. Cortisol-producing ACTs include benign adrenocortical adenoma (ACA), malignant adrenocortical carcinoma (ACC), rare forms of bilat-eral adrenal hyperplasia (BAH) and adrenocortical oncocytoma (ADO) (1). BAHs consist of macronodular and micronodular hyperplasia, such as adrenocorticotropin-independent macronodular adrenocortical hyper-plasia (AIMAH) and primary pigmented nodular adrenocortical disease (PPNAD).

Several genetic alterations have been described in inherited or spo-radic BAHs. The inactivating mutations in PRKAR1A (cAMP-dependent protein kinase regulatory subunit type Iα) are dominant in patients with Carney complex and PPNAD. Germline or somatic mutations in phos-phodiesterase genes (PDE11A and PDE8B) and GNAS are associated with adrenal hyperplasia (2, 3). However, the genetic architecture of adrenal Cushing’s syndrome remains largely uncharacterized, hampering the development of diagnostic and therapeutic approaches for Cushing’s syndrome.

To investigate the genetic lesions in adrenal Cushing’s syndrome, we per-formed whole-exome sequencing of DNA from 49 cortisol-producing ACTs and matched blood pairs, including 39 ACAs, 7 AIMAHs and 3 ADOs (table S1). The histological subtypes of all tumors were confirmed by pathological examinations. The average sequencing depth was 169X (82X-277X), and 95.61% (91.20%-99.50%) of the target regions were covered by at least 10X (table S2 and fig. S1). Meanwhile, we conducted RNA sequencing (RNA-seq) on 44 ACTs, including 36 whole-exome sequenced tumors, and obtained an average of 12.6 (9.4-17.4) Gb of sequenced data per sample (table S3).

We identified a total of 425 candi-date somatic mutations among the 49 samples, including 97 synonymous, 298 missense, 15 nonsense, 3 splicing, 4 frameshift and 8 in-frame, resulting in an average of 8.7 (0-28) somatic muta-tions in exonic regions (tables S4 and S5). We validated 55 out of 57 predict-ed somatic mutations (96.5%) by Sang-er sequencing and also verified a subset of highly expressed mutations using RNA-seq data (table S4). The predomi-nant substitution in these mutations was the C:G > T:A transversion, consistent with most cancer types (fig. S2).

We defined 12 potentially function-al mutated genes, including 8 recurrent-ly mutated genes, 3 consensus cancer genes with potentially damaging muta-tions and the only one mutated gene in AIMAH3 (4). PRKACA (cAMP-dependent protein kinase catalytic sub-unit α) was the only significantly mu-tated gene in our study (q = 3.77e-11)

(4). Strikingly, a hotspot somatic c.T617G/p.L205R mutation in PRKACA was discovered in 55.1% of sequenced ACTs (27/49) and ex-clusively in 69.2% of ACAs (27/39). Further screening revealed this somatic c.T617G/p.L205R mutation in 65.5% of ACAs (57/87) and none in AIMAHs (n = 13), ACCs (n = 16) and ADOs (n = 3) by Sanger se-quencing (fig. S3 and table S6). The L205R mutations were mainly de-tected in adult females (Fisher’s exact test, P = 0.017), and there were no significant differences in serum cortisol, ACTH and urinary free cortisol levels between ACA patients with and without the PRKACA mutation (tables S7 and S8). In addition, only one somatic PRKAR1A mutation was observed in an ACA. Thus, the PRKACA L205R mutation was be-lieved to be the dominant genetic alteration in sporadic ACAs.

Activating CTNNB1 mutations are frequently observed in benign and malignant ACTs (5). Germline APC mutations in patients with fa-milial adenomatous polyposis could result in the emergence of ACT (6). We identified activating CTNNB1 mutations (S33F and S45A) in ACA and ADO, and an APC truncating mutation in ACA (fig. S4 and table S4). In addition, activating mutations in R201 and Q227 of GNAS (en-codes stimulatory G-protein α subunit), resulting in PKA activation, have been reported in McCune-Albright syndrome, and adrenal and pituitary tumors (7, 8). We found 2 activating GNAS mutations (R201H

Activating Hotspot L205R Mutation in PRKACA and Adrenal Cushing’s Syndrome Yanan Cao,1† Minghui He,2† Zhibo Gao,2† Ying Peng,1 Yanli Li,1 Lin Li,2 Weiwei Zhou,1 Xiangchun Li,2 Xu Zhong,1 Yiming Lei2, Tingwei Su,1 Hang Wang2, Yiran Jiang,1 Lin Yang,2 Wei Wei,1 Xu Yang,2 Xiuli Jiang,1 Li Liu,2 Juan He,1 Junna Ye,1 Qing Wei,4 Yingrui Li,2 Weiqing Wang,1* Jun Wang,2,5,6,7,8* Guang Ning1,3* 1Shanghai Clinical Center for Endocrine and Metabolic Diseases, Shanghai Key Laboratory for Endocrine Tumors, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. 2BGI-Shanghai, BGI-Shenzhen, Shenzhen, China. 3Laboratory of Endocrinology and Metabolism, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, China. 4Department of Pathology, Rui-Jin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai, China. 5Department of Biology, University of Copenhagen, Copenhagen, Denmark. 6King Abdulaziz University, Jeddah, Saudi Arabia. 7Macau University of Science and Technology, Macau, China. 8Department of Medicine, University of Hong Kong, Hong Kong.

†These authors contributed equally to this work.

*To whom correspondence should be addressed. E-mail: guangning@medmail.com.cn (G.N.); wangj@genomics.org.cn (J.W.); wqingw@hotmail.com (W.W.)

Adrenal Cushing’s syndrome is caused by excess production of glucocorticoid from adrenocortical tumors and hyperplasias, which lead to metabolic disorders. We performed whole-exome sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing adrenocortical adenomas (ACAs), ACTH-independent macronodular adrenocortical hyperplasia (AIMAH), and adrenocortical oncocytoma (ADO). We identified a hotspot in the PRKACA gene with a c.T617G/p.L205R mutation in 69.2% (27 out of 39) of ACAs and validated in 65.5% of total 87 ACAs. Our data revealed that the activating L205R mutation, which locates in the P+1 loop of PKA catalytic subunit, promoted PKA substrates phosphorylation and target genes expression. Moreover, we discovered recurrently mutated DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight potentially functional mutated genes in adrenal Cushing’s syndrome.

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and R201C) in two PRKACA wild-type ACAs (Fig. 1 and table S4). We also uncovered novel mutated genes in cortisol-producing ACTs. In the ACAs, we identified mutations in cancer-related genes, including ARID1A and STAT3. We also detected recurrently mutated GPR98, co-localizing with PRKACA mutations in ACAs. In the AIMAHs, we iden-tified 2 mutations in the highly conserved methyltransferase domain of DOT1L, a histone H3 lysine 79 methyltransferase (Fig. 1 and fig. S5). DOT1L regulates gene transcription, cell proliferation and mediates leukemic transformation in MLL-rearranged leukemias (9, 10). Moreo-ver, the histone deacetylase HDAC9 was mutated in one AIMAH3 (table S4). These data suggest deregulated chromatin modification might con-tribute to tumorigenesis of AIMAH. A recent study revealed recurrent ARMC5 mutations in AIMAHs (11), whereas DOT1L and HDAC9 muta-tions have not been reported. Functional adrenocortical oncocytoma causing Cushing’s syndrome is exceptionally rare. We found CLASP2 mutated in 2 out of 3 ADOs, in which we predict to serve a functional role in cell division (12). In addition, we also observed PRKAR1A and CTNNB1 mutations in ADOs and PRUNE2 mutations in both ACA and AIMAH (Fig. 1). Taken together, our findings indicate that multiple genes may be responsible for adrenal Cushing’s syndrome.

The L205R mutation in PKA catalytic subunit (PKA C) appears to be a dominant genetic characteristic of ACAs although PKA C mutation has not been associated with a human disease. Structure analyses of PKA C shows that L205 is a component of the P+1 loop (L198-L205), which is in charge of the specific binding between the kinase and its substrates, and mediates the communication between catalytic residues and substrates (13–20). The highly conserved P+1 loop and activation loop constitute a conserved protein kinase core and contain the activa-tion segment (DFG to APE motif) (fig. S6). Previous studies have demonstrated that the Y204A mutation in the P+1 loop altered the en-zyme catalysis and phosphoryl transfer of substrate (16–18). L198, P202 and L205 in the P+1 loop form a hydrophobic binding pocket for ac-commodating hydrophobic side-chain of the P+1 residue in the substrate consensus (17). Crystal structure analyses of the complex between PKA C and regulatory subunit (PKA R), and the PKA C in complex with substrate peptide (Fig. 2) suggested that L205R changed the surface for substrate binding (15, 19, 20). All together, the structure-based analyses indicated that the L205R mutation might alter the PKA activity by af-fecting the substrate contact dynamics and catalytic efficiency of PKA C in a manner that contributes to ACA formation.

We analyzed RNA-seq data and obtained 232 differentially ex-pressed genes between PRKACA mutant and wild-type ACAs (Fig. 3A and table S9) (4). Of note, we didn’t observe significantly expressed change of PRKACA, which was further supported by qPCR quantifica-tion (Fig. 3B). Pathway analysis of these 232 genes using DAVID and WebGestalt (tables S10 and S11) nominated associated GO-terms of “biosynthesis and metabolism of steroid and cholesterol”, and “response to chemical stimulus”. StAR, MC2R, GSTA1, CXCL2 and S100A8/9 in-volved in these two terms were significantly upregulated in PRKACA L205R mutant ACAs (fig. S7). These genes may participate in tumor growth, survival and adrenal steroidogenesis according to previous pub-lication (21–24). Especially, the mRNA and protein levels of StAR were markedly elevated in the PRKACA L205R mutant ACAs compared to normal adrenocortical tissues and PRKACA wild-type tumors (Fig. 3, B and C). Furthermore, Western blot analysis showed significantly in-creased phosphorylation of PKA substrates in ACAs with L205R muta-tion (Fig. 3D). Together with that PKA activation can promote StAR expression, phosphorylate and activate StAR (25), our results indicate that L205R mutation triggers the activation of PKA, which promote tumorigenesis and steroidogenesis through phosphorylation of sub-strates.

To further investigate the function and characteristic features of PRKACA L205R mutation, we performed gain-of-function experiments

in 293T cells. Overexpression of L205R mutants significantly enhanced the phosphorylation of PKA substrates compared to wild-type (Fig. 4A). The activity of wild-type and mutated PKA C could be suppressed by the selective inhibitor H89 (fig. S8). L205R mutants induced more phos-phorylation of CREB (cAMP response element binding protein), which initiates transcription of target genes such as StAR and MC2R (Fig. 4B) (25). In addition, the phosphorylation of PKA C at T197, which is criti-cal for catalytic activity of PKA (26), was facilitated by the L205R mu-tation (Fig. 4C). Co-immunoprecipitation assays showed that mutated PKA C was pulled down by PKA R, indicating that the interaction was not interrupted by the L205R mutation (Fig. 4D).

Furthermore, co-expression of L205R mutants and PKA R exhibited more activity on phosphorylation of PKA substrates and CREB than wild-type in 293T cells, although protein levels of phosphor-PKA C were comparable (Fig. 4, E and F). PKA activity assays demonstrated that the L205R mutants induced a significant increase in PKA activity, whereas the substrate competitive peptide inhibitor (PKI) could block the catalytic activity of mutated PKA C (Fig. 4, G and H). Taken togeth-er, these results indicated that the L205R mutation enhanced the phos-phorylation catalysis of PKA.

The identification of activating mutations in oncogenes is particular-ly valuable in developing targeted therapies for cancer. PKA signaling plays a pivotal role in various physiological and pathological processes, including development, metabolism and tumorigenesis. Although inacti-vating mutations in PRKAR1A and phosphodiesterase genes are known in adrenal hyperplasia, we revealed the activating PRKACA L205R mu-tation and several potential functional mutated genes in ACTs. Future investigations on mutagenesis and hormones or environmental initiators of the L205R mutation may provide clues for prevention or treatment of Cushing’s syndrome.

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Acknowledgments: Supported by grants from Natural Science Foundation of China (30900702, 81130016, and 81270859); Guangdong Innovative Research Team Program (2009010016). The sequencing data have been deposited in the European Genome-phenome Archive (accession no. EGAS00001000712)

Supporting Online Material www.sciencemag.org/cgi/content/full/science.1249480/DC1 Materials and Methods Figs. S1 to S8 Tables S1 to S11 References (27-40)

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Fig. 1. PRKACA L205R mutations in ACAs and landscape of somatic mutations in cortisol-producing ACTs. Candidate cancer genes (4) are listed vertically on the left. Columns represent samples, with the average mutation rate in exon regions at the top. Samples are arranged from left to right by subtypes and mutation numbers. Histological subtypes and gender of each patient are reported at the bottom (green, ACA; red, AIMAH; orange, ADO; purple, female; blue, male). Colored rectangles indicate mutation category observed in the cohort. Asterisks indicate significantly mutated genes in tumor subtypes (PRKACA, 28/39 in ACA; DOT1L, 2/7 in AIMAH; CLASP2, 2/3 in ADO).

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Fig. 2. L205R mutation occurs in the catalytic subunit of PKA (PKA C). Crystal structure of the interaction between catalytic and regulatory subunits of PKA (left, PDB: 2QCS), and a binding substrate peptide (right, PDB: 1L3R). L205R mutation in the P+1 loop is presented in pink. The substrate peptide derived from inhibitor PKI is shown in red.

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Fig. 3. PRKACA L205R mutation activates PKA signaling in ACAs. (A) Heatmap of significantly differentially expressed genes identified by RNA-seq in ACAs. Columns represent samples; genes are in rows. The mutation status of PRKACA for each sample is reported at the top (blue, wild-type; red, L205R). The color of rectangles represents gene expression levels (red, upregulation; green, downregulation; black, no change). Samples are arranged by the clustering of gene expression patterns. (B) Quantitative RT-PCR analysis of PRKACA and StAR in normal human adrenocortical tissues (green), L205R mutant (red) and wild-type ACAs (blue) (normal, n = 10; L205R, n = 20; WT, n = 9). (C) Elevated protein level of StAR in L205R mutant ACAs. (D) The phosphorylation of PKA substrates is enhanced in L205R mutant ACAs. (E) The PKA activity is significantly higher in L205R mutant ACAs (n = 5). Data represent mean ± standard deviation (SD); t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

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Fig. 4. PRKACA L205R mutation promotes PKA activation and substrate phosphorylation. (A) Overexpression of L205R mutated PKA C increases phosphorylation of PKA substrates compared to wild type in 293T cells. (B) Overexpression of L205R mutated PKA C promotes phosphorylation of CREB in 293T cells. (C) The phosphorylation of T197 is elevated in L205R mutated PKA C. (D) Co-immunoprecipitation of wild-type, L205R mutated PKA C and flag-tagged regulatory subunit of PKA (PKA R) in 293T cells. Protein lysates were prepared and immunoprecipitated with anti-flag or control immunoglobulin G, respectively. (E and F) Co-expression of PKA R and PKA C in 293T cells. L205R mutated PKA C leads to more phosphorylation of PKA substrates (E) and CREB (F). (G and H) L205R mutated and wild-type PKA C was overexpressed in 293T cells for PKA activity assay. The activity of L205R mutated PKA C is significantly higher (G). The peptide inhibitor PKI inhibits the activity of L205R mutated PKA C (H).