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Complete mutational spectrum of the autophagy interactome: a novel class of tumor suppressor genes in myeloid neoplasms

Complete mutational spectrum of the autophagy interactome: a novel class of tumor suppressor genes in myeloid neoplasms

Comparative analysis (Sanger, TruSeq, WES, TCGA) found that patients with autophagyrelated genes are more susceptible to acquire mutations in signal transduction pathways (26 mutations), DNA methylation (24 mutations), and RNA-splicing (20 mutations) (Figure 1c and Figure S2). Twenty-five mutations had a cut-off more than 20% variant allele frequency (VAF) ( Figure S3). Targeted gene panel sequencing was applied for 22 genes and confirmed the presence of high variant allele frequency mutations like NOD2 (63%), PIK3C2B (44%), PRKACB (36%) and STAT3 (38%). The majority of mutant patients had a dismal prognosis, suggesting that these patients had cumulative unfavorable mutations like RUNX1, ASXL1, DNMT3A and that these negative prognostic mutations 6 represented driver mutations in these patients. Seven patients receiving hypomethylating agents had no response to therapy (Table S1) Given the recent discovery of familial AML and MDS cases carrying pathogenic germ-line variants, 7,8 we also evaluated whether the same autophagy genes we found to be somatically mutated may also be prone to germ-line alterations. An in house analysis of a database of 263,973 variants obtained from WES was conducted by testing different predictive tools assessing deleterious variants and by calling a variant as deleterious if present as deleterious in all Supplemental methods). We found 4 variants in 3 genes among 4 patients with possible deleterious exonic non-synonymous variants that were predicted to be significantly damaging by all the tested scores: PIK3C2G (rs377020826, c.T3083C, total allele frequency: 0.0001; rs201569993, c.C2938T, total allele frequency: 0.0007), NOD2 (rs104895423, c.T662G, total allele frequency: 0.0006), and HIF1A (c.A1666G; total allele frequency: undetermined) (Table S3). In total, PIK3C2G carried 1 somatic and 2 germ-line variants, NOD2 had 2 somatic and 1 germ-line variants, and HIF1A harbored 1 somatic and 1 germ-line variant. A complete table summarizes the somatic and germ-line status of the variants we identified in the autophagy pathway (Table S4).
As mentioned previously, many types of malignant cells have been reported to exhibit defective or dysregulated autophagy including mutations in BECN1 (17q21), which is monoallelically deleted in 40-75% of patients with breast, ovarian, and prostate cancer. 9 In other tumors, autophagy genes such as SH3GLB1 have been associated with regions of loss of heterozygosity. 10 In our study, we identified mutations in proximal co-factors of BECN1 such as PI3KCIII, vacuolar sorting proteins (Vps34, Vps11) and ATG14. We performed an analysis of the related protein sequences (http://www.uniprot.org/) of the mutated regions and found that several mutations occurred in essential domains/regions involved in proteinprotein interactions, particularly those required for autophagosome formation (ATG14, BECN1, PI3KC3, SEC22B, VPS34/11) (Table 1). Several mutations were found in functional domains of the proteins [pt#32 (DNM2; pleckstrin homology domain), pt#38 (PRKAA2; protein kinase domain)]. Other mutations were detected in regions of interactions between genes highly relevant in the autophagy pathway such as WDFY3 mutation (p.G3362V; pt#2) maps in a region spanning the interaction with SQSTM1 (aminoacid 2586-3526), ATG5 (amino-acid 2981-3526) and the region containing the LC3interacting region (LIR) motif, which allows the targeting of autophagy receptors to LC3 (or other ATG8 family proteins) as a crucial step in the autophagy cascade. SQSTM1 was also found mutated in our cohort (p.D347Y; pt#10) and the mutated region was proximal to the LIR motif (amino-acid 336-341). We also detected a mutation in a Beclin-1-associated autophagy key regulator (ATG14; pt#33), which has been shown to directly interact with Beclin-1 and increases phosphatidylinositol 3-phosphate kinase class III (PI3KC3) activity to initiate autophagosome membrane nucleation by targeting SNARE-associated proteins. 11 We also reviewed extensively the literature to identify possible variants occurring also in other diseases (Table S5).
Our collective findings define for the first time an important and perhaps so far underappreciated role for genetic aberrations in the autophagy network in the pathogenesis of myeloid neoplasms. Our data demonstrate that autophagy are autophagy-related gene mutations are more prevalent in MDS and AML, co-occur with unfavorable mutations (RUNX1, ASXL1, DNMT3A), and are associated with inferior survival. These findings support further investigation of genetic loss of autophagy regulation as a novel cooperative mechanism leading to leukemogenesis that can potentially be therapeutically targeted with synthetic lethal approaches. Results from high-throughput next generation sequencing collected from a larger population of genetically annotated patients with myeloid neoplasms and comprehensive clinical information from different institutional centers will help define the exact frequency of single gene mutations and the precise correlation with the clinical phenotypes.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

(a) Somatic mutations of the autophagy network in patients with myeloid neoplasms.
Whole exome and targeted sequencing identified 55 somatic mutations in autophagy-related genes (a complete list of mutations is presented in Table 1). A schematic diagram illustrates mutations per patients (N=40; pts #1-31; our cohort; pts #32-40, TCGA cohort). For pt #1, exome sequencing was performed at the stage of MDS and AML. Different colors represent the specific pathways where genes were clustered based on their function in the autophagy pathway (yellow: early stage autophagy; gray: vesicles-mediated transport; light blue: lysosomal enzymes: red: protein kinases and dependent kinases family; purple: protein tyrosine phosphatase family; black: phosphatidylinositol-3 phosphatases; green: apoptotic inducers/inhibitors; dark green: insulin receptor/mTOR associated signaling. Clustering was conducted by reviewing previous published reports and by using the reactome pathway database (http://www.reactome.org/). Pink half square triangles represent patients who were hemizygous for specific mutations. Stars indicate genes for which germline variants were also detected. The total number of mutations per pathway is indicated in the right column.