Genetic and Epigenetic Defects in the Autophagy Machinery in Myelodysplastic Syndromes

Autophagy is a degradation process for the turnover of damaged organelles and long-lived proteins that also plays an important role during erythropoiesis. Accordingly, knockout of the essential autophagy gene Atg7 in mice leads to clinico-morphologic features of MDS. To date, no study has determined the prevalence and impact of defects (mutations, aberrant expression) in the autophagy machinery in MDS. We interrogated the occurrence of alterations in 180 autophagy genes by analyzing WES of patients with MDS (N=120). For comparison, we analyzed results from other hematologic neoplasms (N=103) and TCGA (N=202).

We detected somatic mutations in autophagy genes in 40/425 patients (9%). Mutations were enriched in MDS (12%;14/120) and prevalent in higher risk MDS patients (30%;12/40). Mutations were found in: ATG2A, ATG4C, ATG14, ATG16L1, BCL2, CDKN2AIPNL, COG8, DNM1L, DNM2, GYS1, HIF1A, KIF1B, LAMP2, MLST8, MTOR, NOD2, PIK3CB, PIK3C2A/B, PIK3C2G, PPP2R2A/B, PPP2R3A, PRKAA1/2, PRKACB, PRKAG1/G2, PTPN2, RICTOR, RPTOR, SEC22B, SMURF1, SQSTM1, STAT3, SUPT20H, TAB2, TNFSF10/13B, ULK4, USP10, VPS11/33B, VTI1A, WDFY3/4, WAC. Twenty-five mutations had a cut-off >20% VAF. Most patients (31/40;78%) had a sole mutation, 4 patients carried 2 mutations each (ULK4, WDFY3), (KIF1B, SEC22B), (VIT1A, TAB2), (DNM2, WAC). PRKACB mutations were found in 3 patients. NOD2, PTPN2, PRKAG1, SEC22B, ULK4, VPS11 and WAC mutations were found in 2 patients. inces autophagy genes has been Loss of function mutations were observed in ULK4, NOD2 and WAC. Four genes mapped to commonly deleted regions e.g., 5q (CDKN2AIPNL, SQSTM1) or 7q (SMURF1, PRKAG2) and coincided with haploinsufficient expression, while 3 genes had hemizygous configuration (SMURF1, PPP2R3A, PIK3C2G). Reactome analysis clustered the mutations in effectors/inhibitors and early stage. The analysis of 263,973 germline variants detected that PIK3C2G, NOD2 and HIF1A were also associated with exonic germline variants predicted to be significantly deleterious. Mutations or aberrant expression of core components of the autophagy network have been associated with poor outcomes in multiple diseases. In our cohort, 21 patients died. Most (57%;21/37) of patients had abnormal karyotype with 4 patients having complex karyotype including -17 and -7. Among the cases with abnormal karyotype, 4 cases had del(5q). Mutant patients had worse survival trending toward significance compared to WT patients (MUTvs. WT=20 vs. 90; median 14 vs. 20 months; LogR=.09). Among disease groups, autophagy mutations were associated with significantly inferior survival in MDS (MUT vs. WT=13 vs. 61; 17 vs. 35 months; LogR=.018) and MDS/MPN (MUT vs. WT=4 vs. 31; 12 vs. 30 months; LogR=.037). Seven mutant patients who received hypomethylating agents had no response. Comparative analyses (Sanger, TruSeq, WES, TCGA) identified that autophagy gene mutations were significantly associated with TET2 (28%;11/40; P=.02) among other mutations [RUNX1, STAG2 (20%;8/40), SRSF2 (18%;7/40), DNMT3A, ASXL1 (15%;6/40)]. Clonal hierarchy showed that autophagy gene mutations were mainly secondary events, were ancestral events in 7 (ATG2A, DNML1, PRKACB, PRKAG1, PTPN2, SEC22B, STAT3) and co-dominant in 2 patients (NOD2, MLST8). When autophagy genes mutations were secondary, the most represented ancestral mutationswere in splicing factors (N=9; SRSF2, PRPF8, U2AF1) and DNA methylation (N=4; TET2, DNMT3A). RNA sequencing determined that changes in autophagy gene expression are overrepresented in specific MDS subtypes with distinct mutational profiles. The expression levels of 2 ULK family members commonly elevated during erythroid maturation were found in SF3B1^K700E compared to SF3B1^WT MDS patients (N=6; ULK1, FC=2; ULK3, FC=4; P=.05). Erythroid cells of these SF3B1^K700E patients showed increased autophagosomes compared to SF3B1^WT cells. In vivo administration of the mTOR inhibitor, temsirolimus (10 mg/kg i.p. 5d/week for 2 wk) improved the erythropoiesis of a transgenic Sf3b1 mouse model by increasing CD71+ cells (10-20% vs. 5%) and ameliorating anemia (Hgb: 7.9 vs. 6.6 g/dL; P=.07; MCV: 43.5 vs. 42.4 fL; P=.08).

In sum, defects in autophagy genes are present in MDS, co-occur with other mutations and impact survival. Changes in expression levels of autophagy genes may be associated with MDS phenotypes and modulated by autophagy inducing drugs as evidenced in models of SF3B1 mutations.