Elevated Basal Autophagy in SF3B1 Mutated Myelodysplastic Syndromes: Relationship with Survival Outcomes and Therapeutic Implications
Authors: Visconte Valeria , Kelly Kevin R. , Nawrocki Steffan T. , Han Yingchun , Carraway Hetty E. , Advani Anjali S. , Maciejewski Jaroslaw P. , Rogers Heesun J , Sekeres Mikkael A. , Carew Jennifer S. 
Published: October 2019

Abstract
About 60-80% of patients with myelodysplastic syndromes (MDS) manifest with anemia. Red blood cell (RBC) transfusions are the most commonly used therapy to alleviate anemia in patients that are ineligible for other curative approaches. Transfusion-dependent patients frequently develop iron overload, which correlates with infections, mortality, leukemia, and organopathy. At the subcellular level, long-term iron exposure produces iron-catalyzed hydroxyl radicals that induce oxidative damage to mitochondria and disrupt bioenergetic homeostasis. The use of iron-chelating drugs to counter transfusion-related iron overload remains controversial due to the significant side effects that these agents cause. New therapies that effectively address iron overload in transfusion-dependent MDS patients are clearly needed. Mitochondrial dysfunction frequently occurs during MDS cell maturation and leads to abnormal iron distribution. However, the mechanistic basis of this biological phenomenon has not been rigorously studied. We previously linked the presence of Splicing factor 3b subunit 1 (SF3B1) mutations, which are frequent in patients with refractory anemia with ring sideroblasts (RARS), with abnormalities in mitochondrial iron. Transmission electron microscopy and flow cytometry showed that mitochondria of SF3B1 mutant RARS patients have higher iron content than those of wild type (WT) RARS patients and expressed an increased mRNA level of the iron transporter, Mitoferrin 1. Despite these prevalent mitochondrial abnormalities and transfusional dependence, SF3B1 mutant lower-risk MDS patients experienced significantly longer median survival compared to SF3B1 WT lower-risk MDS patients (34 mos vs. 13 mos; P = .002; N=16 vs. 101). Autophagy is an evolutionarily conserved lysosomal mechanism of protein degradation that plays a critical role in the elimination of damaged mitochondria and other organelles. We hypothesized that autophagic clearance of defective mitochondria may contribute to the superior survival of SF3B1 mutant patients suffering from transfusion-mediated iron overload. We conducted RNA sequencing analyses on a group of fresh bone marrow (BM) cells of SF3B1 mutant and WT RARS patients and healthy donors (n = 11) to investigate the basal autophagy status in this distinct patient population. In addition to confirming increased levels of mitochondrial transporters such as Mitoferrin 1 and 2 (FC = 2.0), we detected a striking increase in multiple genes involved in the proximal and distal regulation of autophagy in cells of SF3B1 mutant RARS compared to WT RARS patients. Genes controlling the early stages of autophagy including the protein kinases ULK1 (FC = 2.0) and ULK3 (FC = 3.9; P=.05), ATG complexes ATG2A/B (FC = 1.9), ATG9A (FC = 5.5; P=.05), ATG18 (FC = 4.8; P=.02) and the cysteine proteases ATG4A/C (FC = 2.0) were all elevated in SF3B1 mutant RARS vs. SF3B1 WT RARS patients. Key components of the late stages of the autophagic degradation cascade, including multiple members of the cathepsin family of lysosomal proteases [CTSL1: FC = 20.9; CTSD: FC = 5.8 (P=.06); CTSB: FC = 2.1; CTSE: FC = 5.9 (P=.01); CTSD: FC = 1.8], were also significantly increased. qRT-PCR confirmed higher expression levels in the BM cells of RARS patients carrying sole SF3B1 mutations compared to cells of SF3B1 WT RARS patients. The link between SF3B1, mitochondrial iron, and elevated autophagy was specific as evidenced by the unmutated status and lack of significant mRNA changes in any other splicing factor genes including PRPF8. Our data demonstrate that autophagy may play an important, previously unreported role in SF3B1 mutant RARS. Based on our findings, we hypothesize that SF3B1 mutant RARS cells stimulate autophagy to eliminate damaged mitochondria and alleviate iron overload and that further stimulation of autophagy will diminish the pathogenic effects of chronic transfusions. We are currently investigating the therapeutic benefit and pharmacodynamics of autophagy-modulating drugs (temsirolimus, metformin, arsenic trioxide) in in vitro (primary cells) and in vivo (SF3B1 haploinsufficient mice) models of MDS to facilitate the design of an investigator-initiated clinical trial that will test autophagy modulation as a new precision strategy for the treatment of transfusion-dependent patients with low-risk MDS carrying SF3B1 mutations.

Disclosures
Sekeres: TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Carew:Boehringer Ingelheim: Research Funding.