Abstract
Abstract
Autophagy is a conserved mechanism of protein degradation that plays a physiologic role in iron homeostasis. The RARS subtype of MDS is characterized by inefficient erythropoiesis and the presence of erythroblasts with iron-laden mitochondria (ring sideroblasts, RS). Patients with RARS exhibit an accumulation of iron due to the inability of their erythroid precursors to properly process iron intended for hemoglobin synthesis. RARS erythroid cells display increased autophagy compared to cells from healthy subjects and mice lacking the essential autophagy genes Atg1 and Atg7 develop progressive anemia due to the inefficient removal of defective mitochondria. We previously showed that the presence of SF3B1 mutations, which are frequent in RARS patients, are associated with the presence of increased mitochondrial iron content and low levels of apoptosis. This finding suggests that cell death-independent mechanisms may temper excess iron by triggering the clearance of mitochondria during erythroid maturation. RNA sequencing analyses have shown that the autophagy pathway is upregulated in SF3B1MUT cells. We investigated the role of mitochondrial autophagy in the alleviation of iron-related damage in RARS erythroid cells and the potential therapeutic benefit of autophagy-stimulating agents for the selective improvement of erythropoiesis and iron homeostasis in SF3B1MUT RARS cells.
Transmission electron microscopy (TEM) demonstrated an accumulation of iron in the mitochondria of SF3B1MUT (K700E, n=4) compared to SF3B1WT RARS erythroblasts. Flow cytometry confirmed increased mitochondrial iron in SF3B1MUT (n=10) compared to SF3B1WT (n=10) RARS cells (82% ± 10 vs. 24% ± 5; P = 0.004). Autophagic vacuolization of the cytoplasm and an increased number of autophagosomes were found in SF3B1MUT (K700E) compared to SF3B1WT erythroblasts (n=2). The mRNA expression levels of numerous autophagy genes were elevated in SF3B1MUT vs. SF3B1WT RARS cells: ATG complexes (ATG2A/B, FC=2; ATG4A, FC=2; ATG9A, FC=5; ATG4C, P=0.05; ATG18 (FC=4.8; P= 0.02), autophagy-initiating kinases (ULK1, FC=2; ULK3, FC=3.9; P=0.05), and cathepsins involved in late stage autophagy (CTSL1, FC=20; CTSD, FC=5.8; P=0.05; CTSB, FC=2.1; CTSE, FC=5.9; CTSD, FC=2; P=0.01). qRT-PCR confirmed that SF3B1MUT cells expressed elevated mRNA levels of selected genes. We next administered 3 FDA-approved drugs with established autophagy-stimulating properties [temsirolimus, metformin, arsenic trioxide (ATO)] to a transgenic SF3B1 mouse model (SF3B1+/-) that exhibits anemia, dysplasia, and RS (Visconte, J Hematol Oncol 2014) to evaluate drug-mediated improvement of erythropoiesis and autophagic clearance of excess iron. Eight-month-old SF3B1+/- mice (n = 10 per group) were treated with vehicle control and the following: temsirolimus (10 mg/kg i.p. 5d/week for 2 wk), metformin (250 mg/kg/d, gavage for 2 wk), and ATO (10 mg/kg i.p. 5 d/wk for 2 wk). All agents were well tolerated and triggered morphologic features of autophagy including increased autophagosome-like structures by TEM. No effects on BM cellularity and/or dysplasia were noted, although changes in the morphology of myeloid cells (numerous swollen nuclei) were detected by Wright stain. Mitochondria that were engulfed in autophagosomes were frequently seen. After 2 weeks, temsirolimus-treated SF3B1+/- mice showed an incremental increase in hemoglobin by 1.2 g/dL (7.9 vs. 6.6 g/dL; P=0.07) and in mean corpuscular volume (43.5 vs. 42.4 fL; P=0.08). Erythropoiesis was improved as shown by increased levels of CD71-positive cells by immunohistochemistry in cells post-temsirolimus treatment compared to pre-treatment (10-20% vs. 5%). Increased dividing erythroid cells with binucleation and budding were also observed in cells following temsirolimus treatment. These hematologic changes were not detected with ATO or metformin.
In sum, our data support a role for activated autophagy in the pathogenesis of RARS and indicate that stimulating autophagy with approved existing drugs or novel investigational drugs may yield therapeutic benefit in SF3B1MUT RARS patients.
Disclosures
Kelly: Pharmacyclics: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau. Advani:Pfizer: Consultancy, Research Funding. Carraway:Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Baxalta: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding, Speakers Bureau.
