Activation of Autophagy in Models of SF3B1 Mutant MDS

Background: SF3B1 is a key pre-mRNA splicing factor that is abundantly expressed in normal erythroid cells and whose mutation has been linked with high frequency to myelodysplastic syndromes with ringed sideroblasts (MDS with RS) implying that this gene may be an important driver of the pathogenesis of this subtype. Given that MDS with RS is characterized by ineffective erythropoiesis, we previously investigated the link between SF3B1 aberrations and the regulation of pathways that control erythroid maturation and iron homeostasis. Indeed, knockdown of SF3B1 led to significant impairment of the growth of erythroid cells and delayed erythroid differentiation. MDS erythroid cells were unable to properly process iron, resulting in its accumulation in mitochondria. Iron build-up inhibited the proliferation of erythroid progenitors and induced mitochondrial DNA-damage. During erythroid maturation, autophagy plays critical roles in the clearance of damaged mitochondria and maintaining iron homeostasis. Knockout of the essential autophagy gene, Atg7 leads to anemia due to a delayed clearance of mitochondria with a consequent increase in iron levels, ultimately resulting in increased reactive oxygen species formation. As such, we set out to determine if autophagy upregulation alleviates oxidative stress and thereby increases erythropoiesis in MDS with RS.

Methods: Transmission electron microscopy (TEM) was performed to examine the cytoplasmic vacuolization, autophagosome content, and morphology of vacuoles and mitochondria in SF3B1 mutant (SF3B1^MUT) vs. wild-type (SF3B1^WT) cells. Immunoblotting was used to detect microtubule-associated protein 1 light chain 3 (LC3B), p62, and cathepsin D (marker of autophagic degradation). Whole exome sequencing (WES) was used to detect somatic alterations in autophagy genes and RNA-Sequencing and qPCR were conducted to evaluate differences in mRNA expression levels of autophagy genes. A missense mutation in SF3B1 resulting in K700E amino-acid substitution was introduced using EGE-CRISPR/Cas9-stimulated homology-mediated repair to generate heterozygous K562 cells expressing K700E.Transgenic heterozygous SF3B1 mice were used as an in vivo model. Temsirolimus (CCI-779), an mTOR inhibitor with autophagy-inducing properties, was tested in vitro and in vivo.

Results: We identified numerous autophagosomes, double-membraned autophagic vacuoles, and single-membrane cytoplasmic structures in SF3B1^MUTvs. SF3B1^WT cells. LC3B protein was higher in primary SF3B1^MUTvs. SF3B1^WT cells. WES did not detect any somatic mutations in autophagy genes in the patients analyzed by TEM. RNA-Sequencing and qPCR showed that primary SF3B1^MUT MDS with RS cells displayed increased expression levels of key genes of the ATG-autophagy pathway: [ATG2A/B, ATG3, ATG5 (FC=2), ATG9, (FC=5); P=.05], serine/threonine kinases [ULK1 (FC=2), ULK3 (FC=4); P=.05], and cathepsins [CTSB/D (FC=2), CTSE (FC=6); P=.01]. CRISPR/Cas9 K700E cells showed high autophagosome counts, increased basal expression of LC3B protein, and a 2-fold increase in autophagy genes (ATG5, ATG7, ULK1) compared to parental cells. Treatment with temsirolimus (10-100 nM) for 24 hours induced growth inhibition (IC_50, 50 nM), enhanced the amount of autophagosomes containing degraded organelles, increased the percentage of dividing CD71⁺ cells and induced a 2-fold increase in LC3B in CRISPR/Cas9 K700E cells compared to parental cells. Temsirolimus (10 mg/kg i.p. 5d/wk) administered for two weeks to 6-8-month-old transgenic SF3B1 heterozygous mice (N=20) induced autophagy by increasing the number of autophagosomes and autophagolysosomes containing entrapped mitochondria, increased hemoglobin (7.5 vs. 6.6 g/dL; P=.05), and doubled the percentage of CD71⁺ erythroid cells (5-10%) in temsirolimus-treated vs. untreated SF3B1 mice.

Conclusions: Basal autophagy is significantly upregulated in SF3B1^MUT cells. Further stimulation of autophagic activity using FDA approved pathway-activating drugs may improve rates of erythrocyte production by SF3B1^MUT clones and thus improve control of anemia in MDS with RS cases.