MMP9 Inhibition Rescues the Erythroid Defect in RPS14-Deficient Del(5q) MDS Models
Authors: Youn Minyoung , Huang Haigen , Chen Cheng , Kam Sharon , Wilkes Mark C , Chae Hee-Don , Narla Anupama , Lin Shuo , Sakamoto Kathleen M. 
Published: October 2019

Abstract
The del(5q) myelodysplastic syndrome (MDS) is a unique subtype of MDS characterized by an interstitial deletion of the long arm of chromosome 5 with macrocytic anemia, normal to increased platelet count, and hypoloblated megakaryocytes. Although haploinsufficiency of several genes encoded within the commonly deleted region have been implicated in the hematologic phenotype of del (5q) MDS, the deletion of the RPS14 gene, which encodes a component of the 40S ribosomal subunit, is a key determinant of the severe macrocytic anemia seen in patients. Lenalidomide is FDA-approved and highly effective for the treatment for del(5q) MDS. However, there are rare resistant cases resulting from p53 mutations in del(5q) MDS patients and 50% of patients acquire resistance within 2 to 3 years. Given these limitations of lenalidomide treatment, as well as frequent side effects, we sought to identify novel targets for the treatment of del(5q) MDS patients by performing an in vivo drug screening assay using a zebrafish model of MDS.
We generated stable genetic mutation of zebrafish RPS14 using CRISPR-Cas9 gene targeting technique. This zebrafish model mirrors the anemic phenotype seen in the 5q-syndrome. We found that mmp9 gene expression was abnormally induced by RPS14 deficiency in zebrafish and MMP9 inhibitors partially rescued erythroid defects in RPS14-deficient zebrafish. To study the role of MMP in human hematopoietic stem cells, we treated MMP9 inhibitors in RPS14 or luciferase control knockdown human bone marrow stem and progenitor (BMCD34+) cells for 6 days and then evaluated erythroid development. Our results showed that treatment with MMP9 inhibitors in RPS14 knockdown CD34+ cells significantly increased erythroid colony numbers compared with DMSO control (1.5 to 2.5-fold increase). Similarly, FACS analysis demonstrated increased the CD71+ erythroid population in RPS14 downregulated cells by MMP9 inhibitor treatment (4-fold increase compared to control), but no difference in the CD11b+ myeloid population. This data suggests that MMP9 inhibitors have specific roles in the erythroid development in RPS14 deficient cells.
To understand the mechanism of MMP9 inhibitors, we first examined MMP9 expression in RPS14-deficient cells. After transduction of human BMCD34+ cells with lentivirus expressing RPS14 shRNAs, we sorted GFP+ cells and observed MMP9 levels by RT-qPCR and Western blot analysis. We found a 4-fold increase in the expression levels of MMP9 in the RPS14 knockdown cells, supporting the hypothesis that MMP9 is increased in RPS14-deficient cells ultimately leading to decreased erythropoiesis.
To determine the effects of MMP9 treatment during erythropoiesis, we treated human BMCD34+ cells with recombinant MMP9 protein (rMMP9). Cells were treated with activated rMMP9 for 2 days, and then examined by FACS analysis. Interestingly, we found that rMMP9 treatment decreased the CD71+ erythroid population by 40% compared to vehicle control. We confirmed that enhanced expression of MMP9 is critical to the negative regulation of erythroid development in RPS14 knockdown cells by a performing double knockdown experiment. We co-transduced lentivirus expressing shMMP9-mCherry and shRPS14-GFP shRNAs and examined erythropoiesis by FACS analysis. Our results demonstrated that MMP9 and RPS14 double knockdown partially rescued the decreased CD71+ population in RPS14 knockdown cells. However, MMP9 knockdown alone did not affect CD71+ erythroid development. Taken together, our data suggest that increased MMP9 contributes to the erythroid defect observed in RPS14-deficient cells.
In this study, our results showed that MMP9 inhibitors rescued the erythroid defects in RPS14 downregulated del(5q) MDS models. This suggests a novel role for MMP9 in the pathogenesis of del(5q) MDS and MMP9 inhibitors in the treatment of patients with del(5q) MDS.

Disclosures
No relevant conflicts of interest to declare.