The LSD1 Inhibitor RN1 Rescues Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease resulting from loss-of-function mutations in SEC23B, which encodes a component of coat complex protein II (COPII) vesicles. Mammals express two paralogous genes for SEC23, SEC23A and SEC23B, which encode proteins with ~85% amino acid sequence identity. We have previously shown that the SEC23 paralogs have identical interactomes and that SEC23A overlaps in function with SEC23B. Since the SEC23B/SEC23A expression ratio is disproportionately high in human erythroid cells, we reasoned that the endogenous SEC23A expression level is not sufficiently elevated to compensate for loss of SEC23B in CDAII erythroid cells. Indeed, we demonstrated that increasing SEC23A expression, using CRISPR activation or cDNA expression, completely rescues the SEC23B-null erythroid defect.

In this study, we generated a human erythroid cell line that expresses eGFP from the endogenous genomic locus of SEC23A and performed a small molecule screen to identify compounds that increase the SEC23A-eGFP level. The top compound passing all filters was an LSD1 inhibitor. We validated that LSD1 inhibition dramatically increased SEC23A mRNA and protein levels in primary erythroid cells derived from CD34+ human hematopoietic stem and progenitor cells (HSPCs), at doses that do not impair erythroid cell growth or differentiation. We also showed that LSD1 inhibition rescues the erythroid differentiation defect resulting from SEC23B deletion in primary human erythroid cells.

We validated LSD1 as the target of RN1 by showing that genetic downregulation of LSD1 also led to a profound induction of SEC23A mRNA levels in HPSC-derived erythroid cells. Subsequently, we validated our findings in vivo. We first demonstrated that deletion of Lsd1 in mouse erythroid cells results in increased Sec23a expression. Subsequently, we generated CDAII mice, with inducible deletion of both Sec23b alleles and haploinsufficiency for Sec23a. We showed that RN1 treatment of CDAII mice rescues the erythroid defect observed in these mice. Finally, using CUT&RUN, we found that LSD1 occupies a sequence in the SEC23A promoter, which when mutated, results in increased SEC23A expression and rescue of CDAII.

Taken together, these finding suggest that LSD1 occupies the SEC23A promoter, repressing SEC23A transcription and that in the setting of LSD1 inhibition, SEC23A expression is de-repressed, resulting in CDAII rescue. Therefore, using an unbiased screen, we have identified and validated a novel promising therapeutic strategy for CDAII, using a compound that inhibit LSD1 or a genetic approach based on deleting the SEC23A promoter sequence that is occupied by LSD1.

Khoriaty:Fulcrum therapeutics: Research Funding.