Small molecule OTS167 increases erythropoiesis and improves anemia in diamond blackfan anemia models in vitro and In Vivo Free

Diamond Blackfan Anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies, and an increased risk of developing cancer. Approximately 70% of patients with DBA have a mutation in one of 20 different genes that encode ribosomal proteins (RPs), resulting in abnormal protein translation. The current standard of care for patients with DBA includes steroids, chronic red cell transfusions, or stem cell transplantation, but these are all associated with significant side effects including infections, iron overload, and the risk of graft versus host disease. Thus, the development of more effective and less-toxic therapies is needed to treat the anemia seen in patients with DBA. A recent study from our lab demonstrated that Nemo-Like Kinase (NLK) is hyperactivated in erythroid precursors in human and mouse models of DBA as well as in patient samples (Wilkes, et al., 2020). We hypothesize that NLK plays an important role in the pathogenesis of DBA and is a potential target for DBA therapy.

After screening small molecule compounds that inhibit NLK as an off-target from previously approved or clinically advanced drugs, we found that a known MELK (maternal embryonic leucine zipper kinase) inhibitor, OTS167, improved erythroid expansion by 50-60% in human DBA cell models in vitro. Flow cytometry analysis demonstrated that OTS167 delayed erythroid differentiation and increased early-stage erythroid precursors by enhancing entry into S-phase in the cell cycle. In vivo, injections of OTS167 intraperitoneally in a previously reported mouse model of DBA (Rpl11^wt/LoxpCre-Ert2; Morgado-Palacin, et al., 2015) significantly increased the uncommitted hematopoietic stem and progenitor cell population in the bone marrow with delayed erythroid differentiation. These data suggest that OTS167 slows down erythropoiesis through amplification of self-renewing immature cells prior to differentiating into erythroid cells. Rpl11^wt/LoxpCre-Ert2 mice maintained the hemoglobin levels during 9 months of treatment with OTS167 (20 mg/kg) while vehicle control mice decreased the hemoglobin levels with significance differences from the beginning time point (p< 0.05, treated; N=4, control; N=4). Moreover, we examined the effects of OTS167 in our Rpl11^wt/LoxpMx-Cre mouse model of DBA, which demonstrates lower hemoglobin levels and shorter life span compared with Rpl11^wt/LoxpCre-Ert2 mice. In vivo OTS167 treatment (20mg/kg, every 2 weeks) in Rpl11^wt/LoxpMx-Cre miceimproved the hemoglobin levels from 4 g/dL up to 9 g/dL during 9 months of treatment. The treated mice showed longer survival rate compared with the control mice (p<0.01, treated; N=5, control; N=3). Regarding the molecular mechanisms of action of OTS167, bulk RNA-seq data from human erythroid cells with RP-insufficiency treated with OTS167 or vehicle control demonstrated that OTS167 treatment leds to a 10-fold downregulation of NUPR1 (Nuclear protein 1) which is known to be involved in proliferation of hematopoietic stem cells, oxidative stress and ferroptosis. Gene set enrichment analysis showed that several gene sets related to cell proliferation were significantly enriched for upregulated genes with OTS167 treatment. The gene set related to heme metabolism was most significantly enriched for downregulated genes with OTS167 treatment, which further suggests that OTS167 suppresses erythroid differentiation. Moreover, the gene set related to p53 pathway was enriched for downregulated genes with OTS167 treatment in RPS19- or RPL11-knockdown condition. Based on those preliminary data, we hypothesize that suppression of NUPR1 promotes expansion of HSPCs and downregulates p53 pathway in the bone marrow, which leads to an increase in overall erythroid cell production. Given that DBA is associated with a number of mutations both ribosomal and non-ribosomal, a drug that inhibits NLK activity as a common therapeutic target could be used for treatment of DBA patients with different gene mutations. Even if the drug targeting NLK does not completely rescue the anemia in human and mouse models of DBA, the improvement could be enough to sufficiently raise the hemoglobin to prevent the need for chronic red cell transfusions or steroid treatment. We propose that OTS167 and other NLK inhibitors are promising approaches to treat DBA.