Abstract

DYRK3, a member of the dual-specificity tyrosine phosphorylation-regulated kinase family, is expressed at low levels in erythroid progenitors and is implicated as a negative regulator of erythropoiesis. An appropriate animal model of anemia was sought to investigate the potential utility of DYRK3 inhibitors as a therapy for the treatment of anemia. Treatment of mice with sub-lethal irradiation followed by a single dose of the chemotherapeutic, nucleoside analog carboplatin, is well known to induce severe anemia. In the first instance, an analysis of DYRK3 mRNA levels was performed to ascertain whether the anemia so induced results in increased DYRK3 transcription. In this carboplatin/radiation model, mice were exposed to sub-lethal gamma irradiation (500 rads) followed by a single dose of carboplatin (60 mg/kg). This treatment induced an approximately 50% decrease in hemoglobin with concomitant drops in other erythroid parameters (hematocrit and RBC) by days 15–17. The following parameters were examined in five animals per day: peripheral blood counts, marrow cell count, plasma Epo levels, marrow Ter119+/CD71+ expression and marrow DYRK3 mRNA. As expected, the hemoglobin, hematocrit and red blood cells decreased gradually to nadir on day 15. White blood cells decreased to very low levels within 2 days of carboplatin/radiation treatment and remained suppressed for 11 days. Platelets decreased to nadir at day 7, where they remain until day 10. Plasma Epo levels were low and abruptly increased at day 3–4. The absolute number of Ter119+/CD71+ cells immediately dropped from normal levels at day 1 and then increased at day 6 and then fluctuated between a 30- to 60- fold enhancement from day 8 through 21 when the study was complete. DYRK3 mRNA, as measured by quantitative PCR (Taqman), increased approximately 10-fold at day 7 and remained in that range until day 21. The number of erythroid progenitors measured by flow cytometry,Ter119+/CD71+ cells, and the level of DYRK3 mRNA remained elevated until the end of the experiment at day 21, at which point the hemoglobin had recovered to near normal levels. GSK626616, a potent, low molecular weight inhibitor of DYRK3 kinase activity (IC50 = 0.7 nM), was dosed once daily, i.p., for 17 days in this model. At day 15, the GSK626616-treated mice (0.03 mg/kg) demonstrated a statistically significant increase in hemoglobin, hematocrit, red blood cell and platelet counts compared to anemic animals treated with vehicle alone. In contrast to its effects in anemic mice, this compound demonstrated no increases in any blood parameters in normal mice over a similar timeframe and dosage regimen. This expected behavior is hypothesized to be due to the low level of DYRK3 mRNA in normal, non-anemic mice. The characterization of this carboplatin/radiation mouse model demonstrates that as the hemoglobin decreased, plasma Epo increased at day 3–4, followed by the increase in Ter119+/CD71+ cells at day 6. Following this surge in erythropoiesis, an increase in DYRK3 mRNA expression naturally follows. The subsequent improved erythropoiesis in animals treated with a DYRK3 inhibitor in this model, suggests that DYRK3 kinase mRNA levels could have utility as a biomarker in the identification of an anemic patient population that then may be a candidate for treatment with a DYRK3 inhibitor.

Author notes

Disclosure:Employment: The authors were employees of GSK at the time of these studies. Ownership Interests:; Several of the authors have stock options in GSK. Research Funding: This effort was funded by GSK.