ALBERT

Description: 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.

Description: Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis. Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor. Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied. Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.

Description: Through both in vitro and in vivo validation studies, such as antisense RNA and gene knock-out experiments, DYRK3 has been implicated as a negative regulator of erythropoiesis. DYRK3, a member of the dual-specificity tyrosine phosphorylation-regulated kinase family, is expressed at low levels in erythroid progenitors and plays a regulatory role in their cellular proliferation and differentiation. High-throughput screening at GSK resulted in the identification of a novel series of thiazolidinone inhibitors of DYRK3. Lead optimization efforts then led to the discovery of GSK626616 as a potent, orally bioavailable inhibitor of DYRK3. GSK626616, inhibits DYRK3 in vitro with an IC50of 0.7 nM. This low molecular weight compound (401 Da) inhibits other members of the DYRK family, e.g., DYRK1A and DYRK2, with similar potency and with an approximate 20-fold selectivity versus the next most potently inhibited kinase, casein kinase 2. GSK626616AC, the meglumine, monohydrate salt form, has high oral bioavailability in all pre-clinical species studied (e.g. canine AUC = 23.64 ± 2.84 μghr/mL for a 30 mg/kg dose presented as crystalline material packed within a capsule). In cellular assays, GSK626616 enhances the number of CFU-E stimulated by Epo from human marrow, although it has no CFU-E activity on its own, consistent with the target’s functional role as a negative regulator. GSK626616 is specific for the erythroid lineage and does not stimulate CFU-GM colonies, either alone or in the presence of G-CSF or GM-CSF. There is also no direct effect on megakaryocyte colony growth from progenitor cells. 3H-thymidine incorporation in kit+ murine marrow is also stimulated after 3 days by exposure to GSK626616 in the presence of SCF and EPO. Similarly, 3 day treatment with GSK626616 increased the percentage, as well as the absolute number, of Ter119+/CD71+ erythroid progenitors derived from kit+ mouse marrow in the presence of SCF and Epo. GSK626616 (0.0001 to 30 uM) dosed i.p., daily for 14 days had no effect on the blood counts of normal mice, either alone or in the presence of 200 or 600 U/kg Epo. This was not unexpected based upon the very low levels of DYRK3 expression in the bone marrow of normal, non-anemic mice and on results previously reported for knock-out mice. These mice have normal erythropoiesis, but demonstrate an enhanced recovery from erythropoietic stress (Wojchowski, Blood 2005). Anemic mice, treated with a daily dose of GSK626616, i.p., had a statistically significant increase in hemoglobin compared to those dosed with vehicle alone at day 15 after anemia was induced through carboplatin/radiation treatment. Platelet levels were also elevated compared to vehicle control at day 15. These data suggest that treatment with GSK626616, through inhibition of DYRK3 activity, leads to an increase in the proliferation of kit+ cells producing an increased number of Ter119+/CD71+ erythroblasts, thereby accelerating recovery from the anemic insult in a carboplatin/radiation mouse model. It is hypothesized that this mechanism may only function under anemic conditions when DYRK3 is elevated and therefore, that the effects will be self-regulated as hemoglobin and EPO levels approach the normal range.

Description: INTRODUCTION: Recent discovery of thrombopoietin receptor (Tpo-R; c-mpl) agonists and thrombopoietin mimetics has warranted a better understanding of their effects on solid and liquid tumors. These agents bind to different components of the Tpo-R and therefore signal differently. Previously published data on Tpo-R agonists have shown antiproliferative effects on leukemia and lymphoma cells in vitro (Kalota A, Gewirtz AM. American Association for Cancer Research. 2008. Abstract 2392; Erickson-Miller CL, et al. American Association for Cancer Research. 2008. Abstract 5691). Although the expression of Tpo-R is well documented on cells of the megakaryocyte lineage, there is little quantitative information available on the expression of Tpo-R on tumors. METHODS & RESULTS: To better define Tpo-R expression, we first performed quantitative reverse-transcriptase PCR (qRT-PCR) on 378 tumor cell lines available from the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ). Tpo-R was consistently expressed at low levels, with a mean normalized abundance of 1,447 and a mode of 621. Only 3 cell lines expressed Tpo-R mRNA below the limits of reliable quantitation (SAOS-2, SF-539, and WIDR; bone, brain, and colon tumor cell lines, respectively). In comparison, the erythropoietin receptor (Epo-R) was expressed in low-to-moderate levels (mean, 12,587; mode, 7,811) and ErbB2 was expressed at higher levels (mean, 280,190; mode, 40,828), with expression, as expected, much higher among the breast tumor cell lines. IGF-1R was also expressed at higher levels (mean, 78,977; mode, 56,624). Three cell lines had greater than 9,500 normalized abundance: HEL 92.1.7, KG-1 (2 erythroleukemia cell lines), and NCI-H510 (lung cancer cell line). To determine if these trends also occurred in patient tumor samples, microarray data were examined from 118 breast cancer, 29 non-small cell lung cancer (NSCLC), and 151 renal cell carcinoma (RCC) samples collected prior to treatment in GlaxoSmithKline clinical trials. Robust multiarray average (RMA) analysis was used to determine relative mRNA expression levels. Tpo-R mRNA levels were too low for accurate measurement in all breast cancer and RCC samples, but were detectable at low levels in 14 (48%) NSCLC samples (Table 1). In contrast, Epo-R was expressed in 75% breast cancer samples, in all NSCLC samples, and in 87% RCC samples. ErbB2 was expressed in all breast samples, 97% of the NSCLC, and in 81% RCC samples. IGF-1R was expressed in 86% breast cancer samples, all NSCLC samples, and 54% RCC samples. For breast tumors, the levels of Tpo-R message expression rank as follows: Tpo-R