scholarly journals The Serine Threonine Kinase Inhibitor Ots-167 Improves Erythropoiesis through Suppression of Nlk Activity in Diamond Blackfan Anemia Models

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2179-2179
Author(s):  
Aya Shibuya ◽  
Mark C Wilkes ◽  
Manuel Serrano ◽  
Johan Flygare ◽  
Bert Glader ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Map Kinases ◽  
Kinase Inhibitor ◽  
Cross Reactivity ◽  
Red Cell ◽  
Erythroid Progenitors ◽  
Serine Threonine Kinase ◽  
Hematopoietic Stem ◽  
Threonine Kinase ◽  
Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is associated with a hypoproliferative anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life with the majority of patients carrying mutations in one of at least 17 ribosomal proteins, with RPS19 being the most common. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions, chronic effects from long term corticosteroid use, or complications from stem cell transplantation. The serine threonine kinase Nemo-like Kinase (NLK) is an atypical member of the MAP kinase family of enzymes and has been shown to be chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as in erythroid progenitors from DBA patients. In RPS19-insufficient human hematopoietic stem and progenitor cells, genetic silencing of NLK by shRNA increased erythroid expansion by 220.3% (SD = 6.6%), indicating that aberrant NLK activation may contribute to the pathogenesis of the disease and is a potential target for DBA therapy. A number of clinically approved or advanced compounds have been developed to inhibit MAP kinases with various degrees of cross reactivity among its family members. We therefore screened a number of compounds that inhibit NLK as an off-target and found that these NLK inhibitors improved erythroid expansion in DBA models. Of these inhibitors, OTS-167 performed optimally, improving erythropoiesis by 2-fold at 300nM, with an EC50 of 146nM. Previous studies of OTS-167 in xenograft models of neuroblastoma for one month did not result in neutropenia, suggesting very little to no toxicity to myeloid cells. The goal in treating DBA patients with NLK inhibitors is to sufficiently raise the hemoglobin to prevent the need for chronic red cell transfusions or treatment with steroids. Our results suggest that pharmacologic inhibition of NLK is a potential approach to treat patients with DBA. We are currently investigating other NLK inhibitors in preclinical models for future clinical application. Disclosures Glader: Agios: Consultancy.

scholarly journals Pharmacological Inhibition of Nlk (Nemo-like Kinase) Rescues Erythropoietic Defects in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 754-754
Author(s):  
Mark C Wilkes ◽  
Jun Chen ◽  
Kavitha Siva ◽  
Gianluca Veretti ◽  
Daniel P. Dever ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Ribosomal Proteins ◽  
Kinase Inhibitor ◽  
Combined Treatment ◽  
Human Model ◽  
Translational Efficiency ◽  
Erythroid Progenitors ◽  
Advisory Committees ◽  
Diamond Blackfan Anemia ◽  
Mtor Activity

Abstract Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. The disease typically presents within the first year of life. Approximately 70% of DBA patients possess a mutations in one of at least 12 ribosomal proteins, with RPS19 and RPL11 being the most prevalent, accounting for over 25% and 5% of cases respectively. Current therapies for DBA have undesirable side effects, including iron overload from repeated transfusions or infections from immunosuppressive drugs and stem cell transplantation. Nemo-like Kinase (NLK) is chronically hyper-activated in RPS19- and RPL11-haploinsufficient murine and human models of DBA, as well as erythroid progenitors from DBA patients. In an RPS19-insufficient human model, genetic silencing of NLK (shRNA) increased erythroid expansion by 2.2 fold, indicating aberrant NLK activation contributes to the pathogenesis of the disease. In an independent, high-throughput kinase inhibitor screen examining progenitor expansion in RPS19-insufficient Kit+ murine cells, a number of compounds were identified that increased progenitor expansion. SB431542 and SD208 are recognized TGFβ inhibitor compounds, but were the only TGFβ inhibitors of a panel of 11 that increased progenitor expansion. Both active compounds robustly inhibited NLK activity in vitro and in vivo while the remaining 9 inhibitors had no significant impact on NLK. Both compounds increased erythroid expansion in murine (3.1 and 5.4 fold) and human (3.2 and 6.3 fold) models of DBA with no effect on wild type erythropoiesis (EC50 5 µM and 0.7 µM). No further increase in erythroid expansion was observed when NLK expression was silenced by shRNA. Virtually identical results were observed in CD34+ progenitors from 3 DBA patient bone marrow aspirates with 2.3, 1.9 and 2.1 fold increases in CD235+ erythroblast generation compared to untreated. NLK hyperactivation was limited to differentiating committed erythroid progenitors and was not detected in megakaryocytic, other myeloid progenitors or lymphoblastoid cells lines from DBA patients. During differentiation, non-erythroid lineages upregulate miR181, which results in NLK transcript degradation and loss of NLK expression. The absence of NLK in non-erythroid progenitors prevents NLK activation during ribosomal insufficiency. CRISPR/Cas9 mutation of the miR181 binding site in the NLK 3'UTR in RPS19-insufficient CD34+ HSPCs prevented NLK downregulation, increased NLK activity, and sensitized megakaryocyte and other myeloid lineages (80.5% and 76% reduction relative to controls). This is comparable to the erythroid expansion defect in RPS19-insufficiency (80.7% reduction). In differentiating erythroid progenitors, RPS19 insufficiency increased phosphorylation of the mTORC1 component Raptor (5.3 fold), reducing mTOR activity by 82%. This was restored to basal levels upon pharmacological or genetic inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTORC1 activity with leucine has been proposed to increase translational efficiency in DBA patients. Probably due to NLK phosphorylation of raptor, DBA patients did not respond as anticipated. While leucine treatment did increase mTOR activity in both control (100% to 188%) and RPS19-insufficiency (27 % to 42% of control), the combined treatment of leucine with NLK inhibition resulted in increased mTOR activity to 142% of control and significantly improved erythroid expansion. Identification of aberrantly activated enzymes, such as NLK, that are specifically expressed in erythroid progenitors, offer therapeutic promise as potential druggable targets in the clinical management of DBA that can be used in combination with existing therapies. Disclosures Glader: Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

scholarly journals ROLE OF SERIN/ THREONINE KINASE INHIBITOR IN THERAPY NON-ALCOHOLIC STEATOHEPATITIS AND ALCOHOLIC HEPATITIS

2019 ◽  
Vol 6 (3) ◽  
pp. 101-109
Author(s):  
Novriantika Lestari
Keyword(s):  
Signal Transduction ◽  
Liver Fibrosis ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Binding Activity ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Receptor Binding Activity ◽  
Marked Accumulation

Liver fibrosis is a reversible response to a wound healing with marked accumulation of extracellular matrix which caused by injury to the liver. Liver fibrosis can be caused by various factors including alcohol and non-alcohol steatohepatitis. The process of fibrosis serves to localize the inflammation during chronic exposure. The hepatic stem cell (HSC) has a key role in the pathogenesis of liver fibrosis. The HSC activation is characterized by increased profibrogenic mediators including members of the TGF-? superfamily. In order to enable signal transduction, the mediator needs to bind to its receptors. The serine/ threonine kinase receptor is a receptor that binds to the TGF-? superfamily ligand, including TGF-?, BMP, activin and other mediators. The ligand receptor-binding activity will stimulate signal transduction that will translocate into the nucleus and phosphorylate various transcription factors that play a role in cell proliferation, differentiation, or apoptosis. There is currently no standard therapy for liver fibrosis. Based on the central role of the serine/ threonine kinase receptor in the pathogenesis of liver fibrosis, it is thought that the use of serine/ threonine kinase inhibitors is a promising therapy.

A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole

2021 ◽  
Author(s):  
Kazuya Matsuo ◽  
Sampreeth Thayyil ◽  
Mitsuyasu Kawaguchi ◽  
Hidehiko Nakagawa ◽  
Nobuyuki Tamaoki
Keyword(s):  
Protein Kinase ◽  
Visible Light ◽  
Kinase Inhibitor ◽  
Coiled Coil ◽  
Rho Kinase ◽  
Rock Inhibitor ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Rho Kinase Inhibitor

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase, whose inhibitors are useful for the regulation of actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole...

scholarly journals Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinase Exerts Synergistic Effect against Chronic Myeloid Leukemia Cells

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1544 ◽  
Author(s):  
Sylwia Flis ◽  
Ewelina Bratek ◽  
Tomasz Chojnacki ◽  
Marlena Piskorek ◽  
Tomasz Skorski
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Synergistic Effect ◽  
Tyrosine Kinase ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cells ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Simultaneous Inhibition

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely “cure” the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1-transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells.

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2430-2430
Author(s):  
Zhong-Fa Yang ◽  
Wang Junling ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Bone Marrow Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Hematopoietic Stem ◽  
Threonine Kinase ◽  
Marrow Cells

Abstract Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

Deleterious Consequences of Diamond Blackfan Anemia on Reproductive Health and Pregnancy Outcomes: A Report from the Diamond Blackfan Anemia Registry (DBAR)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4399-4399 ◽  
Author(s):  
Andrea M Tufano ◽  
Diana S Osorio ◽  
Eva Atsidaftos ◽  
Cristina P. Sison ◽  
Jeffrey M Lipton ◽  
...  
Keyword(s):  
Pregnancy Complications ◽  
Placental Abruption ◽  
Average Risk ◽  
Red Cell ◽  
Transfusion Therapy ◽  
Hematopoietic Stem ◽  
Corticosteroid Administration ◽  
Live Births ◽  
Diamond Blackfan Anemia ◽  
Steroid Dependent

Abstract Background: Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by red cell aplasia, birth anomalies, and a predisposition to cancer. Due to a primarily autosomal dominant mode of inheritance, DBA affects males and females at a ratio of 1:1. Treatment consists of corticosteroid administration and chronic red cell transfusion therapy and in some cases hematopoietic stem cell transplantation. Current clinical observations suggest that women with DBA may experience delayed puberty, irregular menstrual cycles, and decreased fertility. Women who do become pregnant may sustain a higher than average risk of pregnancy complications, including miscarriage, placental abruption, and stillbirth, and others of vascular-placental origin (Faivre et al. Haematologica, 2006). Anecdotal reports suggest that DBA women experience changes to treatment response and remission status during periods characterized by hormonal fluctuations; namely, puberty and pregnancy. The etiology of these complications is unclear. Methods: Questionnaires were sent to females greater than 15 years of age (n=224) participating in the Diamond Blackfan Anemia Registry of North America (DBAR), a comprehensive database of 720 patients who are enrolled after obtaining informed consent. The questionnaires ask about menstrual and gynecologic health and pregnancies, focusing on complications, treatment requirements, and outcomes. We grouped patients by steroid and/or transfusion dependence status and/or remission before age 12 to analyze the effects of treatment on menarche. The patients were regrouped based on the majority of their treatment to evaluate for gynecologic health and pregnancy complications. The Fisher’s exact test was used to examine associations between variables. When compared to the general population, normal values were obtained from a variety of sources including the American Academy of Pediatrics, the National Institutes of Aging and the March of Dimes. Results and Conclusions: We reviewed results from 84 women aged 15 to 62 years (median age 28.6 years). Menarche was delayed in both steroid dependent and transfusion dependent girls, with 39.5% and 77.8%, respectively experiencing menarche at age 15 or later (vs 2% normal). Transfusion dependent girls were significantly more likely to have delayed menarche compared to the steroid dependent and remission groups (p<.005). Additionally, transfusion dependent females were significantly more likely to experience premature ovarian failure, compared to those who were either steroid dependent or in remission (32.1% vs 8.6%; p<.025). Of those who report entering menopause, 75% stopped by age 40 or younger (vs average, 51 years). Women who are transfusion dependent were more likely to report having taken medication to regulate their menstrual cycle, as compared to steroid dependent and remission groups (48.1% vs 22.9%; p<.06). Of the 50 pregnancies reported in 23 women, 30 resulted in live births (60%). Of the total live births, 43.3% were preterm (vs 11.5% normal). Interestingly, steroid dependent women as well as those in remission had the highest percentage of preterm deliveries at 66.7% and 63.6%, respectively. 26% of the pregnancies resulted in miscarriage, and 4.3% resulted in stillbirth (vs normal, 15% and 0.6%, respectively) and 4.3% of pregnancies were complicated by placental abruption (vs normal, <1%). During pregnancy, 52.2% of women reported requiring transfusions; specifically, 100% of the women who were steroid dependent at conception required transfusions during pregnancy. 7.4% of women in the study had a hysterectomy, half of whom were under age 40. We also noted an increased incidence of endocrinopathies: transfusion dependent women reported a significantly higher prevalence of thyroid disorders compared to the steroid dependent group (42.9% vs 11.1%; p<.03). Additionally, there was a higher prevalence of diabetes in the transfusion group as compared to the steroid dependent group (14.3% vs 0%, p=NS). These findings suggest an important role of iron overload in menstrual abnormalities, reproductive issues and endocrinopathies in DBA women. Pregnancy complications are in excess of those seen in the normal population. Future analyses will determine whether there is a correlation between menstrual abnormalities and/or pregnancy complications and specific DBA genotypes. Disclosures No relevant conflicts of interest to declare.

scholarly journals SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2189-2189
Author(s):  
Mark C Wilkes ◽  
Aya Shibuya ◽  
Vanessa M Scanlon ◽  
Hee-Don Chae ◽  
Anupama Narla ◽  
...  
Keyword(s):  
Cord Blood ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Culture Media ◽  
Hsp70 Expression ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacological Inhibition of Nemo-like Kinase Rescues mTOR-Mediated Translation and Primes Progenitors for Leucine-Stimulated Erythroid Expansion in Pre-Clinical Models of Diamond Blackfan Anemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 455-455
Author(s):  
Mark C Wilkes ◽  
Jacqueline D Mercado ◽  
Mallika Saxena ◽  
Jun Chen ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fold Increase ◽  
Healthy Controls ◽  
In Vitro Activity ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Mtor Activity

Diamond Blackfan Anemia (DBA) is associated with anemia, congenital abnormalities, and cancer. Current therapies for DBA have undesirable side effects, including iron overload from repeated red cell transfusions or infections from immunosuppressive drugs and hematopoietic stem cell transplantation. Human hematopoietic stem and progenitor cells (HSPCs) from cord blood were transduced with lentiviral shRNA against a number of ribosomal genes associated with DBA, reducing the specific ribosomal protein expression by approximately 50%. During differentiation, these cells demonstrated a DBA-like phenotype with significantly reduced differentiation of erythroid progenitors (over 80%), yet only modest (15-30%) reduction of other hematopoietic lineages. NLK was immunopurifed from differentiating HSPCs and activity was assessed by the extent of in vitro phosphorylation of 3 known NLK substrates NLK, c-Myb and Raptor. As NLK activation requires phosphorylation at Thr298, we also showed that in vitro activity correlated with intracellular NLK phosphorylation by Western blot analysis. Nemo-like Kinase (NLK) was hyperactivated in the erythroid progenitors (but not other lineages), irrespective of the type of ribosomal gene insufficiency. We extended these studies using other sources of HSPCs (fetal liver, whole blood and bone marrow), along with RPS19- and RPL11-insufficient mouse models of the disease, as well as DBA patient samples. NLK was hyperactivated in erythroid progenitors from mice (5.3- and 7.2-fold increase in Raptor phosphorylation in RPS19- and RPL-11 insufficiency respectively) and from humans (7.3- and 9.0-fold in RPS19- and RPL11-insufficiency respectively) as well as HSPCs from three DBA patient (4.8-, 4.1- and 4.2-fold increase above controls). In RPS19-insufficient human HSPCs, genetic silencing of NLK increased erythroid expansion by 2.2-fold (p=0.0065), indicating that aberrant NLK activation contributes to disease pathogenesis. Furthermore, a high-throughput inhibitor screen identified a compound that inhibits NLK (IC50:440nM) and increases erythroid expansion in murine (5.4-fold) and human (6.3-fold) models of DBA without effects on normal erythropoiesis (EC50: 0.7 µM). Identical results were observed in bone marrow CD34+ progenitors from three DBA patients with a 2.3 (p=0.0009), 1.9 (p=0.0007) and 2.1-fold (p=0.0001) increase in CD235+ erythroid progenitor population following NLK inhibition. In erythroid progenitors, RPS19-insufficiency increased phosphorylation of the mTORC1 component Raptor, reducing mTOR in vitro activity by 82%. This was restored close to basal levels (93.8% of healthy control) upon inhibition of NLK. To compensate for a reduction in ribosomes, stimulating mTOR activity with leucine has been proposed to increase translational efficiency in DBA patients. In early clinical trials, not all DBA patients have responded to leucine therapy. We hypothesize that one of the reasons might be due to NLK phosphorylation of Raptor. While leucine treatment increased mTOR activity in both RPS19-insufficient and control cells (164% of healthy controls: p=0.007 and 24% to 42% of healthy controls: p=0.0064), combining leucine with NLK inhibition increased mTOR activity in RPS19-insufficiency from 24% to 142% of control (p=0.0012). This translated to improvements in erythroid expansion of RPS19-insufficient HSPCs from 8.4% to 16.3% with leucine treatment alone, 28.4% with NLK inhibition alone, but 68.6% when leucine and NLK inhibition were combined. This 8.2-fold improvement in erythroid progenitor production indicates that identification of aberrantly activated enzymes, such as NLK, offer therapeutic promise used alone, or in combination with existing therapies, as druggable targets in the clinical management of DBA. Disclosures Glader: Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia

2020 ◽  
Vol 12 (566) ◽  
pp. eabb5831 ◽  
Author(s):  
Alison M. Taylor ◽  
Elizabeth R. Macari ◽  
Iris T. Chan ◽  
Megan C. Blair ◽  
Sergei Doulatov ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Ribosomal Proteins ◽  
P53 Protein ◽  
Erythroid Differentiation ◽  
Normal Activity ◽  
Small Subunit ◽  
Hematopoietic Stem ◽  
Diamond Blackfan Anemia ◽  
Calmodulin Inhibitors

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 (rps29) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration–approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34+ hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34+ cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.

Sign in / Sign up

Export Citation Format

Share Document