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Blood, 15 December 2006, Vol. 108, No. 13, pp. 4170-4177. Prepublished online as a Blood First Edition Paper on August 29, 2006; DOI 10.1182/blood-2006-05-023093.
NEOPLASIA Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitorsFrom the Albert Einstein College of Medicine, Bronx, NY; the University of Texas Southwestern Medical School, Dallas, TX; the Dallas Veterans Affairs Medical Center, Dallas, TX; Scios Inc, Fremont, CA; the Northwestern University Robert H. Lurie Cancer Center, Chicago, IL; and the Moffit Cancer Center, Tampa, FL.
The myelodysplastic syndromes (MDSs) are collections of heterogeneous hematologic diseases characterized by refractory cytopenias as a result of ineffective hematopoiesis. Development of effective treatments has been impeded by limited insights into any unifying pathogenic pathways. We provide evidence that the p38 MAP kinase is constitutively activated or phosphorylated in MDS bone marrows. Such activation is uniformly observed in varied morphologic subtypes of low-risk MDS and correlates with enhanced apoptosis observed in MDS hematopoietic progenitors. Most importantly, pharmacologic inhibition of p38  by a novel small molecule inhibitor, SCIO-469, decreases apoptosis in MDS CD34+ progenitors and leads to dose-dependant increases in erythroid and myeloid colony formation. Down-regulation of the dominant p38 isoform by siRNA also leads to enhancement of hematopoiesis in MDS bone marrow progenitors in vitro. These data implicate p38 MAPK in the pathobiology of ineffective hematopoiesis in lowrisk MDS and provide a strong rationale for clinical investigation of SCIO-469 in MDS.
The myelodysplastic syndromes (MDSs) comprise a spectrum of stem-cell malignancies characterized by cytologic dysplasia and ineffective hematopoiesis.1-3 Although approximately one third of patients may experience progression to acute leukemia, refractory cytopenias are the principal cause of morbidity and mortality. MDS can be divided into low- and high-risk subtypes using the International Prognostic Scoring System (IPSS), based on features such as the number of hematopoietic deficits, the percentage of marrow blasts, and cytogenetic pattern.4 Approximately two thirds of patients present with lower-risk disease (Low and Int-1 IPSS scores) characterized by increased rates of apoptosis in the progenitor and differentiated cell compartments in the marrow.5-8 High intramedullary apoptosis leads to ineffective hematopoiesis and peripheral cytopenias. Higher grade or more advanced disease categories (Int-2 and High IPSS scores) are associated with a significant risk of leukemia transformation with a corresponding lower apoptotic index and higher percentage of marrow blasts.
Cytokines play important roles in the regulation of normal hematopoiesis, and a balance between the actions of hematopoietic growth factors and myelosuppressive factors is required for optimal production of different hematopoietic-cell lineages. Excess production of inhibitory cytokines contributes in part to ineffective hematopoiesis in MDS. Tumor necrosis factor-
We have previously shown that IFN-
Our previous studies also show that p38 MAPK inhibition may have a therapeutic role in acquired aplastic anemia (AA).30 Overproduction of TNF
Cells lines and reagents
Human CD34+ cells were isolated from bone marrows of healthy subjects and patients, after obtaining their informed consent in accordance with the Declaration of Helsinki, for participation in the study protocol approved by the institutional review boards (IRBs) of UT Southwestern Medical School, the Dallas VA Medical Center, the University of Arizona College of Medicine, and the University of South Florida. A portion of human CD34+ cells were also purchased from Cambrex, MA. Erythroid progenitors at the CFU-E level of differentiation were grown in Iscove modified Dulbecco medium (IMDM) enriched with insulin growth factor (IGF), stem-cell factor (SCF), interleukin 3 (IL-3) and erythropoietin (Epo), all of which were obtained from R&D Systems (Minneapolis, MN) as described in our previous studies.18,19,64,36 MDS1 cell line was derived from a patient with MDS with refractory anemia with excess blasts (RAEB) subtype and was provided by Dr Alan List. Human recombinant TNF Cell lysis and immunoblotting Cells were lysed in phosphorylation lysis buffer as previously described.30 In the experiments in which the effects of SCIO-469 were studied, DMSO (diluent)–treated cells were used as control. Immunoblotting was performed as previously described.30 Immunohistochemistry Paraffin-mounted bone marrow core biopsy sections from patients with MDS and control subjects were obtained after informed consent. Control subjects had anemia from non–MDS-related causes. Slides were deparaffinized and hydrated. Mercury pigments from B5 fixative were removed by iodine-sodium thiosulfate sequences. After rinsing 3 times in PBS, all sections were immersed in 3% hydrogen peroxide for 20 minutes at room temperature to completely block endogenous peroxidases. Antigen retrieval (Citrate Buffer, pH 6.0) was used for all these antibodies. To prevent nonspecific binding with primary antibodies, sections were pretreated with 15% normal goat serum. After 3 washes with PBS, the sections were incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were rabbit phospho-p38 monoclonal antibody diluted at 1:50 (Cell Signaling Technology), mouse monoclonal CD34 Ab-1 (Lab Vision, Fremont, CA), and affinity-purified rabbit-activated caspase 3 antibody (R&D Systems) diluted at 1:400. After 3 washes with PBS, the sections for caspase 3 and CD34 staining were then incubated with biotinylated goat antirabbit (Chemicon International, Temecula, CA) and goat antimouse (Chemicon International) secondary antibodies, respectively, at 1:2000 dilution at room temperature for 30 minutes. Normal rabbit or mouse IgG (Santa Cruz Biotechnology) was used as negative control. All sections were then treated with ABC reagents (Vector, Burlingame, CA) and finally stained with diaminobenzidine (Research Genetic, Carlsbad, CA). Following several more rinses, the sections were counterstained with hematoxylin and subsequently mounted with Permount mounting medium. The quantification of phospho-p38 and cleaved–caspase-3 staining was analyzed by counting the total number of positively stained cells and by measuring the intensity of the positively stained cells in 5 hot fields (hot field is defined as area of high density of phospho-p38 or caspase 3 staining) for each patient sample under x400 magnification aided by Image Pro Plus software (Media Cybernetics, Silver Spring, MD). The results were expressed as mean number of positively stained cells per field and mean intensity per field for each individual patient sample. Flow cytometry
Apoptosis. Primary human bone marrow mononuclear cells were obtained from healthy volunteers after IRB approved informed consent. CD34+ cells were obtained after immunomagnetic selection and were suspended in IMDM in the presence and absence of 20 ng/mL TNF MDS bone marrow mononuclear cells were obtained after IRB informed consent. They were cultured in IMDM with 20% FBS in the presence and absence of 500 nM SCIO-469 for 48 hours. Three MDS samples were evaluated by 3-color flow cytometry after staining them with CD34-APC, Annexin V-FITC, and propidium iodide (PI). Four-color flow cytometry was performed in the next 2 samples using CD34-APC, CD71-PE, Annexin V-FITC, and 7AAD. Apoptosis was evaluated in all samples by determining Annexin V positivity in a gated population of CD34+ cells.
Cell proliferation. Purified primary BM CD34+ progenitors (5 x 105; Stem Cell Technologies, Vancouver, BC) were cultured for 6 days in IMDM with 20% FBS and enriched with TPO, Flt3L, and SCF (all from R&D Systems) with or without 20 ng/mL TNF Immunofluorescence. Bone marrow core biopsy sections from patients with MDS were obtained after IRB informed consent. The biopsies were decalcified by prolonged exposure to EDTA. This was done instead of standard acid decalcification to obtain better signals on immunofluorescence. Paraffin blocks were used to prepare sections. Slides were deparaffinized and hydrated. After rinsing 3 times in PBS, all sections were immersed in 3% hydrogen peroxide for 20 minutes at room temperature to completely block endogenous peroxidases. Antigen retrieval (citrate buffer, pH 6.0) was used for all these antibodies. To prevent nonspecific binding with primary antibodies, sections were pretreated with 15% normal goat serum. Cytonin treatment was used for permeabilization. After 3 washes with PBS, the sections were incubated with rabbit phospho-p38 antibody (Cell Signaling) diluted at 1:50 at 4°C overnight. TACS in situ Apoptosis Detection Kit (R&D Systems; catalog no. TA4627) was used to identify apoptotic cells by detecting DNA fragmentation in bone marrow biopsy. Biotinylated nucleotides are incorporated into the 3-OH ends of the DNA fragments by terminal deoxynucleotidyl transferase (TdT) as per the directions of the kit. The biotinylated nucleotides were detected using a streptavidinfluorescein conjugate. After 3 washes with PBS, the sections were then incubated with goat anti–rabbit IgG Alexa Fluor 568 (catalog no. A11011; Molecular Probes) secondary antibodies at 1:200 dilution at room temperature for 30 minutes. Both secondary antibodies alone and primary antibodies alone were used as negative controls. Following several more rinses, the sections were counterstained with DAPI and subsequently mounted with aqueous mounting medium. Fluorescence was analyzed by Olympus Fluorescent microscope under x 60 magnification. siRNA transfections
Small interfering RNA duplexes (siRNAs) against p38 Hematopoietic progenitor-cell assays Hematopoietic progenitor colony formation was determined by clonogenic assays in methylcellulose, as in our previous studies.29,30 All participants in the study signed informed consent, approved by IRB of UT Southwestern and Dallas VA Medical Center. Granulocyte and macrophage colony-forming units (CFU-GMs) and erythroid burst-forming units (BFU-Es) from bone marrow samples were scored on day 14 of culture.
p38 MAPK is constitutively activated in low-grade myelodysplastic syndromes Bone marrows of patients with MDS were assessed for the activation or phosphorylation state of p38 MAPK by immunohistochemistry. Patients were divided into lower (Low and Int-1 IPSS) and higher (Int-2 and High IPSS scores) grade subtypes of MDS (Table 1). MDS bone marrow samples were compared with age-matched controls with non-MDS causes of cytopenias (1 with iron deficiency anemia, 1 with vitamin B12 deficiency, 2 with chemotherapy-related anemia, 2 with chronic renal insufficiency, and 1 with anemia associated with multiple chronic medical problems with high ferritin in the absence of any dysplasia). Notable activation of p38 was seen in bone marrow cells of all patients with low-grade MDS (Figure 1A), with a greater number of phospho-p38–positive staining cells (Figure 1B) and significantly higher intensity of staining (Figure 1C) when compared with controls. Activation of p38 was seen in all subtypes of low-grade MDS examined (1 with refractory anemia [RA], 1 with refractory anemia with ringed sideroblasts [RARS], and 7 with refractory cytopenias with multilineage dysplasia [RCMD]). The level of activation was significantly decreased in high-grade cases and was comparable to controls.
Because p38 MAPK is ubiquitously expressed, we also investigated the phenotypes of bone marrow cells that are expressing the activated kinase. Histologic examination revealed that p38 was activated in hematopoietic progenitors of all lineages, including erythroid and myeloid progenitors and even megakaryocytes. Staining with anti-CD3 antibody revealed very few lymphocytes, most of which appeared to be phospho-p38 negative (data not shown). Immunohistochemical staining with an antibody against total p38 MAPK was also performed to determine any changes in p38 MAPK expression in MDS bone marrows when compared with controls. No significant differences in either staining intensity or in the number of p38-positive cells were seen (Figure 1D; Table 2), suggesting that p38 MAPK is overactivated but not overexpressed in low-grade MDS.
p38 MAPK activation correlates with enhanced apoptosis in MDS bone marrows Increased hematopoietic progenitor-cell apoptosis plays a role in the pathobiology of ineffective hematopoiesis in MDS. Because we observed high levels of p38 activation in low-grade MDS, we sought to determine its relation to the degrees of apoptosis seen in this disease. Immunostaining of MDS bone marrows with antibodies against cleaved caspase 3 and phosphorylated-p38 was performed in serial sections of bone biopsies. We observed that cases of low-grade MDS showed a high percentage of cells positive for cleaved caspase 3 (apoptotic cells) consistent with previous studies6 (Figure 2A). These apoptotic cells show strong activation of p38 MAPK in serial sections (Figure 2B-C). There was a positive correlation between p38 activation and apoptosis (Figure 2B), and it appears that p38 MAPK is activated strongly in progenitors that are undergoing apoptosis (Figure 2C). In contrast, the number of apoptotic cells was decreased in high-grade cases and was comparable with controls. Further determination of this correlation was done by double immunofluorescence staining of MDS bone marrow sections after TdT in situ labeling of apoptotic cells (TUNEL assay) in combination with antibodies against phospho-p38. Merged immunofluorescence showed that cells undergoing apoptosis also displayed high levels of activated p38 (Figure 2D). SCIO-469 reverses TNF-induced apoptosis of normal hematopoietic progenitors
TNF
p38 MAPK inhibition decreases MDS CD34+ progenitor apoptosis
After showing a correlation between MDS progenitor-cell apoptosis and p38 activation, we sought to directly evaluate the functional role of p38 in this phenomenon. Bone marrow mononuclear cells from 5 patients with early or low-grade MDS were cultured in the presence and absence of the selective p38 Down-regulation of p38 MAPK with siRNA promotes hematopoiesis in MDS
To further determine the biologic significance of p38 activation in MDS, siRNAs against p38
Treatment with pharmacologic inhibitors of p38 MAPK leads to increased colony formation from MDS hematopoietic progenitors Having established the potential of p38 inhibition as a therapeutic strategy in MDS, we wanted to determine the efficacy of various small molecule inhibitors of p38 MAPK in this disease. Bone marrow aspirates were collected from 19 patients with a variety of MDS subtypes. Most of the patients had low-grade MDS and did not have increased blast counts (Table 1). Consistent with results seen with siRNAs, treatment with the p38 inhibitor SB203580 (Figure 6) resulted in a striking increase in erythroid (BFU-E) and myeloid (CFU-granulocytic monocytic) colony numbers in all the cases (P < .001). However as expected,35 there were no increases on exposure to MEK inhibitor PD98059 or negative control inactive structural analog SB202474 (Figure 6) (P = .3).
Most interestingly, similar results were observed on treatment with 2 new p38 MAPK inhibitors SCIO-469 and SD-282. These compounds are potent, selective inhibitors of p38 and show at least a 10-fold higher affinity for p38 as compared with p38 .37-40 SCIO-469 is an oral compound presently being used in phase 2 clinical trials for rheumatoid arthritis and other diseases. On exposure to these 2 inhibitors, MDS progenitors showed increases in both erythroid and myeloid colony numbers in vitro (Figure 6; P < .001). Consistent with previous studies, untreated MDS CD34+ cells exhibited poor colony-forming ability in vitro, showing poor hematopoietic potential of these cells. Strikingly, treatment with SCIO-469 at low doses (20-100 nM) was able to increase myeloid and erythroid colony numbers 2- to 3-fold in MDS progenitors, pointing to therapeutic potential of SCIO-469 in early or low-grade MDS.
Myelodysplastic syndromes are groups of clonal hematopoietic disorders characterized by refractory cytopenias with limited treatment options.1 These disorders are common in the elderly and impose a significant burden on health care resources. A stumbling block in the discovery of treatments of this disease has been the heterogeneity observed in subsets of this disease and the lack of a unifying pathophysiologic target. Our work has identified a kinase, p38 MAPK, that is activated in bone marrow cells of a large number of patients with MDS, even in those with different chromosomal alterations. Inhibition of this pathway leads to in vitro stimulation of hematopoiesis, suggesting that p38 MAPK pathway is a functionally important inhibitory pathway in MDS. p38 MAPK is not found to be activated in normal bone marrows, and consequently p38 inhibition does not have significant stimulatory effects on hematopoietic progenitors derived from normal bone marrows. p38 MAPK was originally discovered as a stress-signaling kinase, and work has implicated it as an important mediator of apoptosis in neuronal, cardiac, immune, and other cells.41-44 Studies have shown that activation of the p38 MAPK pathway can oppose the proliferative effects of the Ras-Erk MAPK pathway and can lead to growth arrest and dormancy in tumors.45 Consistent with its cytostatic properties in nonhematopoietic cells, our work has shown an important role for p38 in cytokine-mediated inhibition of human hematopoiesis.29,30 In fact, a recent report implicates p38 in hematopoietic stem death induced by reactive oxygen species and suggests that p38 inhibition may be beneficial in prolonging stem-cell survival in ATM knockout mice.46 This report validates our findings of the important role of this MAP kinase in hematopoiesis, and it appears p38 activation is a downstream effector of many different cell-death pathways in hematopoietic stem cells.
Our data establish a correlation between marrow p38 activation and apoptosis that is characteristic of early-stage MDS. The lack of activation of p38 in nutritional deficiency anemias, chemotherapy-induced anemias, and other non-MDS cytopenias points to the relative specificity of p38 activation in low-risk/Int-1 MDS. At this stage of the disease, both normal and cytogenetically abnormal hematopoietic clones are found to exist in the marrow.47 It has been shown that abnormal MDS progenitor clones are resistant to apoptosis and have higher levels of antiapoptotic proteins Bcl-2 and Bax.6 Thus, it is possible that p38 inhibition may prevent cell death in the susceptible normal progenitors, thereby rescuing normal hematopoiesis in the early or low-grade stage of this disease. Because most MDS cases are low risk, and the morbidity experienced is due to low blood counts, hematopoietic recovery is a major therapeutic goal in treating these patients. The high numbers of apoptotic progenitors seen by us are consistent with similar high percentages seen in other reports in low-grade MDS.5 With disease progression toward high-risk stages, normal progenitors gradually undergo apoptosis, resulting in a bone marrow comprised mainly of the resistant abnormal clones. Thus, examination of the marrow at late stages reveals a low apoptotic index with higher percentages of myeloblasts. The low level of p38 activation correlates with the reduced apoptosis seen at this stage of the disease. Instead, constitutive activation of the antiapoptotic NF  B particularly in MDS progenitors was observed in a majority of high-risk MDS cases.48
MDS is highlighted by a stromal pathology of still unknown causes, which contributes to the pervasive presence of proinflammatory cytokines in the bone marrow. Dysregulation of various cytokines has been implicated in the pathogenesis of MDS.9,11,15-17 TNF
SCIO-469 and SD-282 are novel small molecule inhibitors that occupy the ATP-binding site on the p38 kinase.37-39 Both of these are highly selective for p38
Contribution: T.A.N., M.M., P.P., and L.Z. designed the experiments and performed the research; J.Y.M., E.H., A.N.N., I.K., and L.S.H. performed the research and contributed the p38 inhibitors; M.E., L.S., and A.A.L. performed the research and contributed thepatient samples; L.C.P. contributed to the experimental design; Y.X., R.C., and S.P. contributed the patient samples; A.V. designed and performed the experiments and wrote the manuscript. Conflict-of-interest disclosure: several of the authors (T.A.N., J.Y.M., E.H., I.K., A.N.N., and L.S.H.) are employed by a company or a competitor of a company (SCIOS, Inc) whose potential product was studied in the present work. The remaining authors declare no competing financial interests. T.A.N., M.M., and M.E. contributed equally to this study.
This work was supported by the National Institutes of Health (1R01HL082946-01), by a grant from the Harris Methodist Foundation, by an VISN-17 award, and by the J.P. McCarthy Grant from the Community Foundation for Southwestern Michigan (A.V.).
Submitted May 22, 2006; accepted July 25, 2006.
Prepublished online as Blood First Edition Paper, August 29, 2006; DOI 10.1182/blood-2006-05-023093.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
Correspondence: Amit Verma, Chanin 601, Albert Einstein Cancer Center, 1300 Morris Park Ave, Bronx, NY 10461; e-mail: averma{at}aecom.yu.edu.
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Abstract
Introduction
and -
