Programmed necroptosis is upregulated in low-grade myelodysplastic syndromes and may play a role in the pathogenesis

Abstract

•Programmed necroptosis is increased in MDS as suggested by increased RIPK1 in erythroid precursors.•Programmed necroptosis contributes to ineffective hematopoiesis in MDS.•Inhibiting RIPK1 improves erythroid colony-forming ability of human MDS. Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow (BM). Excessive hematopoietic programmed cell death (PCD) and inflammation have been observed in the bone marrow of patients with MDS, and are thought to play a significant role in the pathogenesis of the disease. Necroptosis is a major pathway of PCD that incites inflammation; however, the role of necroptosis in human MDS has not been extensively investigated. To assess PCD status in newly diagnosed MDS, we performed immunofluorescence staining with computational image analysis of formalin-fixed, paraffin-embedded BM core biopsies using cleaved caspase-3 (apoptosis marker) and necroptosis markers (receptor-interacting serine/threonine-protein kinase 1 [RIPK1], phospho-mixed lineage kinase domain-like protein [pMLKL]). Patients with MDS, but not controls without MDS or patients with de novo acute myeloid leukemia, had significantly increased expression of RIPK1 and pMLKL but not cleaved caspase-3, which was most evident in morphologically low-grade MDS (<5% BM blasts) and in MDS with low International Prognostic Scoring System risk score. RIPK1 expression highly correlated with the distribution of CD71+ erythroid precursors but not with CD34+ blast cells. We found that necroptosis is upregulated in early/low-grade MDS relative to control participants, warranting further study to define the role of necroptosis in the pathogenesis of MDS and as a potential biomarker for the diagnosis of low-grade MDS. Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow (BM). Excessive hematopoietic programmed cell death (PCD) and inflammation have been observed in the bone marrow of patients with MDS, and are thought to play a significant role in the pathogenesis of the disease. Necroptosis is a major pathway of PCD that incites inflammation; however, the role of necroptosis in human MDS has not been extensively investigated. To assess PCD status in newly diagnosed MDS, we performed immunofluorescence staining with computational image analysis of formalin-fixed, paraffin-embedded BM core biopsies using cleaved caspase-3 (apoptosis marker) and necroptosis markers (receptor-interacting serine/threonine-protein kinase 1 [RIPK1], phospho-mixed lineage kinase domain-like protein [pMLKL]). Patients with MDS, but not controls without MDS or patients with de novo acute myeloid leukemia, had significantly increased expression of RIPK1 and pMLKL but not cleaved caspase-3, which was most evident in morphologically low-grade MDS (<5% BM blasts) and in MDS with low International Prognostic Scoring System risk score. RIPK1 expression highly correlated with the distribution of CD71+ erythroid precursors but not with CD34+ blast cells. We found that necroptosis is upregulated in early/low-grade MDS relative to control participants, warranting further study to define the role of necroptosis in the pathogenesis of MDS and as a potential biomarker for the diagnosis of low-grade MDS. Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis manifesting as low blood counts and normo- or hypercellular bone marrow (BM) [1Cazzola M Malcovati L. Myelodysplastic syndromes—Coping with ineffective hematopoiesis.N Engl J Med. 2005; 352: 536-538Crossref PubMed Scopus (300) Google Scholar, 2Malcovati L Nimer SD. Myelodysplastic syndromes: Diagnosis and staging.Cancer Control. 2008; 15: 4-13Crossref PubMed Scopus (46) Google Scholar, 3Raza A Galili N. The genetic basis of phenotypic heterogeneity in myelodysplastic syndromes.Nat Rev Cancer. 2012; 12: 849-859Crossref PubMed Scopus (105) Google Scholar]. Two prominent features of MDS that have been implicated in the pathogenesis of the disease are increased bone marrow cell death associated with increased inflammatory cytokines such as tumor necrosis factor α (TNF-α) [4Kitagawa M Saito I Kuwata T et al.Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes.Leukemia. 1997; 11: 2049-2054Crossref PubMed Scopus (216) Google Scholar, 5Tsimberidou AM Estey E Wen S et al.The prognostic significance of cytokine levels in newly diagnosed acute myeloid leukemia and high-risk myelodysplastic syndromes.Cancer. 2008; 113: 1605-1613Crossref PubMed Scopus (96) Google Scholar, 6Tehranchi R Invernizzi R Grandien A et al.Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes.Blood. 2005; 106: 247-253Crossref PubMed Scopus (87) Google Scholar]. Early MDS studies described increased hematopoietic cell death, especially in low-risk MDS, (refractory anemia and refractory anemia with ringed sideroblasts) [6Tehranchi R Invernizzi R Grandien A et al.Aberrant mitochondrial iron distribution and maturation arrest characterize early erythroid precursors in low-risk myelodysplastic syndromes.Blood. 2005; 106: 247-253Crossref PubMed Scopus (87) Google Scholar] that was proposed to play a significant role in disease pathogenesis [7Steensma DP Tefferi A. The myelodysplastic syndrome(s): a perspective and review highlighting current controversies.Leuk Res. 2003; 27: 95-120Crossref PubMed Scopus (132) Google Scholar]. Recently, inflammatory cytokines such as TNF-α, which have also been implicated as regulatory cues, have been reported to promote the proliferation and PCD of hematopoietic progenitors in MDS, further implicating an inflammatory process as a pathogenic driver of MDS [4Kitagawa M Saito I Kuwata T et al.Overexpression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma by bone marrow cells from patients with myelodysplastic syndromes.Leukemia. 1997; 11: 2049-2054Crossref PubMed Scopus (216) Google Scholar,5Tsimberidou AM Estey E Wen S et al.The prognostic significance of cytokine levels in newly diagnosed acute myeloid leukemia and high-risk myelodysplastic syndromes.Cancer. 2008; 113: 1605-1613Crossref PubMed Scopus (96) Google Scholar,8Hormaechea-Agulla D Matatall KA Le DT et al.Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNgamma signaling.Cell Stem Cell. 2021; 28: 1428-1442Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 9Muto T Walker CS Choi K et al.Adaptive response to inflammation contributes to sustained myelopoiesis and confers a competitive advantage in myelodysplastic syndrome HSCs.Nat Immunol. 2020; 21: 535-545Crossref PubMed Scopus (37) Google Scholar, 10Cai Z Kotzin JJ Ramdas B et al.Inhibition of inflammatory signaling in Tet2 mutant preleukemic cells mitigates stress-induced abnormalities and clonal hematopoiesis.Cell Stem Cell. 2018; 23: 833-849.e5Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar]. Increased programmed cell death (PCD) in early MDS BM studies was interpreted as apoptotic death, based on increased in situ DNA end labeling (TUNEL staining) [11Raza A Gezer S Mundle S et al.Apoptosis in bone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes.Blood. 1995; 86: 268-276Crossref PubMed Google Scholar, 12Shetty V Mundle S Alvi S et al.Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes.Leuk Res. 1996; 20: 891-900Crossref PubMed Scopus (161) Google Scholar, 13Kerbauy DB Deeg HJ. Apoptosis and antiapoptotic mechanisms in the progression of myelodysplastic syndrome.Exp Hematol. 2007; 35: 1739-1746Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar]. Apoptosis is driven by proteases called caspases that break down the cytoskeleton to cause collapse of the cell in a mostly immune silent process [14Bogdanovic AD Jankovic GM Colovic MD Trpinac DP Bumbasirevic VZ. Apoptosis in bone marrow of myelodysplastic syndrome patients.Blood. 1996; 87: 3064Crossref PubMed Google Scholar,15Shetty V Hussaini S Alvi S et al.Excessive apoptosis, increased phagocytosis, nuclear inclusion bodies and cylindrical confronting cisternae in bone marrow biopsies of myelodysplastic syndrome patients.Br J Haematol. 2002; 116: 817-825Crossref PubMed Scopus (22) Google Scholar]. Recently, necroptosis has been identified as an important programmed cell death (PCD) pathway, which is driven by RIP kinases. In contrast to apoptosis, necroptosis is characterized by premature rupture of the plasma membrane, resulting in the release of damage-associated molecular patterns (DAMPs) that elicit an inflammatory response [16Galluzzi L Kepp O Kroemer G. RIP kinases initiate programmed necrosis.J Mol Cell Biol. 2009; 1: 8-10Crossref PubMed Scopus (92) Google Scholar,17Sun L Wang H Wang Z et al.Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.Cell. 2012; 148: 213-227Abstract Full Text Full Text PDF PubMed Scopus (1396) Google Scholar]. The most extensively characterized death receptor-induced programmed necroptosis pathway is initiated by ligation of TNF receptor (TNFR1) with TNF-α. After ligation, TNFR1 recruits tumor necrosis factor receptor type 1-associated death domain (TRADD), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), TNF receptor-associated factor 2 (TRAF2), and cellular inhibitor of apoptosis protein 1 (cIAP1/2) to form complex I at the plasma membrane. RIPK1 undergoes deubiquitylation to allow association with Fas-associated protein with death domain (FADD), TRADD, and RIPK3 in the cytosol to form complex II. FADD mediates the recruitment and activation of pro-caspase-8 [18Kaczmarek A Vandenabeele P Krysko DV. Necroptosis: the release of damage-associated molecular patterns and its physiological relevance.Immunity. 2013; 38: 209-223Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar]. Activation of caspase-8 plays a key role in directing whether a cell will undergo apoptosis or necroptosis. Activated caspase-8 can activate effector caspases such as caspase-3 to initiate apoptosis. In addition, activated caspase-8 cleaves and inactivates RIPK1 and RIPK3, thereby preventing necroptosis [19Lalaoui N Boyden SE Oda H et al.Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.Nature. 2020; 577: 103-108Crossref PubMed Scopus (94) Google Scholar,20Newton K Wickliffe KE Dugger DL et al.Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis.Nature. 2019; 574: 428-431Crossref PubMed Scopus (129) Google Scholar]. However, if caspase-8 enzymatic activity is inhibited (by cFLIP), RIPK1 and RIPK3 are stabilized and form the necroptosome [21Feltham R Vince JE Lawlor KE. Caspase-8: not so silently deadly.Clin Transl Immunology. 2017; 6: e124Crossref PubMed Scopus (75) Google Scholar], allowing interdependent phosphorylation and activation of RIPK1 and RIPK3 within the necroptosome and execution of necroptosis (Figure The to PCD in early studies not necroptosis and apoptosis. apoptotic and cells will increased in situ end increased end labeling and increased V Increased caspase-3 activity has been of BM cells from patients with MDS S P V et al.The relative and of CD34+ cells to undergo apoptosis in myelodysplastic J Hematol. Google Scholar] but not caspase-3 activation is by a of MDS samples evidence of caspase-3 activation E R et abnormalities and response in bone marrow hematopoietic cell of myelodysplastic syndrome Res. 2012; PubMed Scopus Google Scholar]. have that BM from patients with MDS has a to undergo PCD as by DNA However, the signaling PCD and role in the pathogenesis of MDS have not been has a in which BM necroptosis is and we found that the of BM with the of MDS, BM and of the hematopoietic cells et bone marrow necroptosis to and bone marrow in 2019; PubMed Scopus Google Scholar], and that levels of RIPK1 and phospho-mixed lineage kinase domain-like protein key necroptosis signaling increased in a of samples from patients with MDS. 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We further the expression of PCD pathway patients with MDS patients with and found a significant in expression A A analysis of expression of type and 2017; PubMed Scopus Google Scholar]. The expression the apoptosis and necroptosis was as a (Figure in the signaling of increased in MDS, TNFR1 and of its of complex In complex and RIPK3 and was is as of the caspases in the and apoptosis In addition, activated caspase-8 cleaves and inactivates RIPK1 and RIPK3, thereby preventing necroptosis D A. of RIPK1 and 2017; 38: Full Text Full Text PDF PubMed Scopus Google Scholar]. We found that in MDS expression was and RIPK3, and with increased necroptosis In with the apoptosis pathway, the of the and and (Figure of at the however, in of of RIPK1 and pMLKL in for the that programmed is increased in the bone marrow samples from patients with MDS A A analysis of expression of type and 2017; PubMed Scopus Google Scholar]. Early studies of MDS BM have found hematopoietic which is thought to play a significant role in the pathogenesis of the disease V Mundle S Alvi S et al.Measurement of apoptosis, proliferation and three cytokines in 46 patients with myelodysplastic syndromes.Leuk Res. 1996; 20: 891-900Crossref PubMed Scopus (161) Google P A E P V Apoptosis in patients with myelodysplastic syndromes: of marrow cells in patients and with PubMed Scopus Google Scholar]. early studies not apoptotic and cell death, and most performed on hematopoietic and not cell death that the in status of MDS BM P L N A apoptosis a process in myelodysplastic J Haematol. 1996; PubMed Scopus Google Scholar]. In addition, analysis of the expression that expression of the of apoptosis, was significantly in MDS relative to bone immunofluorescence staining that the apoptosis cleaved caspase-3, was not increased in BM biopsy from patients with MDS as with control or de novo In addition, antiapoptotic such as and factor in MDS E L A. and apoptosis in bone marrow from patients with myelodysplastic syndromes with and Res. 1997; 21: PubMed Scopus Google Scholar]. In contrast to the of cleaved caspase-3, we found a of expression for the necroptosis signaling RIPK1 and pMLKL in the The complex has been as a key of the necroptosis pathway D A. of RIPK1 and 2017; 38: Full Text Full Text PDF PubMed Scopus Google Scholar]. The inhibitor but not apoptotic death of the kinase domain of further the of RIPK1 in programmed necroptosis A J M et of kinase as a cellular of Biol. 2008; PubMed Scopus Google Scholar]. In RIPK1 expression was significantly increased in MDS BM relative to BM of control patients or patients with RIPK1 expression was in MDS BM with low-grade and the expression was to that of To is the of the of programmed necrosis apoptosis in BM samples from patients with MDS. is to in which necroptosis signaling significantly increased in the such is the of increased cell Cell and cell proliferation have been in MDS and by de activity is in myelodysplastic syndrome and MDS and B 2021; PubMed Scopus Google Scholar] and S M et of in and prognostic 2017; PubMed Scopus Google Scholar]. studies proliferation in MDS and increased proliferation in was found to and of prognostic significance in the we increased RIPK1 in early MDS, has been found to relative to we not that the increased RIPK1 to increased in MDS bone anemia is with increased cell are associated with increased and activation of inflammatory and we that RIPK1 increased in and important to the necroptosis pathway at the protein in and bone marrow disorders characterized by increased or increased studies have reported increased a form of inflammatory cell death that is the in a of samples from patients with MDS low-risk MDS samples and high-risk MDS A. 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We also found that samples are to RIPK1 with and that with are with a role for necroptosis in MDS cell erythroid cells. based on the expression the most significant apoptosis and necroptosis PCD was expression of in patients with MDS as with with and control (Figure the and apoptotic cleavage of the H H of by caspase mediates the mitochondrial in the pathway of Full Text Full Text PDF PubMed Scopus Google Scholar]. also caspase-8 as a key of the apoptotic death T Vandenabeele P RIP kinases at the of cell death and 2009; Full Text Full Text PDF PubMed Scopus Google Scholar]. We further found in the apoptosis pathway to the of the and and In we found that expression of in the necroptosis pathway was increased in MDS relative to is important to of the analysis with to PCD PCD is to a significant by of PCD In addition, the not low-risk high-risk MDS expression evidence that the necroptosis pathway is upregulated in MDS. with that but not apoptosis, is upregulated in MDS, especially MDS. We that necroptosis and not apoptosis is a prominent of PCD in MDS and that further study of RIPK1 expression as a biomarker for the diagnosis and of early MDS is The of The from and and the of and the and of performed in the at performed in using the Cell and analysis was performed as a of BM using at The abnormalities to the International System for The or at The and at the of from the BM core biopsy samples in study de novo and and from human for immunofluorescence staining and BM samples as of in a to and in a and We most the of the bone marrow that is The human was to the BM as as without to the of on and staining with of BM core biopsy with the RIPK1 pMLKL cleaved caspase-3 Cell RIPK3 and RIPK1 and cleaved caspase-3 by an and a Cell and by or using or with using cells from bone marrow from a by in and on a with and for and as or further for in at a of 2 which is by the with identified and MDS marrow cells from an at a of 2 in which is by the with was or for and cells at for After identified and and on a Cell The the of as from the is performed using R A and for R for of as group of features MDS and de novo was for and for or was to the the or to study three or was to assess the

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