Papers by Lilian Varricchio
Frontiers in Oncology
Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as com... more Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as components of bone marrow (BM) niches that support the development of hematopoietic stem and progenitor cell (HSPC) and provide inflammatory signals. MKs can dynamically change their activities during homeostasis and following stress, thereby regulating hematopoietic stem cell (HSC) function. Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm (MPN) characterized by hyperactivation of JAK/STAT signaling and MK hyperplasia, which is associated with an aberrant inflammatory signature. Since JAK1/2 inhibitor alone is incapable of depleting the malignant HSC clones or reversing BM fibrosis, the identification of mechanisms that cooperate with MF JAK/STAT signaling to promote disease progression might help in developing combination therapies to modify disease outcomes. Chronic inflammation and MK hyperplasia result in an abnormal release of TGFβ1, which plays a critical role ...
Blood, 2021
TGFβ plays a pivotal role in the pathobiology of myelofibrosis (MF) by not only promoting bone ma... more TGFβ plays a pivotal role in the pathobiology of myelofibrosis (MF) by not only promoting bone marrow fibrosis (BMF) but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs). Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to MKs decreased pSMAD2 without affecting total SMAD2/3 and led to increased numbers of MKs. Treatment of MF MNCs with AVID200 also led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. A phase 1b trial of AVID200 (NCT03895112) was performed and completed in IN...
Italian journal of anatomy and embryology, 2015
Calreticulin (CALR) is a multifunctional protein normally found within the lumen of the endoplasm... more Calreticulin (CALR) is a multifunctional protein normally found within the lumen of the endoplasmic reticulum that mediates the cellular response to Ca2+ by chaperoning other proteins to their acting sites. Somatic loss-of-function mutations in the CALR gene were recently discovered in 70% of patients with the Philadelphia-negative myeloproliferative neoplasm (MPN) primary myelofibrosis (PMF) who did not harbor gain-of-function mutations of JAK21,2. Nevertheless, the JAK2 pathway is constitutively activated also in patients carrying CALR mutation and treatments with JAK2 inhibitors are effective not only in MPN patients (PMF and polycythemia vera, PV) harboring JAK2 mutations but also in PMF patients harboring mutations in CALR3. We have previously reported that erythroid cells from PV and PMF patients express abnormal activity of the glucocorticoid receptor (GR), a nuclear receptor whose transcriptional activity plays an important role in the regulation of stress erythropoiesis4,5....
Frontiers in Oncology, 2019
Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT sign... more Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.
Blood, 2009
3899 Poster Board III-835 Glucocorticoids delay the maturation of erythroid cells retaining them ... more 3899 Poster Board III-835 Glucocorticoids delay the maturation of erythroid cells retaining them in the proliferative phase. This effect is mediated by synergistic transcriptional and membrane-associated pathways. The glucocorticorticoid receptor (GR) upon activation forms a transcriptional complex with phosphorylated STAT-5 (P-STAT) which binds and alters the expression of a subset of the erythropoietin (EPO)- and stem cell factor (SCF)-target genes. In addition, the physical interaction between the GR and the EPO receptor (EPO-R) in the cytoplasm suppresses the ability of both receptors to phosphorylate STAT-5 when activated together. In addition to the canonical αGR signalling isoform, cells may express the dominant negative βGR isoform which antagonizes αGR activity. Erythroid cells (EBs) generated from peripheral blood mononuclear cells (PBMC) from patients with Polycythemia Vera (PV) and those obtained from normal donors stimulated with dexamethasone (D), an agonist of GR, and...
Blood, 2011
3395 Stem cell factor (SCF) is a cyokine produced by bone marrow stromal cells that upon engageme... more 3395 Stem cell factor (SCF) is a cyokine produced by bone marrow stromal cells that upon engagement of its cognate receptor cKit and in combination with other hematopoietic growth factors initiates downstream phosphorylation events that generate hematopoietic progenitors and precursors of all types. SCF and cKit are encoded by highly conserved genes. The human (hSCF) and murine (mSCF) forms are highly homologous and are 82% identical at the amino acid levels. Both have been reported to sustain differentiation of human mast cells from cord blood (CB) but mast cells generated with mSCF remain immature (Mitzu et al, PNAS 90:735–739). In addition, anecdotic reports indicate that hSCF is 100-fold less active than mSCF on mouse cells while mSCF and hSCF are equally active on human cells. To clarify if structural differences between the two growth factors have biological significance, we compared cord blood (CB) mononuclear cells and CD34pos cells as the stem cell source for in vitro produ...
Blood, 2016
PV is characterized by the gain-of-function V617F mutation in JAK2, the gene encoding the first s... more PV is characterized by the gain-of-function V617F mutation in JAK2, the gene encoding the first signaling element of the cytokine receptor superfamily. Progenitor cells from PV are more sensitive in vitro to Imatinib, which also inhibits the SCF receptor cKIT, than those from normal sources (Gaikwad, Exp Hemat 2007;35:931) and clinical trials with similar tyrosine kinase inhibitors have been reported to have some efficacy in PV (Nussenzveig, Int J Hematol 2009;90:58; Silver, Leuk Res 2012;36:156). These data led us to examine the mechanism by which this tyrosine kinase inhibitor affects erythropoiesis in PV. We observed that in cultures containing SCF PV progenitor cells generated similar numbers of erythroid cells (Ery) as those from adult (AB) and cord (CB) blood by day 10 [fold increase (FI) ~1-2] but by day 13 PV cells generated significantly greater numbers of Ery than AB and CB [PV FI=11±0.2, p=0.021 vs AB; CB FI=6.2±1.9, p=0.025 vs PV and 0.0055 vs AB; AB FI=2.6±0.5]. Since b...
Blood, 2010
4780 Histone deacetylation maintains chromatin in a condensed configuration preventing gene expre... more 4780 Histone deacetylation maintains chromatin in a condensed configuration preventing gene expression in eukaryotic cells. The deacetylation reaction is catalyzed by enzymes of the histone deacetylase (HDAC) superfamily, which perform their functions as multiprotein complexes including at least 2 HDAC isoforms, DNA docking factors (transcription factors and methyl-binding proteins) and protein kinases (PKC and Erk). The well established role of HDACs in gene silencing has suggested studies to identify HDAC inhibitors (HDACi) that, by re-activating γ-globin expression, might treat the anemia due to insufficient β-globin expression (Cao et al Blood 103:701, 2004). Over the years several HDACi have been documented to induce γ-globin expression in human erythroid cultures, adult baboons, and β-thalassemia and sickle cell patients. Among those, Class I HDACi, and in particular those that inhibit HDAC3, appear to be more potent as γ-globin gene activators (Mankidy et al, Blood 108:3179, ...
Blood, 2012
2339 In mice, loss-of-function studies have identified that glucocorticoid receptor (GR) activati... more 2339 In mice, loss-of-function studies have identified that glucocorticoid receptor (GR) activation mediates the massive red blood cell expansion occurring following stress challenges. This effect involves generation of stress-specific erythroid precursors expressing KIT (the receptor for SCF), CD71 (the transferrin receptor co-expressed in human erythroblasts with CD36) and TER119 (Glycophorin A) that are blocked in maturation and induced into a self-replication state. Studies in culture indicate that GR activation increases also the generation of human erythroid cells. In fact, human CD34poscells from adult (AB) or cord (CB) blood give rise to great numbers of erythroblasts within 14–18 days when cultured with SCF, IL-3, EPO and the GR agonist dexamethasone (DXM) (fold increase: 691±257 vs 396±187 from AB or CB). Whether the effects of GR on human erythropoiesis are mediated by stress-specific erythroid precursors phenotypically and functionally similar to those observed in mice r...
Blood, 2015
Cushing's disease (CD) is a rare endocrine disorder (1.2-2.4/million/year) characterized by c... more Cushing's disease (CD) is a rare endocrine disorder (1.2-2.4/million/year) characterized by chronic excess endogenous glucocorticoids (GC) due to an adrenocorticotropic hormone-secreting pituitary adenoma. Untreated CD results in increased mortality and multiple morbidities (obesity, diabetes, hypertension, cardiovascular disease) and, in one case report, erythrocytosis (Gursoy et al, J End Invest. 2006;29:742). The effects of chronic GC exposure on erythropoiesis in a CD cohort have not yet been studied. We prospectively quantified hematocrit (Hct), hemoglobin (Hb) and platelets (ptl) values in CD patients before (v1) and after surgical remission (v2, mean time since surgery=14.5 months) and in matched healthy controls (HC). Frequency, antigenic profiling and erythroid (Ery) expansion potential of circulating hematopoietic progenitor cells (HPC) in the three cohorts were also evaluated. The subjects analyzed included 28 v1 [6 males, 22 females, mean age=41 years; body mass inde...
Blood, 2016
Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which act... more Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which activates Ca2+signaling and terminal maturation [Miller, Blood 1989;73:1188]. Under stress, GR switches EPO-R signaling to a proliferation mode allowing erythroblast (Ery) amplification [Zhang, Nature 2013;499:92]. However, for recovery from anemia to occur, a mechanism, still to be identified, must counteract GR and switch EPO-R signaling back to a maturation mode. CALR chaperones other proteins to their active sites[Michalak, Biochem J 1999;344: 281]. By importing GR from the nucleus, CALR resets the stress response of murine fibroblasts. This function is regulated by the exposure of its C-terminal domain (C-CALR) sustained by Ca2+ [Holaska, Mol Cell Biol 2002;22:6286]. Whether CALR regulates nuclear import of GR in human Ery and how this regulation is modulated by Ca2+and EPO is unknown. Studies were performed to fill this gap. By WB, human Ery expanded in vitro from adult blood expresse...
Blood, 2015
Calreticulin (CALR) mutations disrupting domains of the protein that regulate its intracellular t... more Calreticulin (CALR) mutations disrupting domains of the protein that regulate its intracellular trafficking were discovered in patients with myeloproliferative neoplasms (MPN) without JAK2 mutations and are associated with JAK2 activation (Klampt et al, NEJM 2013;369:2379; Nangalia et al, NEJM 2013;369:2391). Whether JAK2 mutations are associated with altered CALR functions is unknown. We previously reported that erythroid cells (Erys) generated in-vitro from JAK2+-polycythemia vera (PV) patients do not respond to dexamethasone (Dex) because in these cells the glucocorticoid receptor (GRα) is constitutively retained in the nucleus (Varricchio et al, Blood 2011;118:425). This was explained by the high frequency found in PV patients of the A3669G GR polymorphism that increases expression of GRβ, the isoform responsible for nuclear retention of GRα. The A3669G frequency is also increased in primary myelofibrosis (PMF) and, when associated with JAK2 mutations, predicts poor survival (Po...
Blood, 2009
642 Ex-vivo generated erythroblasts (EBs) represent alternative transfusion products. Adult blood... more 642 Ex-vivo generated erythroblasts (EBs) represent alternative transfusion products. Adult blood (AB) contains numbers of progenitor cells comparable to those present in cord blood (CB) (106 vs 1.8×106 CD34pos cells in average AB and CB donations) but generates lower numbers of erythroblasts (EBs) (∼4.8×108 vs 6.6×1010, respectively) and, in spite of its numerous advantages, is not considered suitable for ex-vivo EB production. To assess the potential of AB to generate EBs ex-vivo, the growth factors [stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO)] and optimal concentration and addition schedule of dexamethasone (DXM) and estradiol (ES) sustaining maximal EB amplification from AB mononuclear cells (MNC) were defined using media with serum previously defined as human erythroid massive amplification culture (HEMAser). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated low numbers (fold increase ∼2) of EBs at all stages of maturation. Conce...
Frontiers in Cell and Developmental Biology, 2017
Calreticulin is a Ca 2+-binding chaperone protein, which resides mainly in the endoplasmic reticu... more Calreticulin is a Ca 2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca 2+ , calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.
Haematologica, 2019
ongenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K... more ongenital dyserythropoietic anemia type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. Molecular characteristics of this disease have not been clarified, partly due to its rarity. We expanded erythroid cells from a patient's peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain patients' clinical and phenotypic features, and elucidate the dominant character of the mutation.
F1000 - Post-publication peer review of the biomedical literature, 2017
Experimental Hematology, 2016
American journal of blood research, 2014
Glucocorticoids are endogenous steroid hormones that regulate several biological functions includ... more Glucocorticoids are endogenous steroid hormones that regulate several biological functions including proliferation, differentiation and apoptosis in numerous cell types in response to stress. Synthetic glucocorticoids, such as dexamethasone (Dex) are used to treat a variety of diseases ranging from allergy to depression. Glucocorticoids exert their effects by passively entering into cells and binding to a specific Glucocorticoid Receptor (GR) present in the cytoplasm. Once activated by its ligand, GR may elicit cytoplasmic (mainly suppression of p53), and nuclear (regulation of transcription of GR responsive genes), responses. Human GR is highly polymorphic and may encode > 260 different isoforms. This polymorphism is emerging as the leading cause for the variability of phenotype and response to glucocorticoid therapy observed in human populations. Studies in mice and clinical observations indicate that GR controls also the response to erythroid stress. This knowledge has been ex...
F1000 - Post-publication peer review of the biomedical literature, 2011
F1000 - Post-publication peer review of the biomedical literature, 2013
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Papers by Lilian Varricchio