Eduardo Anguita

Detailed Characterization of Mesenchymal Stem/Stromal Cells from a Large Cohort of AML Patients Demonstrates a Definitive Link to Treatment Outcomes

Summary Bone marrow mesenchymal stem/stromal cells (BM-MSCs) are key components of the hematopoietic niche thought to have a direct role in leukemia pathogenesis. BM-MSCs from patients with acute myeloid leukemia (AML) have been poorly characterized due to disease heterogeneity. We report a functional, genetic, and immunological characterization of BM-MSC cultures from 46 AML patients, stratified by molecular/cytogenetics into low-risk (LR), intermediate-risk (IR), and high-risk (HR) subgroups. Stable MSC cultures were successfully established and characterized from 40 of 46 AML patients irrespective of the risk subgroup. AML-derived BM-MSCs never harbored tumor-specific cytogenetic/molecular alterations present in blasts, but displayed higher clonogenic potential than healthy donor (HD)-derived BM-MSCs. Although HD- and AML-derived BM-MSCs equally provided chemoprotection to AML cells in vitro, AML-derived BM-MSCs were more immunosuppressive/anti-inflammatory, enhanced suppression of lymphocyte proliferation, and diminished secretion of pro-inflammatory cytokines. Multivariate analysis revealed that the level of interleukin-10 produced by AML-derived BM-MSCs as an independent prognostic factor negatively affected overall survival. Collectively our data show that AML-derived BM-MSCs are not tumor related, but display functional differences contributing to therapy resistance and disease evolution.

Human promoter mutations unveil Oct-1 and GATA-1 opposite action on Gfi1b regulation

Growth factor-independence 1b (Gfi1b) is a zinc finger transcription factor essential for erythroid and megakaryocytic development. To better understand Gfi1b regulation and to know the implication of the level of expression of this gene in human pathology, we have searched for promoter punctual sequence variations in 214 patients with different hematological diseases. We found two previously unknown congenital mutations at evolutionary conserved GATA and octamer-binding (Oct) transcription factor sites. The Oct site mutation was also found in five relatives of the patient. The GATA motif mutation reduced promoter activity by 50% in vitro, while homozygous patients with the octamer site mutation showed a four-to-five times increase of Gfi1b RNA in platelets. Electrophoretic mobility shift analyses demonstrated that different protein complexes bind to both sites and that binding is reduced by the mutations. Finally, we found that GATA-1 and Oct-1 are the main components of each complex. This study provides evidences of a new mechanism for Gfi1b repression. This is also the first report of Gfi1b mutations with a functional implication; further investigation and follow-up will clarify the involvement of these mutations in hematological disease.

Increasing TIMP3 expression by hypomethylating agents diminishes soluble MICA, MICB and ULBP2 shedding in acute myeloid leukemia, facilitating NK cell-mediated immune recognition

Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza®, AZA) and decitabine (Dacogen®, DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.

Transcription Factor GFI1B in Health and Disease

Many human diseases arise through dysregulation of genes that control key cell fate pathways. Transcription factors (TFs) are major cell fate regulators frequently involved in cancer, particularly in leukemia. The GFI1B gene, coding a TF, was identified by sequence homology with the oncogene growth factor independence 1 (GFI1). Both GFI1 and GFI1B have six C-terminal C2H2 zinc fingers and an N-terminal SNAG (SNAIL/GFI1) transcriptional repression domain. Gfi1 is essential for neutrophil differentiation in mice. In humans, GFI1 mutations are associated with severe congenital neutropenia. Gfi1 is also required for B and T lymphopoiesis. However, knockout mice have demonstrated that Gfi1b is required for development of both erythroid and megakaryocytic lineages. Consistent with this, human mutations of GFI1B produce bleeding disorders with low platelet count and abnormal function. Loss of Gfi1b in adult mice increases the absolute numbers of hematopoietic stem cells (HSCs) that are less quiescent than wild-type HSCs. In keeping with this key role in cell fate, GFI1B is emerging as a gene involved in cancer, which also includes solid tumors. In fact, abnormal activation of GFI1B and GFI1 has been related to human medulloblastoma and is also likely to be relevant in blood malignancies. Several pieces of evidence supporting this statement will be detailed in this mini review.

A novel ex vivo high-throughput assay reveals antiproliferative effects of idelalisib and ibrutinib in chronic lymphocytic leukemia

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect.

Splenectomy in ITP: we keep removing a healthy functional organ

Dear Editor, Lozano et al. [1] present the real life scenery of immune thrombocytopenia (ITP) with some unexpected data we, in actual fact, are used to encountering in clinics. They show the unorthodox, but not surprising finding that ITP secondline treatment with the use of thrombopoietin (TPO) receptor agonists (TOP-RAs) exceeds for more than twice the splenectomy in adult patients at the Spanish hospitals, in contrast with present guidelines [1, 2]. ITP is a disease we find every day in outpatient care units. It represents 1.6–4.7 cases/10/year, with up to 60% increased mortality rate, mainly due infection and bleeding [3]. Higher incidence with advancing age makes it to become an ever increasing health problem in Europe. Guidelines recommend corticosteroids and intravenous immunoglobulin—the last for acute bleeding—as first-line treatment followed by splenectomy as second-line and the use of rituximab and TPO-RA in case of failure or contraindication [2]. This hierarchy has been recently questioned. Splenectomy produces 80 % responses, most of them durable. However, this method produces more complications than other abdominal surgeries and implies an increased risk of infection, mainly by encapsulated bacteria, but also virus and others, for life [2, 4, 5]. Vaccination can reduce the incidence of those, but recent reports reveal that vaccination is disappointingly forgotten or misused in this condition. Recently, Moulis et al. [6] show that only a 58 % of splenectomized ITP patients in France receive pneumococcus vaccination (52 % only in the indicated timing). This is a major problem in a situation with a 3.25 % of postsplenectomy sepsis, most of them by Streptococcus pneumoniae (57 % of infections and 59 % of deaths) with increased risk for over 10 years of the surgery, and possibly lifelong [4, 5]. Also thrombotic disease, solid cancers, and blood malignancies have been found at a higher level [5]. To increase the problem, age ≥65 years negatively affects safety and efficacy outcomes of splenectomy [7]. The use of rituximab, anti CD20, as second-line treatment has been viewed as a hope to spare the spleen. Nevertheless, it has been shown not to significantly reduce the rate of treatment failure compared with placebo, no difference in overall and complete response. Longterm net rate of sustained responses of a 10 % difference could be not clinically important and would not justify the use of rituximab [8]. TOP-RAs achieve 80–95 % response, with comparable effect for both approved TPO-RAs and higher response in non-splenectomized patients. TPO-RAs are safe and useful in elderly and children [9, 10]. Furthermore, loss of response, 4–9 %, can be treated changing the TPO-RA. In multivariate analysis the only independent negative factor for response was splenectomy. Recently, discontinuation of these drugs with remission continuation has been described [11]. When economical aspects are also analyzed, ITP represents a high burden, with particular impact of splenectomy in the ITP derived costs, in this context a TPO receptor agonist has been shown to be cost effective in both splenectomized and no splenectomized patients [3]. Also, ITP involves an important psychological impact that is worsened with the remove of the spleen. TPO-ARs are also better tolerated for patients. * Eduardo Anguita eduardo.anguita@salud.madrid.org

Caracterización molecular de dos nuevas mutaciones de α° talasemia en 2 familias españolas (mutación -- ED y --GP)

BACKGROUND AND OBJECTIVESnThe two structural genes encoding the human α-globin chains are located on the short arm of chromosome 16. Normal individuals have four genes α (αα/αα). α-thalassemias are usually produced by the deletion of one, two, three, or four α genes. Deletion of both α genes within the same chromosome (α° thalassemia) is commonly observed in individuals from the Mediterranean basin and Southeast Asia.nnnMATERIAL AND METHODSnWe study two natural families of Madrid with microcytic hypochromic anemia. The DNA extracted from peripheral blood leukocytes was digested with different restriction enzymes and hybridization with probes of gene cluster α. The ends of the deletion were characterized by combining the techniques of Southern blot, PCR and FISH.nnnRESULTSnWe present two new mutations of α° thalassemia in two Spanish families, not previously described in the literature. The deletion (--(ED)) is ∼80 kb with the break point 5 in the coordinate +100 (± 3 kb), whereas the end 3HVR places in the coordinate 178±750 bp. The second deletion (--(GP)) is more extensive, with loss of 145 kb, placing the deletion in the end 5 between the coordinates 34 and 37, respecting therefore the telomere. In the centromeric region the breakpoint places as the previous one in the coordinate 178±1.4 bp.nnnCONCLUSIONSnIn both mutations both alpha genes were deleted, the gene θ and the region HS40. The exact identification of these deletions is essential to determine the function of the genes α with a view to a possible genetic diagnosis.