NG2 antigen is a therapeutic target for MLL-rearranged B-cell acute lymphoblastic leukemia

Altres ajuts: This work has been supported by the Asociación Española Contra el Cáncer (AECC), Beca FERO, and OM are supported by postdoctoral fellowships from the AECC scientific foundation and the Catalunya Government (Beatriu de Pinos, BP00048), respectively. PM also acknowledges the financial support from the Obra Social La Caixa-Fundaciò Josep Carreras and "Premio Miguelín".B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with cure rates of ∼80%. MLL-rearranged (MLLr) B-ALL (MLLr-B-ALL) has, however, an unfavorable prognosis with common therapy refractoriness and early relapse, and therefore new therapeutic targets are needed for relapsed/refractory MLLr-B-ALL. MLLr leukemias are characterized by the specific expression of chondroitin sulfate proteoglycan-4, also known as neuron-glial antigen-2 (NG2). NG2 was recently shown involved in leukemia invasiveness and central nervous system infiltration in MLLr-B-ALL, and correlated with lower event-free survival (EFS). We here hypothesized that blocking NG2 may synergize with established induction therapy for B-ALL based on vincristine, glucocorticoids, and l-asparaginase (VxL). Using robust patient-derived xenograft (PDX) models, we found that NG2 is crucial for MLLr-B-ALL engraftment upon intravenous (i.v.) transplantation. In vivo blockade of NG2 using either chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significant mobilization of MLLr-B-ALL blasts from bone marrow (BM) to peripheral blood (PB) as demonstrated by cytometric and 3D confocal imaging analysis. When combined with either NG2 antagonist, VxL treatment achieved higher rates of complete remission, and consequently higher EFS and delayed time to relapse. Mechanistically, anti-NG2 MoAb induces neither antibody-dependent cell-mediated not complement-dependent cytotoxicity. NG2 blockade rather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to chemotherapy. We provide a proof of concept for NG2 as a therapeutic target for MLLr-B-ALL

Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

Altres ajuts: Funding This work was supported by the Obra Social La Caixa (LCF/PR/HR19/52160011), the Spanish Cancer Association and Leo Messi Foundation to PM.Background There are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19 - either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22 + CD19 - B-ALL relapses and CD19 - preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied. Methods Here, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays. Results Conformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy. Conclusions We report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22 high and CD22 low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22-CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL

Robustness of dead Cas9 activators in human pluripotent and mesenchymal stem cells

Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.We thank CERCA/Generalitat de Catalunya and Fundació Josep Carreras-Obra Social la Caixa for their institutional support. We thank Jose Luis Sardina (IJC, Barcelona) for technical assistance with the teratoma assays. Financial support for this work was obtained from the Catalunya Goverment (SGR330 and PERIS 2017-2019), the Spanish Ministry of Economy and Competitiveness (SAF2016-80481-R), the European Research Council (CoG-2014-646903), and the Fundación Leo Messi to P.M.; the Spanish Association against Cancer (AECC-CI-2015) and the Health Institute Carlos III (ISCIII/FEDER, PI17/01028) to C.B.; the Biotechnology and Biological Sciences Research Council (BBRSC) to L.M.F. and A.F.; and the Spanish National Research and Development Plan (ISCIII/FEDER, PI17/02303) and the AEI/MICIU EXPLORA Project (BIO2017-91272-EXP) to S.R.-P. P.M. is an investigator of the Spanish Cell Therapy Cooperative Network (TERCEL). R.T.-R. is supported by a postdoctoral fellowship from the Asociación Española Contra el Cáncer (AECC).S

The insecticides permethrin and chlorpyrifos show limited genotoxicity and no leukemogenic potential in human and murine hematopoietic stem progenitor cells

Altres ajuts: European Food and Safety Authority, EFSA.PRAS.2018.04-CT1; Consejo Nacional de Ciencia y Tecnología, CB-2012-01-183467; Centro de Investigación Biomédica en Red en Cáncer, PID2019-104695RBI00; Asociación Española Contra el Cáncer, AECC INVES211226MOLI

Enhanced hemato-endothelial specification during human embryonic differentiation through developmental cooperation between AF4-MLL and MLL-AF4 fusions.

The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.Wellcome Trust, CRUK, Bloodwise, ERC, Generalitat de Catalunya, Spanish Ministry of Economy and Competitiveness, Spanish Association Against cancer, Health Institute Carlos III, NIHR GOSH BRC, Great Ormond Steet Hospital Children's Charity, Deutsche José Carreras Leukämie Stiftung, Obra Social La Caixa-Fundaciò Josep Carreras, Spanish Association of Cancer Researc

Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.This research was supported by the European Research Council (H2020) (CoG-2014-646903), the Agencia Estatal de Investigacion/European Re- ´ gional Development Fund (SAF2016-80481-R and SAF2016-75442-R), and the Catalunya Government (SGR330 and PERIS 2017) (P.M.), as well as the Asociacion Española Contra el C ´ ancer, Beca FERO, and the ´ ISCIII/FEDER (PI17/01028) (C.B.). P.M. also acknowledges institutional support from the Obra Social La Caixa-Fundacio Josep Carreras. J.G.P. ` holds a Miguel Servet contract (CP15/00014), and O.B.-L. is supported by an AGAUR-FI fellowship from the Catalan Government. P.M. is an investigator of the Spanish Cell Therapy cooperative network (TERCEL).Peer reviewe

Impaired Condensin Complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, and high-hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD is an initiating oncogenic event affiliated to childhood B-ALL, the mitotic and chromosomal defects associated to HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated to chromosome alignment defects at the metaphase plate leading to robust chromosome segregation defects and non-modal karyotypes. Mechanistically, biochemical, functional and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness and mis-localization of the chromosome passenger complex proteins Aurora B Kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and impaired spindle assembly checkpoint (SAC) thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated to defective condensin complex, AURKB and SAC

Caracterització de la nova línia nod.Ace2-/-: estudi de l’aparició de diabetis i la progressió de la nefropatia diabètica

L’enzim convertidor d’angiotensina (ECA) promou la formació de l’angiotensina II (Ang-II). Contràriament, l’ECA2, una monocarboxipeptidasa que presenta un 40% d’homologia amb l’ECA, degrada l’Ang-II (accions vasoconstrictores) a angiotensina 1-7 (Ang-(1-7) (accions vasodilatadores). L’eix ECA2/Ang-(1-7) desencadena accions oposades a les de l’eix ECA/Ang-II. A nivell clínic, el tractament amb inhibidors de l’ECA i/o bloquejants dels receptors d’angiotensina en pacients amb nefropatia diabètica (ND) ha demostrat disminuir l’albuminúria i frenar la progressió cap a malaltia renal crònica (MRC). Donat aquest fet i que l’ECA2 és un regulador negatiu del sistema renina angiotensina (SRA), hipotetitzem que la manca d’ECA2 contribuirà a un augment de l’aparició de la diabetis i a una major progressió de la ND en un model experimental espontani de diabetis tipus 1, la soca no obesa diabètica (NOD). Amb aquesta finalitat s’ha generat la nova línia de ratolins NOD.ACE2-/- i els respectius controls. L’estudi de la diabetis en els animals NOD.ACE2-/- s’ha observat: 1) viabilitat, fertilitat i, fenotípicament, una alteració de la coloració del pelatge i menor pes corporal, 2) major aparició de diabetis acompanyada d’una alteració en l’homeòstasi de la glucosa i 3) augment en la sensibilitat a la insulina exògena. En l’estudi de la ND, els animals NOD.ACE2-/- mostren: 1) hipertrofia glomerular, expansió de la matriu mesangial i pèrdua podocitària sense alteracions en la filtració glomerular ni l’albuminúria, 2) menor activitat renal cortical d’ECA. En resum, la manca d’ECA2 en la soca NOD contribueix a un empitjorament en l’homeòstasi de la glucosa i de la insulina així com alteracions estructurals a nivell del glomèrul que inclouen: augment de l’àrea glomerular, expansió de la matriu mesangial i pèrdua podocitària.Angiotensin converting enzyme (ACE) promotes angiotensin II (Ang II) formation. Conversely, ACE2, a monocarboxipeptidase that shares 40% homology with ACE, degrades promotes angiotensin 1-7 (Ang- (1-7) (vasodilator actions) formation from Ang-II (vasoconstrictor actions). ACE2/Ang-(1-7) axis actions opposite ACE/Ang-II axis actions. ACE inhibitors and/or angiotensin receptor blockers treatment in patients with diabetic nephropathy (DN) have shown to blunt albuminuria progression to chronic kidney disease (CKD). Given this and the fact that ACE2 acts as a negative regulator of the renin angiotensin system (RAS), we hypothesize that ECA2 loss contribute to an increase in diabetis development and a greater progression of DN in an experimental model of spontaneous type 1 diabetes, the non obese diabetic (NOD) mice. With this aim, we have generated a new experimental model, the NOD.ACE2-/- mice and their respective controls. The study of diabetes onset in NOD.ACE2-/- showed: 1) viability, fertility and, phenotypically, a change in coat color and lower body weight, 2) higher diabetis incidence accompanied by altered glucose homeostasis and 3) increase in exogenous insulin sensitivity. Within the DN study, NOD.ACE2-/- mice showed: 1) glomerular hypertrophy, mesangial matrix expansion and podocyte loss without changes in glomerular filtration rate nor albuminuria, 2) lower renal cortical activity.In summary, loss of ACE2 in the NOD strain contributes to a glucose homeostasis and insulin impairment involving structural alterations within the glomerulus, including: increased glomerular area, mesangial matrix expansion and podocyte loss

Characterization of ACE and ACE2 Expression within Different Organs of the NOD Mouse

Renin angiotensin system (RAS) is known to play a key role in several diseases such as diabetes, and renal and cardiovascular pathologies. Its blockade has been demonstrated to delay chronic kidney disease progression and cardiovascular damage in diabetic patients. In this sense, since local RAS has been described, the aim of this study is to characterize angiotensin converting enzyme (ACE) and ACE2 activities, as well as protein expression, in several tissues of the non-obese diabetic (NOD) mice model. After 21 or 40 days of diabetes onset, mouse serums and tissues were analyzed for ACE and ACE2 enzyme activities and protein expression. ACE and ACE2 enzyme activities were detected in different tissues. Their expressions vary depending on the studied tissue. Thus, whereas ACE activity was highly expressed in lungs, ACE2 activity was highly expressed in pancreas among the studied tissues. Interestingly, we also observed that diabetes up-regulates ACE mainly in serum, lung, heart, and liver, and ACE2 mainly in serum, liver, and pancreas. In conclusion, we found a marked serum and pulmonary alteration in ACE activity of diabetic mice, suggesting a common regulation. The increase of ACE2 activity within the circulation in diabetic mice may be ascribed to a compensatory mechanism of RAS

Caracterització de la nova línia nod.Ace2-/-: estudi de l'aparició de diabetis i la progressió de la nefropatia diabètica

L'enzim convertidor d'angiotensina (ECA) promou la formació de l'angiotensina II (Ang-II). Contràriament, l'ECA2, una monocarboxipeptidasa que presenta un 40% d'homologia amb l'ECA, degrada l'Ang-II (accions vasoconstrictores) a angiotensina 1-7 (Ang-(1-7) (accions vasodilatadores). L'eix ECA2/Ang-(1-7) desencadena accions oposades a les de l'eix ECA/Ang-II. A nivell clínic, el tractament amb inhibidors de l'ECA i/o bloquejants dels receptors d'angiotensina en pacients amb nefropatia diabètica (ND) ha demostrat disminuir l'albuminúria i frenar la progressió cap a malaltia renal crònica (MRC). Donat aquest fet i que l'ECA2 és un regulador negatiu del sistema renina angiotensina (SRA), hipotetitzem que la manca d'ECA2 contribuirà a un augment de l'aparició de la diabetis i a una major progressió de la ND en un model experimental espontani de diabetis tipus 1, la soca no obesa diabètica (NOD). Amb aquesta finalitat s'ha generat la nova línia de ratolins NOD.ACE2-/- i els respectius controls. L'estudi de la diabetis en els animals NOD.ACE2-/- s'ha observat: 1) viabilitat, fertilitat i, fenotípicament, una alteració de la coloració del pelatge i menor pes corporal, 2) major aparició de diabetis acompanyada d'una alteració en l'homeòstasi de la glucosa i 3) augment en la sensibilitat a la insulina exògena. En l'estudi de la ND, els animals NOD.ACE2-/- mostren: 1) hipertrofia glomerular, expansió de la matriu mesangial i pèrdua podocitària sense alteracions en la filtració glomerular ni l'albuminúria, 2) menor activitat renal cortical d'ECA. En resum, la manca d'ECA2 en la soca NOD contribueix a un empitjorament en l'homeòstasi de la glucosa i de la insulina així com alteracions estructurals a nivell del glomèrul que inclouen: augment de l'àrea glomerular, expansió de la matriu mesangial i pèrdua podocitària.Angiotensin converting enzyme (ACE) promotes angiotensin II (Ang II) formation. Conversely, ACE2, a monocarboxipeptidase that shares 40% homology with ACE, degrades promotes angiotensin 1-7 (Ang- (1-7) (vasodilator actions) formation from Ang-II (vasoconstrictor actions). ACE2/Ang-(1-7) axis actions opposite ACE/Ang-II axis actions. ACE inhibitors and/or angiotensin receptor blockers treatment in patients with diabetic nephropathy (DN) have shown to blunt albuminuria progression to chronic kidney disease (CKD). Given this and the fact that ACE2 acts as a negative regulator of the renin angiotensin system (RAS), we hypothesize that ECA2 loss contribute to an increase in diabetis development and a greater progression of DN in an experimental model of spontaneous type 1 diabetes, the non obese diabetic (NOD) mice. With this aim, we have generated a new experimental model, the NOD.ACE2-/- mice and their respective controls. The study of diabetes onset in NOD.ACE2-/- showed: 1) viability, fertility and, phenotypically, a change in coat color and lower body weight, 2) higher diabetis incidence accompanied by altered glucose homeostasis and 3) increase in exogenous insulin sensitivity. Within the DN study, NOD.ACE2-/- mice showed: 1) glomerular hypertrophy, mesangial matrix expansion and podocyte loss without changes in glomerular filtration rate nor albuminuria, 2) lower renal cortical activity.In summary, loss of ACE2 in the NOD strain contributes to a glucose homeostasis and insulin impairment involving structural alterations within the glomerulus, including: increased glomerular area, mesangial matrix expansion and podocyte loss