Optimization of universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of acute myeloid leukemia.

Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes (HLA-IKO/TCRKO CAR-T cells) for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile to non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, the production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allows the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment.

Percepciones y sentimientos de niños argentinos pospandemia por COVID-19 / Perceptions and feelings of Argentine children post pandemic COVID-19

La mortalidad y formas graves atribuibles al COVID-19 en población pediátrica han sido bajas en comparación con los adultos. No obstante, los niños, niñas y adolescentes (NNA) constituyen un universo especialmente afectado por la pandemia, en cuanto a restricción y limitación de sus derechos. La Subcomisión de Derechos de la Niñez y Adolescencia y el Comité de Pediatría Social de la Sociedad Argentina de Pediatría entendieron trascendente recolectar sus voces para relevar el impacto que la transición de la situación de pandemia por COVID-19 a la actual "nueva normalidad" ha producido en los sentimientos, emociones y deseos de los NNA en Argentina. Realizamos un estudio cualitativo, descriptivo, basado en encuesta anónima, entre NNA argentinos de 6 a 18 años residentes en Argentina. El análisis incluyó 1537 entrevistas efectivas. Los resultados permitieron sugerir recomendaciones para desarrollar estrategias de afrontamiento, contención y acompañamiento de los NNA en la pospandemia.

The frequency of the severity of the different expressions of SARS-COV-2 disease, and its mortality in the pediatric population have been low unlike in the adult population. However, children and adolescents have been very affected by this virus, through the restriction and limitations of their rights. The Subcommittee on the Rights of the Childhood and Adolescence and the Committee of Social Pediatrics of the Sociedad Argentina de Pediatría understood that it was very important to gather their voices to weigh the impact of the transition from COVID pandemic to this "new normalcy" and the effects on argentine children and adolescents' rights, emotions and desires, especially those related to accessibility to education and health. We carried on a qualitative descriptive narrative transversal phenomenological research, through an open anonymous survey, among children and adolescents between 6 and 18 years old, living in Argentina; 1537 surveys were collected. The research findings allowed elaborate recommendations to develop strategies to face, protect and accompany the children and adolescents during the post-pandemic.

[Perceptions and feelings of Argentine children post pandemic COVID-19]. / Percepciones y sentimientos de niños argentinos pospandemia por COVID-19.

The frequency of the severity of the different expressions of SARS-COV-2 disease, and its mortality in the pediatric population have been low unlike in the adult population. However, children and adolescents have been very affected by this virus, through the restriction and limitations of their rights. The Subcommittee on the Rights of the Childhood and Adolescence and the Committee of Social Pediatrics of the Sociedad Argentina de Pediatría understood that it was very important to gather their voices to weigh the impact of the transition from COVID pandemic to this "new normalcy" and the effects on argentine children and adolescents' rights, emotions and desires, especially those related to accessibility to education and health. We carried on a qualitative descriptive narrative transversal phenomenological research, through an open anonymous survey, among children and adolescents between 6 and 18 years old, living in Argentina; 1537 surveys were collected. The research findings allowed elaborate recommendations to develop strategies to face, protect and accompany the children and adolescents during the post-pandemic.

La mortalidad y formas graves atribuibles al COVID-19 en población pediátrica han sido bajas en comparación con los adultos. No obstante, los niños, niñas y adolescentes (NNA) constituyen un universo especialmente afectado por la pandemia, en cuanto a restricción y limitación de sus derechos. La Subcomisión de Derechos de la Niñez y Adolescencia y el Comité de Pediatría Social de la Sociedad Argentina de Pediatría entendieron trascendente recolectar sus voces para relevar el impacto que la transición de la situación de pandemia por COVID-19 a la actual "nueva normalidad" ha producido en los sentimientos, emociones y deseos de los NNA en Argentina. Realizamos un estudio cualitativo, descriptivo, basado en encuesta anónima, entre NNA argentinos de 6 a 18 años residentes en Argentina. El análisis incluyó 1537 entrevistas efectivas. Los resultados permitieron sugerir recomendaciones para desarrollar estrategias de afrontamiento, contención y acompañamiento de los NNA en la pospandemia.

CAR density influences antitumoral efficacy of BCMA CAR T cells and correlates with clinical outcome.

Identification of new markers associated with long-term efficacy in patients treated with CAR T cells is a current medical need, particularly in diseases such as multiple myeloma. In this study, we address the impact of CAR density on the functionality of BCMA CAR T cells. Functional and transcriptional studies demonstrate that CAR T cells with high expression of the CAR construct show an increased tonic signaling with up-regulation of exhaustion markers and increased in vitro cytotoxicity but a decrease in in vivo BM infiltration. Characterization of gene regulatory networks using scRNA-seq identified regulons associated to activation and exhaustion up-regulated in CARHigh T cells, providing mechanistic insights behind differential functionality of these cells. Last, we demonstrate that patients treated with CAR T cell products enriched in CARHigh T cells show a significantly worse clinical response in several hematological malignancies. In summary, our work demonstrates that CAR density plays an important role in CAR T activity with notable impact on clinical response.

Effect of a Diet Supplemented with Sphingomyelin and Probiotics on Colon Cancer Development in Mice.

Previous studies have reported that dietary sphingomyelin could inhibit early stages of colon cancer. Lactic acid-producing bacteria have also been associated with an amelioration of cancer symptoms. However, little is known about the potential beneficial effects of the combined administration of both sphingomyelin and lactic acid-producing bacteria. This article analyzes the effect of a diet supplemented with a combination of the probiotics Lacticaseibacillus casei and Bifidobacterium bifidum (108 CFU/ml) and sphingomyelin (0.05%) on mice with 1,2-dimethylhydrazine (DMH)-induced colon cancer. Thirty-six BALB/c mice were divided into 3 groups: one healthy group (group C) and two groups with DMH-induced cancer, one fed a standard diet (group D) and the other fed a diet supplemented with sphingomyelin and probiotics (DS). The number of aberrant crypt foci, marker of colon cancer development, was lower in the DS. The dietary supplementation with the synbiotic reversed the cancer-induced impairment of galactose uptake in enterocyte brush-border-membrane vesicles. These results confirm the beneficial effects of the synbiotic on the intestinal physiology of colon cancer mice and contribute to the understanding of the possible mechanisms involved.

CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation.

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.

C3G downregulation induces the acquisition of a mesenchymal phenotype that enhances aggressiveness of glioblastoma cells.

Glioblastoma (GBM) is the most aggressive tumor from the central nervous system (CNS). The current lack of efficient therapies makes essential to find new treatment strategies. C3G, a guanine nucleotide exchange factor for some Ras proteins, plays a dual role in cancer, but its function in GBM remains unknown. Database analyses revealed a reduced C3G mRNA expression in GBM patient samples. C3G protein levels were also decreased in a panel of human GBM cell lines as compared to astrocytes. Based on this, we characterized C3G function in GBM using in vitro and in vivo human GBM models. We report here that C3G downregulation promoted the acquisition of a more mesenchymal phenotype that enhanced the migratory and invasive capacity of GBM cells. This facilitates foci formation in anchorage-dependent and -independent growth assays and the generation of larger tumors in xenografts and chick chorioallantoic membrane (CAM) assays, but with a lower cell density, as proliferation was reduced. Mechanistically, C3G knock-down impairs EGFR signaling by reducing cell surface EGFR through recycling inhibition, while upregulating the activation of several other receptor tyrosine kinases (RTKs) that might promote invasion. In particular, FGF2, likely acting through FGFR1, promoted invasion of C3G-silenced GBM cells. Moreover, ERKs mediate this invasiveness, both in response to FGF2- and serum-induced chemoattraction. In conclusion, our data show the distinct dependency of GBM tumors on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibition protocols. Hence, patients with a low C3G expression might not respond to EGFR inhibitors.

The novel proautophagy anticancer drug ABTL0812 potentiates chemotherapy in adenocarcinoma and squamous nonsmall cell lung cancer.

Around 40% of newly diagnosed lung cancer patients are Stage IV, where the improvement of survival and reduction of disease-related adverse events is the main goal for oncologists. In this scenario, we present preclinical evidence supporting the use of ABTL0812 in combination with chemotherapy for treating advanced and metastatic Nonsmall cell lung adenocarcinomas (NSCLC) and squamous carcinomas. ABTL0812 is a new chemical entity, currently in Phase 1b/2a clinical trial for advanced squamous NSCLC in combination with paclitaxel and carboplatin (P/C), after successfully completing the first-in-human trial where it showed an excellent safety profile and signs of efficacy. We show here that ABTL0812 inhibits Akt/mTOR axis by inducing the overexpression of TRIB3 and activating autophagy in lung squamous carcinoma cell lines. Furthermore, treatment with ABTL0812 also induces AMPK activation and ROS accumulation. Moreover, combination of ABTL0812 with chemotherapy markedly increases the therapeutic effect of chemotherapy without increasing toxicity. We further show that combination of ABTL0812 and chemotherapy induces nonapoptotic cell death mediated by TRIB3 activation and autophagy induction. We also present preliminary clinical data indicating that TRIB3 could serve as a potential novel pharmacodynamic biomarker to monitor ABTL0812 activity administered alone or in combination with chemotherapy in squamous NSCLC patients. The safety profile of ABTL0812 and its good synergy with chemotherapy potentiate the therapeutic potential of current lines of treatment based on chemotherapy regimens, arising as a promising option for improving these patients therapeutic expectancy.

Tumor-Associated Fibroblasts Promote HER2-Targeted Therapy Resistance through FGFR2 Activation.

PURPOSE: Despite the therapeutic success of existing HER2-targeted therapies, tumors invariably relapse. This study aimed at identifying new mechanisms responsible for HER2-targeted therapy resistance. EXPERIMENTAL DESIGN: We have used a platform of HER2-targeted therapy-resistant cell lines and primary cultures of healthy and tumor-associated fibroblasts (TAF) to identify new potential targets related to tumor escape from anti-HER2 therapies. RESULTS: We have shown that TAFs promote resistance to HER2-targeted therapies. TAFs produce and secrete high levels of FGF5, which induces FGFR2 activation in the surrounding breast cancer cells. FGFR2 transactivates HER2 via c-Src, leading to resistance to HER2-targeted therapies. In vivo, coinoculating nonresistant cell lines with TAFs results in more aggressive and resistant tumors. Resistant cells activate fibroblasts and secrete FGFR ligands, creating a positive feedback loop that fuels resistance. FGFR2 inhibition not only inhibits HER2 activation, but also induces apoptosis in cells resistant to HER2-targeted therapies. In vivo, inhibitors of FGFR2 reverse resistance and resensitize resistant cells to HER2-targeted therapies. In HER2 patients' samples, α-SMA, FGF5, and FGFR2 contribute to poor outcome and correlate with c-Src activation. Importantly, expression of FGF5 and phospho-HER2 correlated with a reduced pathologic complete response rate in patients with HER2-positive breast cancer treated with neoadjuvant trastuzumab, which highlights the significant role of TAFs/FGF5 in HER2 breast cancer progression and resistance. CONCLUSIONS: We have identified the TAF/FGF5/FGFR2/c-Src/HER2 axis as an escape pathway responsible for HER2-targeted therapy resistance in breast cancer, which can be reversed by FGFR inhibitors.

Histamine receptor 1 inhibition enhances antitumor therapeutic responses through extracellular signal-regulated kinase (ERK) activation in breast cancer.

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine triggers cell proliferation, embryonic development, and tumor growth. We recently established that HRH1 is up-regulated in basal and human epidermal growth factor receptor 2 (HER2)-enriched human breast tumors and that its expression correlates with a worse prognosis. Nevertheless, the functional role of HRH1 in basal and HER2-targeted therapy-resistant breast cancer (BC) progression has not yet been addressed. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that the inhibition of HRH1 activity in basal BC cells leads to sub-G0 cell accumulation, suppresses proliferation, promotes cell motility and triggers the activation of extracellular signal-regulated kinase (ERK) signaling, initiating the mitochondrial apoptotic pathway. Furthermore, HER2-targeted therapy-resistant cells express higher levels of HRH1 and are more sensitive to terfenadine treatment. Moreover, in vivo experiments showed that terfenadine therapy reduced the tumor growth of basal and trastuzumab-resistant BC cells. In conclusion, our results suggest that targeting HRH1 is a promising new clinical approach to consider that could enhance the effectiveness of current therapeutic treatment in patients with basal and BC tumors resistant to HER2-targeted therapies.

The oncolytic adenovirus Δ24-RGD in combination with cisplatin exerts a potent anti-osteosarcoma activity.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24-RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24-RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24-RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24-RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24-RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24-RGD for this devastating disease.

A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool.

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. METHODS: To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. RESULTS: We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. CONCLUSIONS: Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Ácidos grasos trans en alimentos consumidos habitualmente por los jóvenes en Argentina

Resumen Introducción: en nuestros jóvenes, la alimentación inadecuada, uno de los factores de riesgo para las enfermedades cardiovasculares, se observa desde la infancia y tiende a persistir en la etapa adulta. El proceso de aterosclerosis se inicia en la infancia, con la formación de las estrías grasas en la pared vascular. Entre los alimentos que repercuten negativamente se encuentran los ácidos grasos saturados y los insaturados con configuración trans. El objetivo fue investigar la presencia de ácidos grasos trans en alimentos de consumo frecuente en jóvenes en Argentina, para proponer estrategias tendientes a disminuir su disponibilidad y consumo. Material y métodos: se estudiaron margarinas, mantecas, mayonesas, galletitas, alfajores y productos de copetín. Se determinó el contenido lipídico por el método de Folch. La composición en ácidos grasos se determinó por cromatografía gaseosa. Resultados: se encontró una importante presencia de ácidos grasos trans en los alimentos, aun en aquellos promocionados como con bajo contenido de colesterol. En algunos casos, el contenido de ácidos grasos saturados y trans es superior al de insaturados cis. El contenido de ácidos grasos poliinsaturados omega-6 es importante en algunos alimentos y, en general, el contenido de ácidos grasos poliinsaturados omega-3 es bajo. Conclusiones: debido al alto contenido de ácidos grasos trans encontrado en los alimentos analizados, además de una relación omega-6/omega-3 no deseada, se hace necesaria la implementación de estrategias que mejoren la calidad nutricional de los alimentos que consumen nuestros jóvenes.

Summary Introduction: in our youths, inadequate nutrition, a risk factor for cardiovascular disease, is observed since childhood and trends to persist in the adult age. This leads to the atherosclerotic process starting in childhood, with formation of fatty streaks on the vessel wall. Saturated fatty acids and unsaturated fatty acids with trans configuration are some of the nutrients that have a negative influence on health. The objective of this study was to investigate the trans fatty acid content in frequently consumed foods among young people in Argentina,and to propose strategies leading to decrease its availability and consumption. Materials and methods: the products studied were margarine, butter, mayonnaise, cookies, “alfajores” (a typical sweet in Argentina, made of two cookies joined with jam) and snack products. The lipid content was measured by the Folch technique. The fatty acid composition was assessed by gas chromatography. Results: the results showed a large amount of trans fatty acids present in the foods evaluated,including those products advertised as having low cholesterol content.In some cases,the contents of saturated and trans fatty acids was higher than that of unsaturated cis fatty acids.Omega-6 polyunsaturated fatty acid content in some foods was high and omega-3 polyunsaturated fatty acids content was low. Conclusions: given the high contents of trans fatty acids found in the analysed foods,together with an unwanted omega-6/omega-3 ratio,the imple- mentation of strategies improving the nutritional quality of food eaten by our youths becomes necessary.

Acidos grasos trans en alimentos consumidos habitualmente por los jóvenes en Argentina / Trans-fatty acids usually ingested by youths in Argentina

Introducción. En nuestros jóvenes, la alimentacióninadecuada, uno de los factores de riesgo para lasenfermedades cardiovasculares, se observa desdela infancia y tiende a persistir en la etapa adulta. Elproceso de aterosclerosis se inicia en la infancia,con la formación de las estrías grasas en la paredvascular. Entre los alimentos que repercuten negativamentese encuentran los ácidos grasos saturadosy los insaturados con configuración trans. Elobjetivo fue investigar la presencia de ácidos grasostrans en alimentos de consumo frecuente en jóvenesen Argentina, para proponer estrategias tendientesa disminuir su disponibilidad y consumo.Material y métodos. Se estudiaron margarinas, mantecas,mayonesas, galletitas, alfajores y productosde copetín. Se determinó el contenido lipídico porel método de Folch. La composición en ácidos grasosse determinó por cromatografía gaseosa.Resultados. Se encontró una importante presenciade ácidos grasos trans en los alimentos, aun enaquellos promocionados como con bajo contenidode colesterol. En algunos casos, el contenido deácidos grasos saturados y trans es superior al deinsaturados cis. El contenido de ácidos grasospoliinsaturados omega-6 es importante en algunosalimentos y, en general, el contenido de ácidosgrasos poliinsaturados omega-3 es bajo.Conclusiones. Debido al alto contenido de ácidosgrasos trans encontrado en los alimentos analizados,además de una relación omega-6/omega-3 no deseada, se hace necesaria la implementación de estrategias que mejoren la calidad nutricional de los alimentos que consumen nuestros jóvenes.(AU)

Ácidos grasos trans en alimentos consumidos habitualmente por los jóvenes en Argentina / Trans-fatty acids usually ingested by youths in Argentina

Introducción. En nuestros jóvenes, la alimentacióninadecuada, uno de los factores de riesgo para lasenfermedades cardiovasculares, se observa desdela infancia y tiende a persistir en la etapa adulta. Elproceso de aterosclerosis se inicia en la infancia,con la formación de las estrías grasas en la paredvascular. Entre los alimentos que repercuten negativamentese encuentran los ácidos grasos saturadosy los insaturados con configuración trans. Elobjetivo fue investigar la presencia de ácidos grasostrans en alimentos de consumo frecuente en jóvenesen Argentina, para proponer estrategias tendientesa disminuir su disponibilidad y consumo.Material y métodos. Se estudiaron margarinas, mantecas,mayonesas, galletitas, alfajores y productosde copetín. Se determinó el contenido lipídico porel método de Folch. La composición en ácidos grasosse determinó por cromatografía gaseosa.Resultados. Se encontró una importante presenciade ácidos grasos trans en los alimentos, aun enaquellos promocionados como con bajo contenidode colesterol. En algunos casos, el contenido deácidos grasos saturados y trans es superior al deinsaturados cis. El contenido de ácidos grasospoliinsaturados omega-6 es importante en algunosalimentos y, en general, el contenido de ácidosgrasos poliinsaturados omega-3 es bajo.Conclusiones. Debido al alto contenido de ácidosgrasos trans encontrado en los alimentos analizados,además de una relación omega-6/omega-3 no deseada, se hace necesaria la implementación de estrategias que mejoren la calidad nutricional de los alimentos que consumen nuestros jóvenes.