Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in Multiple Myeloma.

Hematological toxicity is a common side effect of CAR-T therapies, particularly severe in relapsed/refractory multiple myeloma (MM) patients. In this study, we analyzed a cohort of 48 patients treated with BCMA CAR-T cells to characterize the kinetics of cytopenia, identify predictive factors and determine potential mechanism underlying these toxicities. The overall incidence of cytopenia was 95.7%, and grade>3 thrombocytopenia and neutropenia, one month after infusion, was observed in 57% and 53% of the patients, being still present after one year in 4 and 3 patients respectively. Presence of cytopenia at baseline and high peak inflammatory markers highly correlated with cytopenia persisting up to three months. To determine potential mechanisms underpinning cytopenias, we evaluated the paracrine effect of BCMA CAR-T cells on HSPCs differentiation using an ex-vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-T cells (spCAR) halted HSPCs differentiation, promoting more immature phenotypes, with reduced expression of granulocytic, monocytic and erythroid markers, which could be prevented with a combination of IFNγ, TNFα/ß, TGFß, IL-6 and IL-17 inhibitors. Single-cell RNA-seq demonstrated upregulation of transcription factors associated with early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and decreased activity of key regulons involved in neutrophil and monocytic maturation (ID2, MAFB). Our results suggest that CAR-T cell activation negatively influences hematopoietic differentiation through paracrine effects inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.

The Altered Transcriptome and DNA Methylation Profiles of Docetaxel Resistance in Breast Cancer PDX Models.

Taxanes are standard therapy in clinical practice for metastatic breast cancer; however, primary or acquired chemoresistance are a common cause of mortality. Breast cancer patient-derived xenografts (PDX) are powerful tools for the study of cancer biology and drug treatment response. Specific DNA methylation patterns have been associated to different breast cancer subtypes but its association with chemoresistance remains unstudied. Aiming to elucidate docetaxel resistance mechanisms, we performed genome-wide DNA methylation in breast cancer PDX models, including luminal and triple-negative breast cancer (TNBC) models sensitive to docetaxel, their matched models after emergence of chemoresistance and residual disease after short-term docetaxel treatment. We found that DNA methylation profiles from breast cancer PDX models maintain the subtype-specific methylation patterns of clinical samples. Two main DNA methylation clusters were found in TNBC PDX and remain stable during the emergence of docetaxel resistance; however, some genes/pathways were differentially methylated according to docetaxel response. A DNA methylation signature of resistance able to segregate TNBC based on chemotherapy response was identified. Transcriptomic profiling of selected sensitive/resistant pairs and integrative analysis with methylation data demonstrated correlation between some differentially methylated and expressed genes in docetaxel-resistant TNBC PDX models. Multiple gene expression changes were found after the emergence of docetaxel resistance in TNBC. DNA methylation and transcriptional changes identified between docetaxel-sensitive and -resistant TNBC PDX models or residual disease may have predictive value for chemotherapy response in TNBC. IMPLICATIONS: Subtype-specific DNA methylation patterns are maintained in breast cancer PDX models. While no global methylation changes were found, we uncovered differentially DNA methylated and expressed genes/pathways associated with the emergence of docetaxel resistance in TNBC.

Root phosphoenolpyruvate carboxylase and NAD-malic enzymes activity increase the ammonium-assimilating capacity in tomato.

Plant ammonium tolerance has been associated with the capacity to accumulate large amounts of ammonium in the root vacuoles, to maintain carbohydrate synthesis and especially with the capacity of maintaining high levels of inorganic nitrogen assimilation in the roots. The tricarboxylic acid cycle (TCA) is considered a cornerstone in nitrogen metabolism, since it provides carbon skeletons for nitrogen assimilation. The hypothesis of this work was that the induction of anaplerotic routes of phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH) and malic enzyme (NAD-ME) would enhance tolerance to ammonium nutrition. An experiment was established with tomato plants (Agora Hybrid F1) grown under different ammonium concentrations. Growth parameters, metabolite contents and enzymatic activities related to nitrogen and carbon metabolism were determined. Unlike other tomato cultivars, tomato Agora Hybrid F1 proved to be tolerant to ammonium nutrition. Ammonium was assimilated as a biochemical detoxification mechanism, thus leading to the accumulation of Gln and Asn as free amino acids in both leaves and roots as an innocuous and transitory store of nitrogen, in addition to protein synthesis. When the concentration of ammonium in the nutrient solution was high, the cyclic operation of the TCA cycle seemed to be interrupted and would operate in two interconnected branches to provide α-ketoglutarate for ammonium assimilation: one branch supported by malate accumulation and by the induction of anaplerotic PEPC and NAD-ME in roots and MDH in leaves, and the other branch supported by stored citrate in the precedent dark period.