T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy affecting both children and adults. Given its persistently poor prognosis, there is a critical need to identify additional factors involved in T-ALL oncogenesis and progression. CD9, a membrane protein of the tetraspanin family implicated in diverse cellular processes, has been associated with prognosis in several cancers, yet its role in T-ALL remains poorly understood. In this study, using a mouse model first, we found that CD9 overexpression is associated with leukemic T cells that have migrated outside the thymus into peripheral tissues. Then, analysis of a human T-ALL cohort shows that CD9 expression is heterogeneous, tends to increase at relapse and is enriched in the TAL1⁺ molecular subtype. We further demonstrate that CD9⁺ cells display enhanced migratory capacity compared with CD9⁻ counterparts, and that CD9 levels affect extracellular vesicle biogenesis. Altogether, our findings support a role for CD9 in T-ALL leukemogenesis and highlight its potential involvement in relapse.

ABSTRACT:T-cell acute lymphoblastic leukemia (T-ALL) is a malignant proliferation of T-cell progenitors originating in the thymus. T-ALL is a heterogenous disease involving the dysregulation of various oncogenes/tumor-suppressor (TS) genes. Loss of the TS gene phosphatase and TENsin homolog (PTEN) is a recurrent alteration, which is often associated with a mature T-ALL subgroup expressing a T-cell receptor. Herein, we used a single-cell RNA-sequencing approach to investigate the impact of the absence of PTEN on pathological development of mouse thymocytes. First, our differential gene expression analysis of tumor cells vs physiologic cells uncovers an ectopic expression, in leukemic cells, of the gene encoding Bex1. Then, to determine the relevance of our observation in humans, we queried a public RNA-sequencing database from the TARGET-TCGA (Therapeutically Applicable Research to Generate Effective Treatments-The Cancer Genome Atlas) project. We show that BEX1, BEX2, and BEX5 genes are ectopically expressed in T-ALL samples and we further found that ectopic BEX expression is mainly restricted to the T-ALL subgroup overexpressing TAL1 oncogene. Proximity ligation assays demonstrated the nuclear colocalization of brain-expressed X-linked 1/2 (BEX1/2) proteins with T-cell acute lymphocytic leukemia protein 1 (TAL1) in T-ALL cells. To investigate their functional role, we generated Jurkat cells with a triple knockout of BEX1, BEX2, and BEX5 using CRISPR-CRISPR-associated protein 9. This genetic inactivation led to reduced cell proliferation, a loss of histone H3 lysine 4 monomethylation (H3K4me1) marks notably at genomic regions enriched for E-box motifs, and dysregulation of several TAL1 target genes. Collectively, our findings suggest that BEX1 and BEX2 may contribute to human T-ALL oncogenesis by acting as cofactors within the TAL1 complex.

PTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ + T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Pten del thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Pten del thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Pten del blasts were unable to release calcium ions (Ca 2+ ) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized.

In the thymus, T cell progenitors differentiate in order to generate naive T lymphocytes which migrate in the periphery where they will fulfill their function in the adaptive immune response. During thymopoiesis, genomic alterations in thymocytes can promote leukemia development. Among recurrent alteration is PTEN inactivation, which is associated to MYC overexpression. Herein, we used conditional Pten and Myc knockout mouse models and single-cell RNA-sequencing approach, to investigate the impact of MYC loss on physio-pathological development of PTEN-proficient or PTEN-deficient T lymphocytes. First, our results confirm that MYC is mandatory for PTEN loss-mediated leukemogenesis, while it is not required for terminal steps of thymopoiesis. In contrast, we uncovered that Myc ablation in CD4+CD8+ thymocytes disrupts T lymphocytes homeostasis in the spleen, notably by drastically reducing the number of MYC-deficient effector/memory T cells. Collectively, our data show that besides naive T cells proliferation, MYC is essential for effector/memory differentiation.

Multiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here, we use antibodies tagged with a hashtag oligonucleotide (Ab-HTO) to label each sample, and 10× Genomics technology to analyze single-cell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein, we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development. For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19 neg ) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19 neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19 neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19 neg subclones, raising the possibility to assess the risk of targeted therapy failure.

Specific antigen recognition by T cell receptor (TCR) activates TCR signaling pathway, leading to T cell proliferation and differentiation into effector and memory cells. Herein, we describe protocols for TCR stimulation assays, including procedures for the isolation and enrichment of mouse splenic T cells for ex vivo TCR stimulation with anti-CD3/CD28 antibodies, and the use of ovalbumin-OT-II mouse model for in vivo TCR stimulation. We applied this protocol to show that MYC protein is essential for T cell proliferation and differentiation.For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).