Calcium Signaling Is Impaired in PTEN-Deficient T Cell Acute Lymphoblastic Leukemia.

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 TCRαß+ T-ALL development. Herein, we explored the functional interaction between TCRαß 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 Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel 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 Ptendel blasts were unable to release calcium ions (Ca2+) 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.

MYC deficiency impairs the development of effector/memory T lymphocytes.

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.

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy.

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 (CD19neg) 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 CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

A novel flavanone derivative inhibits dengue virus fusion and infectivity.

Dengue infection is a global burden affecting millions of world population. Previous studies indicated that flavanones were potential dengue virus inhibitors. We discovered that a novel flavanone derivative, 5-hydroxy-7-methoxy-6-methylflavanone (FN5Y), inhibited DENV2 pH-dependent fusion in cell-based system with strong binding efficiency to DENV envelope protein at K (P83, L107, K128, L198), K' (T48, E49, A50, L198, Q200, L277), X' (Y138, V354, I357), and Y' (V97, R99, N103, K246) by molecular dynamic simulation. FN5Y inhibited DENV2 infectivity with EC50s (and selectivity index) of 15.99 ±â€¯5.38 (>6.25), and 12.31 ±â€¯1.64 (2.23) µM in LLC/MK2 and Vero cell lines, respectively, and inhibited DENV4 at 11.70 ±â€¯6.04 (>8.55) µM. CC50s in LLC/MK2, HEK-293, and HepG2 cell lines at 72 h were higher than 100 µM. Time-of-addition study revealed that the maximal efficacy was achieved at early after infection corresponded with pH-dependent fusion. Inactivating the viral particle, interfering with cellular receptors, inhibiting viral protease, or the virus replication complex were not major targets of this compound. FN5Y could become a potent anti-flaviviral drug and can be structurally modified for higher potency using simulation to DENV envelope as a molecular target.