Generation of a patient-derived induced pluripotent cell line (SCTCi016-A) carrying a homozygous variant in RPE65.

Leber congenital amaurosis (LCA) can be caused by mutations in more than 20 different genes. One of these, RPE65, encodes a protein essential for the visual cycle that is expressed in retinal pigment epithelium cells. In this work, we describe the generation and characterization of the human iPSC line SCTCi16-A. This hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) from a patient affected with LCA caused by the homozygous c.11+5G>A variant in the RPE65 gene. Reprograming was conducted using episomal vectors containing OCT3/4, SOX2, KLF4, L-MYC, and LIN28.

Exploiting the passenger ACO1-deficiency arising from 9p21 deletions to kill T-cell lymphoblastic neoplasia cells.

Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.

Downregulation of specific FBXW7 isoforms with differential effects in T-cell lymphoblastic lymphoma.

FBXW7 is a driver gene in T-cell lymphoblastic neoplasia acting through proteasome degradation of key proto-oncogenes. FBXW7 encodes three isoforms, α, ß and γ, which differ only in the N-terminus. In this work, massive sequencing revealed significant downregulation of FBXW7 in a panel of primary T-cell lymphoblastic lymphomas characterised by the absence of mutations in its sequence. We observed that decreased expression mainly affected the FBXW7ß isoform and to a lesser extent FBXW7α and may be attributed to the combined effect of epigenetic changes, alteration of upstream factors and upregulation of miRNAs. Transient transfections with miRNA mimics in selected cell lines resulted in a significant decrease of total FBXW7 expression and its different isoforms separately, with the consequent increment of critical substrates and the stimulation of cell proliferation. Transient inhibition of endogenous miRNAs in a T-cell lymphoblastic-derived cell line (SUP-T1) was capable of reversing these proliferative effects. Finally, we show how FBXW7 isoforms display different roles within the cell. Simultaneous downregulation of the α and γ isoforms modulates the amount of CCNE1, whilst the ß-isoform alone was found to have a prominent role in modulating the amount of c-MYC. Our data also revealed that downregulation of all isoforms is a sine qua non condition to induce a proliferative pattern in our cell model system. Taking these data into account, potential new treatments to reverse downregulation of all or a specific FBXW7 isoform may be an effective strategy to counteract the proliferative capacity of these tumour cells.