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LSD1 is a histone lysine demethylase proposed as therapeutic target in cancer. Chemical modifications applied at C2, C4 and/or C7 positions of the quinazoline core of the previously reported dual LSD1/G9a inhibitor 1 led to a series of non-covalent, highly active, and selective LSD1 inhibitors (2-4 and 6-30) and to the dual LSD1/G9a inhibitor 5 that was more potent than 1 against LSD1. In THP-1 and MV4-11 leukemic cells, the most potent compounds (7, 8, and 29) showed antiproliferative effects at sub-micromolar level without significant toxicity at 1μM in non-cancer AHH-1cells. In MV4-11cells, the new derivatives increased the levels of the LSD1 histone mark H3K4me2 and induced the re-expression of the CD86 gene silenced by LSD1, thereby confirming the inhibition of LSD1 at cellular level. In breast MDA-MB-231 as well as in rhabdomyosarcoma RD and RH30cells, taken as examples of solid tumors, the same compounds displayed cell growth arrest in the same IC50 range, highlighting a crucial anticancer role for LSD1 inhibition and suggesting no added value for the simultaneous G9a inhibition in these tumor cell lines.

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer. ispartof: CELL DEATH AND DIFFERENTIATION vol:29 issue:3 pages:614-626 ispartof: location:England status: published

The role of proNGF, the precursor of Nerve Growth Factor (NGF), on the biology of adult neural stem cells (aNSCs) is still unclear. Here I analyzed adult hippo-campal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. I found in-creased proliferation of progenitors but a reduced neurogenesis in the AD11 DG- hippocampus (HP-DG). Also in vitro, AD11 hippocampal neural stem cells (NSCs) pro-liferated more but were unable to differentiate into morphologically mature neu-rons. By treating wild-type (WT) hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR) I demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) neural stem cells through the induction of cyclin D1 expression. These cells express high level of p75NTR, as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly-dissociated HP-DG or selected in vitro from NSCs by LIF (leukemia inhibitory factor). Clonogenic assay per-formed in the absence of mitogens showed that RGLs respond to proNGF-KR by re-activating their proliferation and thus leading to neurospheres formation. The mito-genic effect of proNGF was further exploited in the expansion of mouse induced Neural Stem Cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, I demonstrated that proNGF acts as mitogen on hippo-campal and induced neural stem cells. The role of proNGF, the precursor of nerve growth factor (NGF), in the biology of adult neural stem cells (aNSCs) is still unclear. Here, we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalance of proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 dentate gyrus (DG)-hippocampus (HP). Also in vitro, AD11 hippocampal neural stem cells (NSCs) proliferated more, but were unable to differentiate into morphologically mature neurons. By treating wild-type hippocampal progenitors with the uncleavable form of proNGF (proNGF-KR), we demonstrated that proNGF acts as mitogen on aNSCs at low concentration. The mitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high levels of p75NTR, as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly dissociated HP-DG or selected in vitro from NSCs by leukemia inhibitory factor. Clonogenic assay performed in the absence of mitogens showed that RGLs respond to proNGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of proNGF was further exploited in the expansion of mouse-induced neural stem cells (iNSCs). Chronic exposure of iNSCs to proNGF-KR increased their proliferation. Altogether, we demonstrated that proNGF acts as mitogen on hippocampal and iNSCs. Stem Cells 2019;37:1223–1237.

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Biallelic mutations in the NBN gene are responsible for the Nijmegen breakage syndrome (NBS), a rare autosomal recessive disorder characterized by chromosome instability and hypersensitivity to ionising radiation (IR). Epidemiological data evidence that the NBN gene can be considered a susceptibility factor for cancer development, as demonstrated by the fact that almost 40% of NBS patients have developed a malignancy before the age of 21. Interestingly, also NBN heterozygotes, which are clinically asymptomatic, display an elevated risk to develop some types of malignant tumours, especially breast, prostate and colorectal cancers, lymphoblastic leukaemia, and non-Hodgkin’s lymphoma (NHL). So far, nine mutations in the NBN gene have been found, at the heterozygous state, in cancer patients. Among them, the 657del5, the I171V and the R215W mutations are the most frequently described. The pathogenicity of these mutations is presumably connected with their occurrence in the highly conserved BRCT tandem domains of the NBN protein, which are present in a large superfamily of proteins and are recognized as major mediators of processes related to cell-cycle checkpoints and DNA repair. This review will focus on the current state-of-knowledge regarding the correlation between carriers of NBN gene mutations and the proneness to the development of malignant tumours.

The Nijmegen breakage syndrome (NBS) is a genetic disorder. caused by mutations in NBN gene and characterized by chromosomal. instability and hypersensitivity to ionizing radiations (IR).. The N-terminus of nibrin (NBN) contains a tandem breast cancer. 1 (BRCA1) carboxy-terminal (BRCT) domain that represents one. of the major mediators of phosphorylation-dependent protein-protein. interactions in processes related to cell cycle checkpoint and. DNA repair functions. Patients with NBS compound heterozygous. for the 657del5 hypomorphic mutation and for the Arg215Trp. missense mutation (corresponding to the 643C[T gene mutation). display a clinical phenotype more severe than that of patients. homozygous for the 657del5 mutation. Here, we show that both. the 657del5 and Arg215Trp mutations, occurring within the tandem. BRCT domains of NBN, although not altering the assembly. of the MRE11\\\/RAD50\\\/NBN (MRN) complex, affect the MRE11. IR-induced nuclear foci (IRIF) formation and the DNA doublestrand. break (DSB) signaling via the phosphorylation of both. ataxia-telangiectasia-mutated (ATM) kinase and ATM downstream. targets (e.g., SMC1 and p53). Remarkably, data obtained. indicate that the cleavage of the BRCT tandem domains of NBN. by the 657del5 mutation affects the DNA damage response less. than the Arg215Trp mutation. Indeed, the 70-kDa NBN fragment,. arising from the 657del5 mutation, maintains the capability. to interact with MRE11 and c-H2AX and to form IRIF. Altogether,. the role of the tandem BRCT domains of NBN in the. localization of the MRN complex at the DNA DSB and in the. activation of the damage response is highlighted.