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In view of our recent findings that a truncated form of the envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) was efficiently incorporated into MoMLV particles, we studied the generation of Moloney murine leukemia virus (MoMLV)/simian immunodeficiency virus (SIV) pseudotypes. Unlike HIV-1, both the wild-type SIV Env and a truncated form, which lacks most of the cytoplasmic domain of the transmembrane glycoprotein, were incorporated into MoMLV particles and generated infectious retroviral vectors which could transduce CD4+ sMAGI macaque cells. The infection depended on target cell CD4 expression, and was neutralized by both soluble CD4 and sera from SIV-infected macaques. We also observed pseudotype-mediated gene transfer of a green fluorescent protein marker into the CD4+ CEMX174 and C8166 lymphoid cell lines. More importantly, primary human lymphocytes were also successfully transduced ex vivo by MoMLV/SIV pseudotypes, albeit at lower efficiency, and gene transfer was specifically restricted to the CD4+ subset. These findings demonstrate that MoMLV/SIV pseudotypes can be used to transduce cells which are susceptible to SIV infection, and thus might be advantageously employed in animal models for direct in vivo delivery of gene therapy-based approaches. [ABSTRACT FROM AUTHOR]

The aim of our study is to investigate the microRNA expression profile to discriminate the inflammatory infiltrate observed in monitoring endomyocardial biopsies (EMBs) in post-transplanted patients with or without Cytomegalovirus (CMV) viremia. We identified 3 groups of patients in the setting of viremia and inflammatory infiltrate, between January 2018 and December 2020: a control group (CG, n=5) with patients negative at EMB for rejection and CMV infection, a rejection group (RG, n=5) with patients positive at EMB for Acute Cellular Rejection (ACR >=3A-2R ISHLT grade) but negative for CMV viremia and an infection group (IG, n=6) with patients positive at EMB for ACR (>=1A-1R ISHLT grade) in the setting of CMV viremia positivity (>1000 copies/mL). We extracted total RNA from FFPE-EMBs and performed a microRNA (miRNA) profiling by a microarray platform (Clariom-S miRNA 4.0 chip) without any bias of miRNA selection. Then, we analysed the miRNA profile using TAC (Transcriptome Analysis Console) software and matched them with the mRNA profile on the same samples. Out of the 2578 miRNAs tested for each patient, 513 miRNAs where differentially expressed (gene level fold-change <-2 or >2, p-value <0.05) between CG and IG (477 upregulated and 36 downregulated), 111 genes between CG and RG (62 upregulated and 49 downregulated) and 386 miRNAs between IG and RG (32 upregulated and 354 downregulated). Among these 386 miRNAs, 29 emerged only in IG vs RG comparison. Starting from these results, we further investigated hsa-mir-8075, mir-93-5p, mir-3651, mir-345-5p and mir-6722, the more significant differentially expressed miRNAs. The screening of PubTator, GeneCards and PubMed databases revealed that these miRNAs are mainly involved in PI3K/Akt pathway, STAT, HIF1α, leading to cell proliferation and apoptosis regulation, as well as mitochondria stress response. Moreover, most of these genes are connected to IL7 pathway, a key positive regulator of T cell proliferation. IL7R resulted differentially expressed in IG vs RG transcriptome analysis performed on the same samples. This mirnome analysis defined a molecular signature distinctive for Infection in post-cardiac transplant follow-up, which could interfere with cell proliferation, apoptosis, and T cell differentiation. [ABSTRACT FROM AUTHOR]

Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4 + CD8 + (DP) T cells that are notably, Notch3 high CXCR4 high . Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4 - CD8 - (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ + CXCR4 - cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis., Methods: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy., Results: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation., Conclusion: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Digital pathology has the potential to quantify tumor markers accurately and reproducibly with various cellular and subcellular localizations in tissues, thus filling a need in cancer research. As a case study, we quantified the percentage of necrosis, microvessels density, and monocarboxylate transporter 4 (MCT4) expression in two ovarian cancer patient-derived xenograft (PDX) models subcutaneously injected in NOD/SCID mice. PDX models were treated with bevacizumab, an antiangiogenic drug, that targets vascular endothelial growth factor A (VEGF-A). Specific signal analysis algorithms allowed us to study morphologic, vascular, and metabolic modifications induced by antiangiogenic therapy by a quantitative, reproducible, and reliable approach., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy. Despite recent advances in treatments with intensified chemotherapy regimens, relapse rates and associated morbidities remain high. In this context, metabolic dependencies have emerged as a druggable opportunity for the treatment of leukemia. Here, we tested the antileukemic effects of MB1-47, a newly developed mitochondrial uncoupling compound. MB1-47 treatment in T-ALL cells robustly inhibited cell proliferation via both cytostatic and cytotoxic effects as a result of compromised mitochondrial energy and metabolite depletion, which severely impaired nucleotide biosynthesis. Mechanistically, acute treatment with MB1-47 in primary leukemias promoted adenosine monophosphate-activated serine/threonine protein kinase (AMPK) activation and downregulation of mammalian target of rapamycin (mTOR) signaling, stalling anabolic pathways that support leukemic cell survival. Indeed, MB1-47 treatment in mice harboring either murine NOTCH1-induced primary leukemias or human T-ALL patient-derived xenografts (PDXs) led to potent antileukemic effects with a significant extension in survival without overlapping toxicities. Overall, our findings demonstrate a critical role for mitochondrial oxidative phosphorylation in T-ALL and uncover MB1-47-driven mitochondrial uncoupling as a novel therapeutic strategy for the treatment of this disease., (© 2021 by The American Society of Hematology.)

Notch1 is a crucial oncogenic driver in T-cell acute lymphoblastic leukemia (T-ALL), making it an attractive therapeutic target. However, the success of targeted therapy using γ-secretase inhibitors (GSIs), small molecules blocking Notch cleavage and subsequent activation, has been limited due to development of resistance, thus restricting its clinical efficacy. Here, we systematically compare GSI resistant and sensitive cell states by quantitative mass spectrometry-based phosphoproteomics, using complementary models of resistance, including T-ALL patient-derived xenografts (PDX) models. Our datasets reveal common mechanisms of GSI resistance, including a distinct kinase signature that involves protein kinase C delta. We demonstrate that the PKC inhibitor sotrastaurin enhances the anti-leukemic activity of GSI in PDX models and completely abrogates the development of acquired GSI resistance in vitro. Overall, we highlight the potential of proteomics to dissect alterations in cellular signaling and identify druggable pathways in cancer.

Folate metabolism enables cell growth by providing one-carbon (1C) units for nucleotide biosynthesis. The 1C units are carried by tetrahydrofolate, whose production by the enzyme dihydrofolate reductase is targeted by the important anticancer drug methotrexate. 1C units come largely from serine catabolism by the enzyme serine hydroxymethyltransferase (SHMT), whose mitochondrial isoform is strongly upregulated in cancer. Here we report the SHMT inhibitor SHIN2 and demonstrate its in vivo target engagement with 13 C-serine tracing. As methotrexate is standard treatment for T-cell acute lymphoblastic leukemia (T-ALL), we explored the utility of SHIN2 in this disease. SHIN2 increases survival in NOTCH1-driven mouse primary T-ALL in vivo. Low dose methotrexate sensitizes Molt4 human T-ALL cells to SHIN2, and cells rendered methotrexate resistant in vitro show enhanced sensitivity to SHIN2. Finally, SHIN2 and methotrexate synergize in mouse primary T-ALL and in a human patient-derived xenograft in vivo, increasing survival. Thus, SHMT inhibition offers a complementary strategy in the treatment of T-ALL.

The Notch pathway is highly active in almost all patients with T-cell acute lymphoblastic leukemia (T-ALL), but the implication of Notch ligands in T-ALL remains underexplored. Methods: We used a genetic mouse model of Notch ligand delta like 4 (DLL4)-driven T-ALL and performed thymectomies and splenectomies in those animals. We also used several patient-derived T-ALL (PDTALL) models, including one with DLL4 expression on the membrane and we treated PDTALL cells in vitro and in vivo with demcizumab, a blocking antibody against human DLL4 currently being tested in clinical trials in patients with solid cancer. Results: We show that surgical removal of the spleen abrogated T-ALL development in our preclinical DLL4-driven T-ALL mouse model. Mechanistically, we found that the spleen, and not the thymus, promoted the accumulation of circulating CD4 + CD8 + T cells before T-ALL onset, suggesting that DLL4-driven T-ALL derives from these cells. Then, we identified a small subset of T-ALL patients showing higher levels of DLL4 expression. Moreover, in mice xenografted with a DLL4-positive PDTALL model, treatment with demcizumab had the same therapeutic effect as global Notch pathway inhibition using the potent γ-secretase inhibitor dibenzazepine. This result demonstrates that, in this PDTALL model, Notch pathway activity depends on DLL4 signaling, thus validating our preclinical mouse model. Conclusion: DLL4 expression in human leukemic cells can be a source of Notch activity in T-ALL, and the spleen plays a major role in a genetic mouse model of DLL4-driven T-ALL., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

In pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug's safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials., (© 2020 by The American Society of Hematology.)

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression.

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors., (Copyright© 2020 Ferrata Storti Foundation.)

The classical cancer stem cell (CSC) model places CSCs at the apex of a hierarchical scale, suggesting different genetic alterations in non-CSCs compared to CSCs, since an ill-defined number of cell generations and time intervals separate CSCs from the more differentiated cancer cells that form the bulk of the tumor. Another model, however, poses that CSCs should be considered a functional state of tumor cells, hence sharing the same genetic alterations. Here, we review the existing literature on the genetic landscape of CSCs in various tumor types and as a case study investigate the genomic complexity of DNA obtained from matched CSCs and non-CSCs from five ovarian cancer patients, using a genome-wide single-nucleotide polymorphism (SNP) microarray., Competing Interests: The authors declare no conflict of interest.