Canonical NF-κB promotes lung epithelial cell tumour growth by downregulating the metastasis suppressor CD82 and enhancing epithelial-to-mesenchymal cell transition

Copyright: © 2021 by the authors. Background: The development of non-small cell lung cancer (NSCLC) involves the progressive accumulation of genetic and epigenetic changes. These include somatic oncogenic KRAS and EGFR mutations and inactivating TP53 tumour suppressor mutations, leading to activation of canonical NF-κB. However, the mechanism(s) by which canonical NF-κB contributes to NSCLC is still under investigation. Methods: Human NSCLC cells were used to knock-down RelA/p65 (RelA/p65KD) and investigate its impact on cell growth, and its mechanism of action by employing RNA-seq analysis, qPCR, immunoblotting, immunohistochemistry, immunofluorescence and functional assays. Results: RelA/p65KD reduced the proliferation and tumour growth of human NSCLC cells grown in vivo as xenografts in immune-compromised mice. RNA-seq analysis identified canonical NF-κB targets mediating its tumour promoting function. RelA/p65KD resulted in the upregulation of the metastasis suppressor CD82/KAI1/TSPAN27 and downregulation of the proto-oncogene ROS1, and LGR6 involved in Wnt/β-catenin signalling. Immunohistochemical and bioinformatics analysis of human NSCLC samples showed that CD82 loss correlated with malignancy. RelA/p65KD suppressed cell migration and epithelial-to-mesenchymal cell transition (EMT), mediated, in part, by CD82/KAI1, through integrin-mediated signalling involving the mitogenic ERK, Akt1 and Rac1 proteins. Conclusions: Canonical NF-κB signalling promotes NSCLC, in part, by downregulating the metastasis suppressor CD82/KAI1 which inhibits cell migration, EMT and tumour growth.Institutional Program Grant for the Development of Research Institutes “Advanced research activities in biomedical and agro-alimentary technologies, ARABAT (BITAD)” (MIS5002469) of the operational program “Competitiveness, Entrepreneurship and Innovation” (NSRF2014-20, EU-ERDF); research grant in Biomedical Sciences from FONDATION SANTÉ; STAVROS NIARCHOS Foundation-FORTH Fellowship for PhD candidates of the program ARCHERS: Advancing young researchers’ human capital in cutting edge technologies in the preservation of cultural heritage and the tackling of societal challenges; Biomedical Research Division, IMBB-FORTH; University of Ioannina Research Committee

The structure-function relationship of oncogenic LMTK3

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therap

A phase I study of the oral gamma secretase inhibitor R04929097 in combination with gemcitabine in patients with advanced solid tumors (PHL-078/CTEP 8575)

PURPOSE: To establish the recommended phase II dose of the oral γ-secretase inhibitor RO4929097 (RO) in combination with gemcitabine; secondary objectives include the evaluation of safety, tolerability, pharmacokinetics, biomarkers of Notch signaling and preliminary anti-tumor activity. METHODS: Patients with advanced solid tumors were enrolled in cohorts of escalating RO dose levels (DLs). Tested RO DLs were 20 mg, 30 mg, 45 mg and 90 mg. RO was administered orally, once daily on days 1-3, 8-10, 15-17, 22-24. Gemcitabine was administered at 1,000 mg/m(2) on d1, 8, and 15 in 28 d cycles. Dose limiting toxicities (DLTs) were assessed by CTCAE v4. Serial plasma was collected for RO (total and unbound) and gemcitabine pharmacokinetic analysis. Biomarkers of Notch signaling were assessed by immunohistochemistry in archival tissue. Antitumor activity was evaluated (RECIST 1.1). RESULTS: A total of 18 patients were enrolled to establish the recommended phase II dose. Of these, 3 patients received 20 mg RO, 7 patients received 30 mg RO, 6 patients received 45 mg RO and 2 patients received 90 mg RO. DLTs were grade 3 transaminitis (30 mg RO), grade 3 transaminitis and maculopapular rash (45 mg RO), and grade 3 transaminitis and failure to receive 75 % of planned RO doses secondary to prolonged neutropenia (90 mg); all were reversible. The maximum tolerated dose was exceeded at 90 mg RO. Pharmacokinetic analysis of both total and free RO confirmed the presence of autoinduction at 45 and 90 mg. Median levels of Notch3 staining were higher in individuals who received fewer than 4 cycles (p = 0.029). Circulating angiogenic factor levels did not correlate with time to progression or ≥ grade 3 adverse events. Best response (RECIST 1.1) was partial response (nasopharyngeal cancer) and stable disease > 4 months was observed in 3 patients (pancreas, tracheal, and breast primary cancers). CONCLUSIONS: RO and gemcitabine can be safely combined. The recommended phase II dose of RO was 30 mg in combination with gemcitabine 1,000 mg/m(2). Although RO exposure was limited by the presence of autoinduction, RO levels achieved exceeded the area under the concentration-time curve for 0-24 h (AUC(0-24)) predicted for efficacy in preclinical models using daily dosing. Evidence of clinical antitumor activity and prolonged stable disease were identified

ASXL2 is essential for haematopoiesis and acts as a haploinsufficient tumour suppressor in leukemia

Additional sex combs-like (ASXL) proteins are mammalian homologues of additional sex combs (Asx), a regulator of trithorax and polycomb function in Drosophila. While there has been great interest in ASXL1 due to its frequent mutation in leukemia, little is known about its paralog ASXL2, which is frequently mutated in acute myeloid leukemia patients bearing the RUNX1-RUNX1T1 (AML1-ETO) fusion. Here we report that ASXL2 is required for normal haematopoiesis with distinct, non-overlapping effects from ASXL1 and acts as a haploinsufficient tumour suppressor. While Asxl2 was required for normal haematopoietic stem cell self-renewal, Asxl2 loss promoted AML1-ETO leukemogenesis. Moreover, ASXL2 target genes strongly overlapped with those of RUNX1 and AML1-ETO and ASXL2 loss was associated with increased chromatin accessibility at putative enhancers of key leukemogenic loci. These data reveal that Asxl2 is a critical regulator of haematopoiesis and mediates transcriptional effects that promote leukemogenesis driven by AML1-ETO

NOTCH1 Signaling Promotes Human T-Cell Acute Lymphoblastic Leukemia Initiating Cell Regeneration in Supportive Niches

Leukemia initiating cells (LIC) contribute to therapeutic resistance through acquisition of mutations in signaling pathways, such as NOTCH1, that promote self-renewal and survival within supportive niches. Activating mutations in NOTCH1 occur commonly in T cell acute lymphoblastic leukemia (T-ALL) and have been implicated in therapeutic resistance. However, the cell type and context specific consequences of NOTCH1 activation, its role in human LIC regeneration, and sensitivity to NOTCH1 inhibition in hematopoietic microenvironments had not been elucidated.We established humanized bioluminescent T-ALL LIC mouse models transplanted with pediatric T-ALL samples that were sequenced for NOTCH1 and other common T-ALL mutations. In this study, CD34(+) cells from NOTCH1(Mutated) T-ALL samples had higher leukemic engraftment and serial transplantation capacity than NOTCH1(Wild-type) CD34(+) cells in hematopoietic niches, suggesting that self-renewing LIC were enriched within the NOTCH1(Mutated) CD34(+) fraction. Humanized NOTCH1 monoclonal antibody treatment reduced LIC survival and self-renewal in NOTCH1(Mutated) T-ALL LIC-engrafted mice and resulted in depletion of CD34(+)CD2(+)CD7(+) cells that harbor serial transplantation capacity.These results reveal a functional hierarchy within the LIC population based on NOTCH1 activation, which renders LIC susceptible to targeted NOTCH1 inhibition and highlights the utility of NOTCH1 antibody targeting as a key component of malignant stem cell eradication strategies

Acute loss of TET function results in aggressive myeloid cancer in mice

TET-family dioxygenases oxidize 5-methylcytosine (5mC) in DNA, and exert tumour suppressor activity in many types of cancers. Even in the absence of TET coding region mutations, TET loss-of-function is strongly associated with cancer. Here we show that acute elimination of TET function induces the rapid development of an aggressive, fully-penetrant and cell-autonomous myeloid leukaemia in mice, pointing to a causative role for TET loss-of-function in this myeloid malignancy. Phenotypic and transcriptional profiling shows aberrant differentiation of haematopoietic stem/progenitor cells, impaired erythroid and lymphoid differentiation and strong skewing to the myeloid lineage, with only a mild relation to changes in DNA modification. We also observe progressive accumulation of phospho-H2AX and strong impairment of DNA damage repair pathways, suggesting a key role for TET proteins in maintaining genome integrityopen0

Notch Ankyrin Repeat Domain Variation Influences Leukemogenesis and Myc Transactivation

, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival., a direct Notch target that has an important role in Notch-associated T-ALL.We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis

Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation

The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using γ-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, γ-secretase inhibitor-treated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that γ-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that γ-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21cip1 protein, a cell cycle suppressor, was upregulated by γ-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma