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Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential and therefore poses the need to identify new therapeutic combinations that could improve treatment outcomes. Despite the human kinome has proved to be an undisputable source of druggable targets, our knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterised. Using a kinome-wide RNAi screen, we found that abrogation of pantothenate kinase 4 (PANK4) enhances the antiproliferative effects of TMZ in GBM in vitro. Further validation of our top-hit across various TMZ-resistant GBM cell models, patient-derived GBM cell lines and tissue samples, as well as in vivo studies, corroborated the potential translational significance of our findings. We showed that PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Using a Tandem Mass Tag (TMT)-based quantitative proteomic approach, a comprehensive global protein dynamics analysis was undertaken to identify key response signatures upon PANK4 knockdown, in the presence or absence of TMZ. We revealed that silencing of PANK4 leads to a marked downregulation of a subset of proteins involved in cellular detoxification. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a synthetic vulnerability, focal point that can lead to critical cellular damage, accumulation of toxic metabolites, and subsequent cell death. Taken together, we unveil a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM.

Correction to: Mol Cancer 20, 53 (2021)\ud \ud https://doi.org/10.1186/s12943-021-01345-3\ud \ud Following the publication of the original article [1], the authors noticed errors on the figures introduced during the production step. Below are the errors:\ud \ud Fig. 2a: The labels for the y- and x-axes have been translocated upwards and need to be realigned with the axes. The x-axis should read: -Log2 (fold change).\ud \ud Fig. 2d: the quantification values in the NUSAP1 blots, for T47D and MDA-MB-231 cells, are no longer visible.\ud \ud The original article has been corrected.

In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment.

Broncho-pulmonary neuroendocrine neoplasms (BP-NENs) are neoplasms orphan of an efficient therapy. Available medical treatments derived from clinical trials are not specific for the management of this malignancy. Sunitinib is a multi-receptor tyrosine-kinases (RTKs) inhibitor that has already shown its efficacy in NENs, but there are no available data about its action in BP-NENs. Therefore, our aim was to understand the effects of RTKs inhibition promoted by sunitinib in order to evaluate new putative targets useful in malignancy treatment. Since our results underlined a role for EGFR and IGF1R in modulating sunitinib antiproliferative action, we investigated the effects of erlotinib, an EGFR inhibitor, and linsitinib, an IGF1R inhibitor, in order to understand their function in regulating cells behaviour. Cell viability and caspase activation were evaluated on two immortalised human BP-NEN cell lines and primary cultures. Our results showed that after treatment with sunitinib and/or IGF1, EGF and VEGF, the antiproliferative effect of sunitinib was counteracted by EGF and IGF1 but not by VEGF. Therefore, we evaluated with AlphaScreen technology the phosphorylated EGFR and IGF1R levels in primary cultures treated with sunitinib and/or EGF and IGF1. Results showed a decrease of p-IGF1R after treatment with sunitinib and an increase after co-treatment with IGF1. Then, we assessed cell viability and caspase activation on BP-NEN cell lines after treatment with linsitinib and/or erlotinib. Results demonstrate that these two agents have a stronger antiproliferative effect compared to sunitinib. In conclusion, our results suggest that IGF1R and EGF1R could represent putative molecular targets in BP-NENs treatment.

Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments., Highlights • hMOB2 promotes homologous recombination (HR) double-strand break (DSB) repair. • hMOB2 is required for the stabilisation of RAD51 on damaged chromatin. • Low hMOB2 levels potentiate the anti-tumour effects of the DNA-damaging agents. • hMOB2 as a predictive biomarker to PARP inhibition and patient stratification.

The structure of oncogenic LMTK3 determines its role and functions allowing drug inhibition as a new therapeutic strategy., 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 therapy.

As there is growing evidence for the tumor microenvironment’s role in tumorigenesis, we investigated the role of fibroblast-expressed kinases in triple-negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of cancer progression. Although PIK3Cδ was expressed in primary fibroblasts derived from TNBC patients, it was barely detectable in breast cancer (BC) cell lines. Genetic and pharmacological gain- and loss-of-function experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. Integrated secretomics and transcriptomics analyses revealed a paracrine mechanism via which f-PIK3Cδ confers its protumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, that led to upregulation of NR4A1 in TNBC cells, where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, along with a decrease in tumor metastasis, emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA data sets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease-free survival, highlighting it as a therapeutic target for TNBC.

As there is growing evidence for the tumor microenvironment’s (TME) role in tumorigenesis, we sought to investigate the role of fibroblast-expressed kinases in triple negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of progression. Although PIK3Cδ has been mainly described in leucocytes, we detected high expression in primary fibroblasts derived from TNBC patients, while PIK3Cδ was undetectable in cancer epithelial cell lines. Genetic and pharmacologic gain- and loss-of functions experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. By employing an integrated secretomics and transcriptomics analysis, we revealed a paracrine mechanism via which f-PIK3Cδ confers its pro-tumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, which subsequently led to upregulation of NR4A1 in TNBC cells where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, in addition to a decrease on tumor metastasis emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA datasets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease free survival, highlighting it as a therapeutic target for TNBC.

Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells\ud overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that\ud doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels.\ud Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and postchemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer.

Although splicing is widespread and evolves rapidly among species, the mechanisms driving this evolution, as well as its functional implications, are not yet fully understood. We analyzed the evolution of splicing patterns based on transcriptome data from five tissues of humans, chimpanzees, rhesus macaques, and mice. In total, 1,526 exons and exon sets from 1,236 genes showed significant splicing differences among primates. More than 60% of these differences represent constitutive-to-alternative exon transitions while an additional 25% represent changes in exon inclusion frequency. These two dominant evolutionary patterns have contrasting conservation, regulation, and functional features. The sum of these features indicates that, despite their prevalence, constitutive-to-alternative exon transitions do not substantially contribute to long-term functional transcriptome changes. Conversely, changes in exon inclusion frequency appear to be functionally relevant, especially for changes taking place in the brain on the human evolutionary lineage.

Background: Kinases represent one of the main therapeutic targets in cancer treatment as their impairing relates to tumor growth and progression. Despite increasing evidence that the tumor microenvironment (TME) signaling influences the behavior of surrounding cancer epithelial cells, still relatively little is known about what changes in stromal cells influence tumor cells' behavior and how they affect their response to target therapy. TNBC patients are still lacking an effective therapy, as not much is known about the biology of this BC tumor subtype. Clinical trials focus mainly on targeting epithelial cancer cells by using a combination of kinase inhibitors and standard chemotherapy however these therapeutic regimens are not considering the action of stromal cells that may influence response to treatment. Aims: • Identify fibroblasts-expressed kinases that modulate tumor cells' invasion • Characterize the mechanism by which these kinases promote/reduce tumor cell invasion. Methods: Human Mammary Fibroblast (HMF) and normal lung fibroblast (MRC5) cells were used in a high-throughput siRNA kinome screening targeting 710 Kinases. 24 hours after transfection, fibroblasts were co-cultured with MDA-MB-231 in 3D for spheroid formation. Matrigel and chemoattractants were then added to promote invasion. Spheroids pictures taken at different time-points were analyzed and results were expressed as changes in spheroid surface formation (ΔRatio= ΔCtrl/ΔKinase). Results: PIK3Cδ was among the most prominent targets, whose silencing decreased TNBC invasion. PIK3Cδ is essential for regulating chemokine production in leukocytes and promotes migration during inflammation, while PIK3Cδ inhibitors (CAL -101) interfere in tumour-stroma interactions without directly killing cancer cells. Despite PIK3Cδ being expressed mainly in leucocytes, we detected high PIK3Cδ protein expression in fibroblast cell lines and primary fibroblasts derived from TNBC patients; still, PIK3Cδ was hardly detectable in breast cancer cell lines. CAL-101 treatment affected fibroblasts viability, while it had limited/no effects on breast cancer cells. Moreover, CAL-101 resulted in reduced phosphorylation of AKT, a PIK3Cδ target. Pretreatment of fibroblasts with CAL-101 significantly decreased TNBC cells' invasion in 2D & 3D co-culture experiments. Interestingly, using transwell systems we saw that TNBC co-culturing increased PIK3Cδ expression in fibroblasts, suggesting a feedback loop that ‘fuels' tumor progression. Additional proteomic/genomic studies, provided us with evidence about the PIK3Cδ-mediated paracrine signaling leading to TNBC invasion. Conclusion: Using a novel 3D co-culture invasion assay, we identified stromal PIK3Cδ as a key mediator of TNBC invasion. Our results suggest that targeting PIK3Cδ in the TME may represent a novel transformative strategy for TNBC therapy. Citation Format: Teresa Gagliano, Viviana Vella, Kamila Bienkowska, Angeliki Ditsiou, Georgios Giamas. Fibroblast-kinome siRNA screening identifies PIK3Cδ as a mediator of Triple-negative breast cancer (TNBC) invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2087.

This abstract was not presented at the symposium.