Genomic profiling of tumours in patients in clinical trials enables rapid testing of multiple hypotheses to confirm which genomic events determine likely responder groups for targeted agents. A key ...challenge of this new capability is defining which specific genomic events should be classified as 'actionable' (that is, potentially responsive to a targeted therapy), especially when looking for early indications of patient subgroups likely to be responsive to new drugs. This Opinion article discusses some of the different approaches being taken in early clinical development to define actionable mutations, and describes our strategy to address this challenge in early-stage exploratory clinical trials.
Triple-negative breast cancer (TNBC) represents a subgroup of breast cancers (BC) associated with the most aggressive clinical behavior. No targeted therapy is currently available for the treatment ...of patients with TNBC. In order to discover potential therapeutic targets, we searched for protein kinases that are overexpressed in human TNBC biopsies and whose silencing in TNBC cell lines causes cell death. A cohort including human BC biopsies obtained at Institut Curie as well as normal tissues has been analyzed at a gene-expression level. The data revealed that the human protein kinase monopolar spindle 1 (hMPS1), also known as TTK and involved in mitotic checkpoint, is specifically overexpressed in TNBC, compared to the other BC subgroups and healthy tissues. We confirmed by immunohistochemistry and reverse phase protein array that TNBC expressed higher levels of TTK protein compared to the other BC subgroups. We then determined the biological effects of TTK depletion by RNA interference, through analyses of tumorigenic capacity and cell viability in different human TNBC cell lines. We found that RNAi-mediated depletion of TTK in various TNBC cell lines severely compromised their viability and their ability to form colonies in an anchorage-independent manner. Moreover, we observed that TTK silencing led to an increase in H2AX phosphorylation, activation of caspases 3/7, sub-G1 cell population accumulation and high annexin V staining, as well as to a decrease in G1 phase cell population and an increased aneuploidy. Altogether, these data indicate that TTK depletion in TNBC cells induces apoptosis. These results point out TTK as a protein kinase overexpressed in TNBC that may represent an attractive therapeutic target specifically for this poor prognosis associated subgroup of breast cancer.
The canonical Wnt/β-catenin pathway is activated in triple-negative breast cancer (TNBC). The activation of this pathway leads to the expression of specific target genes depending on the cell/tissue ...context. Here, we analyzed the transcriptome of two different TNBC cell lines to define a comprehensive list of Wnt target genes. The treatment of cells with Wnt3a for 6h up-regulated the expression (fold change > 1.3) of 59 genes in MDA-MB-468 cells and 241 genes in HCC38 cells. Thirty genes were common to both cell lines. Beta-catenin may also be a transcriptional repressor and we found that 18 and 166 genes were down-regulated in response to Wnt3a treatment for 6h in MDA-MB-468 and HCC38 cells, respectively, of which six were common to both cell lines. Only half of the activated and the repressed transcripts have been previously described as Wnt target genes. Therefore, our study reveals 137 novel genes that may be positively regulated by Wnt3a and 104 novel genes that may be negatively regulated by Wnt3a. These genes are involved in the Wnt pathway itself, and also in TGFβ, p53 and Hedgehog pathways. Thorough characterization of these novel potential Wnt target genes may reveal new regulators of the canonical Wnt pathway. The comparison of our list of Wnt target genes with those published in other cellular contexts confirms the notion that Wnt target genes are tissue-, cell line- and treatment-specific. Genes up-regulated in Wnt3a-stimulated cell lines were more strongly expressed in TNBC than in luminal A breast cancer samples. These genes were also overexpressed, but to a much lesser extent, in HER2+ and luminal B tumors. We identified 72 Wnt target genes higher expressed in TNBCs (17 with a fold change >1.3) which may reflect the chronic activation of the canonical Wnt pathway that occurs in TNBC tumors.
PTEN-null tumors become dependent on the PI3Kβ isoform and can be targeted by molecules such as the selective PI3Kβ inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, ...the consequences of inhibiting PI3Kβ are poorly defined.
To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.
AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kβ also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.
This study identifies novel mechanistic biomarkers of PI3Kβ inhibition in PTEN-null tumors supporting the concept that targeting PI3Kβ may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents.
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We report the preparation and pharmacological profile of new c-Src inhibitors elaborated around a 3-amino-thieno2,3-
bpyridine nucleus.
Among the recently investigated targets for cancer therapy is ...the c-Src non-receptor tyrosine kinase. Indeed research around deregulated activity of this enzyme has proven its role in tumor progression, while the beneficial effects of c-Src inhibitors in several pathological models has also been demonstrated.
We report here the preparation and pharmacological profile of a novel series of c-Src inhibitors that was elaborated around a 3-amino-thieno2,3-
bpyridine discovered during an HTS campaign.
c-Src enzyme inhibition and c-Src inhibition were investigated in a series of related compounds derived from the initial hit. Molecular modeling as well as X-ray studies on one active compound allowed us to hypothesize on ligand orientation and interactions within the ATP hydrophobic pocket.
Design and synthesis of structural analogs then led to new ligands possessing quite efficient enzymatic and c-Src inhibition.
The structure–activity elements disclosed in this study shed light on the role played by substituents on the thienopyridine ring as well as the impact of other aromatic moieties in the molecule when interacting with the enzyme.
Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In ...contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs.
In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines.
The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition.
These data provide insight into the molecular pathogenesis of BLCs and implicate the PTEN-dependent activated Akt signalling pathway as a potential therapeutic target for the management of patients with poor prognosis BLCs.
Elk-1, a member of the TCF family of Ets domain proteins, contains a C-terminal transcriptional activation domain with multiple copies of the MAPK core consensus sequence S/T-P. This region is ...phosphorylated by MAP kinases in vitro and in vivo, but the extent and kinetics of phosphorylation at the different sites have not been investigated in detail. We prepared antisera against the phosphorylated forms of residues T353, T363, T368, S383, S389 and T417. The antisera specifically recognize the phosphorylated Elk-1 C terminus and are specific for their cognate sites, as assessed by peptide competition and mutagenesis experiments. Analysis of cells stably expressing Elk-1 in vivo shows that following serum or TPA stimulation, residues T353, T363, T368, S383, S389 and T417 become phosphorylated with similar kinetics. Mutation of any one site does not prevent phosphorylation of the others. Mutation to alanine of S383, F378 or W379, which virtually abolishes transcriptional activation by Elk-1, does not affect phosphorylation of any sites tested. Analysis of Elk-1 using two-dimensional gel electrophoresis shows that following ERK activation Elk-1 receives at least six phosphates in addition to those present prior to stimulation. We propose that the Elk-1 C-terminal regulatory domain becomes stoichiometrically phosphorylated following growth factor stimulation.
c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma ...cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.
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