Colorectal cancer (CRC) is the second cause of cancer-related death where the CpG-island methylation pathways (CIMP) is associated to KRAS/BRAF mutations, two oncogenes rewiring cell metabolism, worst prognosis and resistance to classical chemotherapies. Despite this, the question of a possible metabolic rewiring in CIMPs has never been asked. Here we asked if metabolic dysregulations were associated to the methylation status of tumours by evaluating the transcriptome of CRC tumours. CIMP-high patients were found to present a hyper-metabolism activating mainly carbohydrates, folates, sphingolipids and arachidonic acid metabolic pathways. A third of these genes had epigenetic targets of Myc in their proximal promoter, activating carboxylic acid, tetrahydrofolate interconversion, nucleobase, and oxoacid metabolisms. Amongst the Myc signature, the expression of GAPDH, TYMS, DHFR, TK1 was enough to predict methylation levels, microsatellite instability (MSI) and mutations in the mismatch repair (MMR) machinery, which are strong indicators of responsiveness to immunotherapies. Finally, we discovered that CIMP patients harboured an increase in genes involved in the one-carbon metabolism, a pathway critical to provide nucleotides for cancer growth and methyl donors for DNA methylation which was associated to worse prognosis. Transcriptomics could hence become a tool to help clinicians stratify their patients better.

(1) Background: Triple negative breast cancer (TNBC) is a distinct subgroup of breast cancer presenting high level of recurrence and neo-adjuvant chemotherapy is beneficial in its therapy management. Anti-PD-L1 immunotherapy improves effect of neo-adjuvant therapy in TNBC. (2) Methods: Immune-modulation and ferroptosis related R-packages were developed for integrative transcriptome to analyze transcriptome datasets: TNBC cells stimulated with ferroptosis inducers (GSE173905, GSE154425), breast tumors with clinical data (TCGA and METABRIC cohorts). Protein level validation was investigated through protein atlas proteome experiments. (3) Results: Erastin/RSL3 ferroptosis inducers up-regulate CD274 in TNBC cells (MDA-MB-231 and HCC38). In breast cancer, CD274 expression is associated to overall survival. Breast tumors presenting high expression of CD274 up-regulated some ferroptosis drivers associated to prognosis: IDO1, IFNG and TNFAIP3. CD274-ferroptosis-driver score computed on breast tumor transcriptome stratified patients on their prognosis: low score was observed in basal subgroup with higher level of recurrent risk scores: oncotypeDx, ggi and gene70 scores. In METABRIC cohort, CD274, IDO1, IFNG and TNFAIP3 were found overexpressed in TNBC subgroup. CD274-ferroptosis-driver score was found associated to overall survival independently of TNM classification and age diagnosis. Tumor expression of CD274, TNFAIP3, IFNG and IDO1 in biopsy of breast ductal carcinoma was confirmed at protein level (4) Conclusion: Ferroptosis inducers up-regulate PD-L1 in TNBC cells, known as effective target of immunotherapy in high risk early TNBC which received neo-adjuvant therapy. Basal and TNBC tumors highly expressed CD274 and ferroptosis drivers: IFNG, TNFAIP3 and IDO1. CD274-ferroptosis driver score is associated to prognosis and to the risk of recurrence in breast cancer. A potential synergy of ferroptosis inducers with anti-PD-L1 immunotherapy is suggested in recurrent TNBC.

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in woman and is a high cause of mortality of them in reason to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy and inhibit distant metastases so potentially acts on tumor micro-environment; (2) Methods: Ferroptosis/Extracellular matrix remodeling literature text-mining results were integrated in breast cancer transcriptome cohort according their distant relapse free survival (DRFS) under adjuvant therapy (anthracyclin+taxanes) and also in MDA-MB-231 transcriptome functional experiments with ferroptosis activations (GSE173905); (3) Results: Ferroptosis/Extracellular matrix remodeling text-mining identified 910 associated genes in at list 10 articles. Univariate Cox analyses censored on breast cancer (GSE25066) selected 252 individual significant genes and 171 of them found with an adverse expression. Functional enrichment of these 171 adverse genes predicted basal breast cancer signatures. By text-mining some ferroptosis significant adverse selected genes shared citations in domain of ECM remodeling such as: TNF, IL6, SET, CDKN2A, EGFR, HMGB1, KRAS, MET, LCN2, HIF1A, TLR4. A molecular score based on expression the eleven genes was found predictive of worst prognosis breast cancer at univariate level: basal subtype, short DRFS, high grade values 3 and 4, estrogen and progesterone receptors negative and nodal stages 2 and 3. This eleven gene signature was validated as regulated by ferroptosis inductors (erastin and RSL3) in triple negative breast cancer cellular model MDA-MB-231.; (4) Conclusions: Crosstalk between ECM remodeling-Ferroptosis functionalities allowed to define a molecular score which have been characterized as an independent adverse parameter in prognosis of breast cancer patients. Gene signature of this molecular score have been validated to be regulated by erastin/RSL3 ferroptosis activators. This molecular score could be promising to evaluate ECM impact of ferroptosis target therapies in breast cancer.

Introduction: Previous studies have suggested that the tyrosine kinase receptor RET plays a sig-nificant role in the hematopoietic potential in mice and could also be used to expand cord-blood derived hematopoietic stem cells (HSCs). The role of RET in the human iPSC-derived hematopoi-esis has not been tested so far. Methods: To test the implication of RET on the hematopoietic potential of iPSCs, we activated its pathway with the lentiviral overexpression of RETWT or RETC634Y mutation in normal iPSCs. An iPSC derived from a patient harboring the RETC634Y mutation (iRETC634Y) and its CRISPR-corrected isogenic control iPSC (iRETCTRL) were also used. Hematopoietic potential was tested using 2D cul-tures and evaluated regarding the phenotype and the clonogenic potential of generated cells. Results: Hematopoietic differentiation from iPSCs with RET overexpression (WT or C634Y) led to a significant reduction in the number and in the clonogenic potential of HSCs (CD34+/CD38-/CD49f+) as compared to control iPSCs. Similarly, the hematopoietic potential of iRETC634Y was reduced as compared to iRETCTRL. Transcriptomic analyses revealed a specific ac-tivated expression profile for iRETC634Y compared to its control with evidence of overexpression of genes which are part of the MAPK network with negative hematopoietic regulator activities. Conclusion: RET activation in iPSCs is associated with an inhibitory activity in iPSC-derived hematopoiesis, potentially related with MAPK activation.