PI3K-driven HER2 expression is a potential therapeutic target in colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells are responsible for tumour spreading and relapse. Human epidermal growth factor receptor 2 (HER2) expression is a negative prognostic factor in colorectal cancer (CRC) and a potential target in tumours carrying the gene amplification. Our aim was to define the expression of HER2 in colorectal cancer stem cells (CR-CSCs) and its possible role as therapeutic target in CRC resistant to anti- epidermal growth factor receptor (EGFR) therapy. DESIGN: A collection of primary sphere cell cultures obtained from 60 CRC specimens was used to generate CR-CSC mouse avatars to preclinically validate therapeutic options. We also made use of the ChIP-seq analysis for transcriptional evaluation of HER2 activation and global RNA-seq to identify the mechanisms underlying therapy resistance. RESULTS: Here we show that in CD44v6-positive CR-CSCs, high HER2 expression levels are associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which promotes the acetylation at the regulatory elements of the Erbb2 gene. HER2 targeting in combination with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MEK) inhibitors induces CR-CSC death and regression of tumour xenografts, including those carrying Kras and Pik3ca mutation. Requirement for the triple targeting is due to the presence of cancer-associated fibroblasts, which release cytokines able to confer CR-CSC resistance to PI3K/AKT inhibitors. In contrast, targeting of PI3K/AKT as monotherapy is sufficient to kill liver-disseminating CR-CSCs in a model of adjuvant therapy. CONCLUSIONS: While PI3K targeting kills liver-colonising CR-CSCs, the concomitant inhibition of PI3K, HER2 and MEK is required to induce regression of tumours resistant to anti-EGFR therapies. These data may provide a rationale for designing clinical trials in the adjuvant and metastatic setting.

Cancer-associated fibroblasts as abettors of tumor progression at the crossroads of EMT and therapy resistance.

In the last decades, the role of the microenvironment in tumor progression and therapeutic outcome has gained increasing attention. Cancer-associated fibroblasts (CAFs) have emerged as key players among stromal cells, owing to their abundance in most solid tumors and their diverse tumor-restraining/promoting roles. The interplay between tumor cells and neighboring CAFs takes place by both paracrine signals (cytokines, exosomes and metabolites) or by the multifaceted functions of the surrounding extracellular matrix. Here, we dissect the most recent identified mechanisms underlying CAF-mediated control of tumor progression and therapy resistance, which include induction of the epithelial-to-mesenchymal transition (EMT), activation of survival pathways or stemness-related programs and metabolic reprogramming in tumor cells. Importantly, the recently unveiled heterogeneity in CAFs claims tailored therapeutic efforts aimed at eradicating the specific subset facilitating tumor progression, therapy resistance and relapse. However, despite the large amount of pre-clinical data, much effort is still needed to translate CAF-directed anti-cancer strategies from the bench to the clinic.

Capturing colorectal cancer inter-tumor heterogeneity in patient-derived xenograft (PDX) models.

Patient-derived xenograft (PDX) models have become an important asset in translational cancer research. However, to provide a robust preclinical platform, PDXs need to accommodate the tumor heterogeneity that is observed in patients. Colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features. Surprisingly, using a set of CRC patients, we revealed the partial representation of tumor heterogeneity in PDX models. The epithelial subtypes, the largest subgroups of CRC subtype, were very ineffective in establishing PDXs, indicating the need for further optimization to develop an effective personalized therapeutic approach to CRC. Moreover, we showed that tumor cell proliferation was associated with successful PDX establishment and able to distinguish patient with poor clinical outcomes within CMS2 group.

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. DESIGN: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. RESULTS: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. CONCLUSIONS: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Microenvironment in neuroblastoma: isolation and characterization of tumor-derived mesenchymal stromal cells.

BACKGROUND: It has been proposed that mesenchymal stromal cells (MSCs) promote tumor progression by interacting with tumor cells and other stroma cells in the complex network of the tumor microenvironment. We characterized MSCs isolated and expanded from tumor tissues of pediatric patients diagnosed with neuroblastomas (NB-MSCs) to define interactions with the tumor microenvironment. METHODS: Specimens were obtained from 7 pediatric patients diagnosed with neuroblastoma (NB). Morphology, immunophenotype, differentiation capacity, proliferative growth, expression of stemness and neural differentiation markers were evaluated. Moreover, the ability of cells to modulate the immune response, i.e. inhibition of phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cytotoxic function, was examined. Gene expression profiles, known to be related to tumor cell stemness, Wnt pathway activation, epithelial-mesenchymal transition (EMT) and tumor metastasis were also evaluated. Healthy donor bone marrow-derived MSCs (BM-MSC) were employed as controls. RESULTS: NB-MSCs presented the typical MSC morphology and phenotype. They showed a proliferative capacity superimposable to BM-MSCs. Stemness marker expression (Sox2, Nanog, Oct3/4) was comparable to BM-MSCs. NB-MSC in vitro osteogenic and chondrogenic differentiation was similar to BM-MSCs, but NB-MSCs lacked adipogenic differentiation capacity. NB-MSCs reached senescence phases at a median passage of P7 (range, P5-P13). NB-MSCs exhibited greater immunosuppressive capacity on activated T lymphocytes at a 1:2 (MSC: PBMC) ratio compared with BM-MSCs (p = 0.018). NK cytotoxic activity was not influenced by co-culture, either with BM-MSCs or NB-MSCs. Flow-cytometry cell cycle analysis showed that NB-MSCs had an increased number of cells in the G0-G1 phase compared to BM-MSCs. Transcriptomic profiling results indicated that NB-MSCs were enriched with EMT genes compared to BM-MSCs. CONCLUSIONS: We characterized the biological features, the immunomodulatory capacity and the gene expression profile of NB-MSCs. The NB-MSC gene expression profile and their functional properties suggest a potential role in promoting tumor escape, invasiveness and metastatic traits of NB cancer cells. A better understanding of the complex mechanisms underlying the interactions between NB cells and NB-derived MSCs should shed new light on potential novel therapeutic approaches.

Intragenic G-quadruplex structure formed in the human CD133 and its biological and translational relevance.

Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression.In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.

Lipid droplets: a new player in colorectal cancer stem cells unveiled by spectroscopic imaging.

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

Cancer-initiating cells from colorectal cancer patients escape from T cell-mediated immunosurveillance in vitro through membrane-bound IL-4.

Cancer-initiating cells (CICs) that are responsible for tumor initiation, propagation, and resistance to standard therapies have been isolated from human solid tumors, including colorectal cancer (CRC). The aim of this study was to obtain an immunological profile of CRC-derived CICs and to identify CIC-associated target molecules for T cell immunotherapy. We have isolated cells with CIC properties along with their putative non-CIC autologous counterparts from human primary CRC tissues. These CICs have been shown to display "tumor-initiating/stemness" properties, including the expression of CIC-associated markers (e.g., CD44, CD24, ALDH-1, EpCAM, Lgr5), multipotency, and tumorigenicity following injection in immunodeficient mice. The immune profile of these cells was assessed by phenotype analysis and by in vitro stimulation of PBMCs with CICs as a source of Ags. CICs, compared with non-CIC counterparts, showed weak immunogenicity. This feature correlated with the expression of high levels of immunomodulatory molecules, such as IL-4, and with CIC-mediated inhibitory activity for anti-tumor T cell responses. CIC-associated IL-4 was found to be responsible for this negative function, which requires cell-to-cell contact with T lymphocytes and which is impaired by blocking IL-4 signaling. In addition, the CRC-associated Ag COA-1 was found to be expressed by CICs and to represent, in an autologous setting, a target molecule for anti-tumor T cells. Our study provides relevant information that may contribute to designing new immunotherapy protocols to target CICs in CRC patients.

Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells.

Glioblastoma is a highly angiogenetic malignancy, the neoformed vessels of which are thought to arise by sprouting of pre-existing brain capillaries. The recent demonstration that a population of glioblastoma stem-like cells (GSCs) maintains glioblastomas indicates that the progeny of these cells may not be confined to the neural lineage. Normal neural stem cells are able to differentiate into functional endothelial cells. The connection between neural stem cells and the endothelial compartment seems to be critical in glioblastoma, where cancer stem cells closely interact with the vascular niche and promote angiogenesis through the release of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (refs 5-9). Here we show that a variable number (range 20-90%, mean 60.7%) of endothelial cells in glioblastoma carry the same genomic alteration as tumour cells, indicating that a significant portion of the vascular endothelium has a neoplastic origin. The vascular endothelium contained a subset of tumorigenic cells that produced highly vascularized anaplastic tumours with areas of vasculogenic mimicry in immunocompromised mice. In vitro culture of GSCs in endothelial conditions generated progeny with phenotypic and functional features of endothelial cells. Likewise, orthotopic or subcutaneous injection of GSCs in immunocompromised mice produced tumour xenografts, the vessels of which were primarily composed of human endothelial cells. Selective targeting of endothelial cells generated by GSCs in mouse xenografts resulted in tumour reduction and degeneration, indicating the functional relevance of the GSC-derived endothelial vessels. These findings describe a new mechanism for tumour vasculogenesis and may explain the presence of cancer-derived endothelial-like cells in several malignancies.

Human NK cells selective targeting of colon cancer-initiating cells: a role for natural cytotoxicity receptors and MHC class I molecules.

Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.

Autoimmune thyroid disease: new models of cell death in autoimmunity.

Autoimmunity to thyroid antigens leads to two distinct pathogenic processes with opposing clinical outcomes: hypothyroidism in Hashimoto's thyroiditis and hyperthyroidism in Graves' disease. The high frequency of these diseases and easy accessibility of the thyroid gland has allowed the identification of key pathogenic mechanisms in organ-specific autoimmune diseases. In early investigations, antibody- and T-cell-mediated death mechanisms were proposed as being responsible for autoimmune thyrocyte depletion. Later, studies on apoptosis have provided new insights into autoimmune target destruction, indicating the involvement of death receptors and cytokine-regulated apoptotic pathways in the pathogenesis of thyroid autoimmunity.

Tumor and its microenvironment: a synergistic interplay.

The mutual and interdependent interaction between tumor and its microenvironment is a crucial topic in cancer research. Recently, it was reported that targeting stromal events could improve efficacies of current therapeutics and prevent metastatic spreading. Tumor microenvironment is a "complex network" of different cell types, soluble factors, signaling molecules and extracellular matrix components, which orchestrate the fate of tumor progression. As by definition, cancer stem cells (CSCs) are proposed to be the unique cell type able to maintain tumor mass and survive outside the primary tumor at metastatic sites. Being exposed to environmental stressors, including reactive oxygen species (ROS), CSCs have developed a GSH-dependent antioxidant system to improve ROS defense capability and acquire a malignant phenotype. Nevertheless, tumor progression is dependent on extracellular matrix remodeling, fibroblasts and macrophages activation in response to oxidative stress, as well as epithelial mesenchymal transition (EMT)-inducing signals and endothelial and perivascular cells recruitment. Besides providing a survival advantage by inducing de novo angiogenesis, tumor-associated vessels contribute to successful dissemination by facilitating tumor cells entry into the circulatory system and driving the formation of pre-metastatic niche. In this review, we focus on the synergistic effect of hypoxia inducible factors (HIFs) and vascular endothelial growth factors (VEGFs) in the successful outgrowth of metastasis, integrating therefore many of the emerging models and theories in the field.

p63 isoforms regulate metabolism of cancer stem cells.

p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.

The miRNA Contribution in Adipocyte Maturation.

Mesenchymal stem cells, due to their multipotent ability, are considered one of the best candidates to be used in regenerative medicine. To date, the most used source is represented by the bone marrow, despite the limited number of cells and the painful/invasive procedure for collection. Therefore, the scientific community has investigated many alternative sources for the collection of mesenchymal stem cells, with the adipose tissue representing the best option, given the abundance of mesenchymal stem cells and the easy access. Although adipose mesenchymal stem cells have recently been investigated for their multipotency, the molecular mechanisms underlying their adipogenic potential are still unclear. In this scenario, this communication is aimed at defining the role of miRNAs in adipogenic potential of adipose-derived mesenchymal stem cells via real-time PCR. Even if preliminary, our data show that cell culture conditions affect the expression of specific miRNA involved in the adipogenic potential of mesenchymal stem cells. The in vitro/in vivo validation of these results could pave the way for novel therapeutic strategies in the field of regenerative medicine. In conclusion, our research highlights how specific cell culture conditions can modulate the adipogenic potential of adipose mesenchymal stem cells through the regulation of specific miRNAs.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells.

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

Differentiation, phenotype, and function of interleukin-17-producing human Vγ9Vδ2 T cells.

In healthy adults, the major peripheral blood γδ T-cell subset expresses the Vγ9Vδ2 TCR and displays pleiotropic features. Here we report that coculture of naive Vγ9Vδ2 T cells with phosphoantigens and a cocktail of cytokines (IL-1-ß, TGF-ß, IL-6, and IL-23), leads to selective expression of the transcription factor RORγt and polarization toward IL-17 production. IL-17(+) Vγ9Vδ2 T cells express the chemokine receptor CCR6 and produce IL-17 but neither IL-22 nor IFN-γ; they have a predominant terminally differentiated (CD27(-)CD45RA(+)) phenotype and express granzyme B, TRAIL, FasL, and CD161. On antigen activation, IL-17(+) Vγ9Vδ2 T cells rapidly induce CXCL8-mediated migration and phagocytosis of neutrophils and IL-17-dependent production of ß-defensin by epithelial cells, indicating that they may be involved in host immune responses against infectious microorganisms. Accordingly, an increased percentage of IL-17(+) Vγ9Vδ2 lymphocytes is detected in the peripheral blood and at the site of disease in children with bacterial meningitis, and this pattern was reversed after successful antibacterial therapy. Most notably, the phenotype of IL-17(+) Vγ9Vδ2 T cells in children with meningitis matches that of in vitro differentiated IL-17(+) Vγ9Vδ2 T cells. Our findings delineate a previously unknown subset of human IL-17(+) Vγ9Vδ2 T lymphocytes implicated in the pathophysiology of inflammatory responses during bacterial infections.

Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation.

Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.

EBF1, MYO6 and CALR expression levels predict therapeutic response in diffuse large B-cell lymphomas.

Background: Diffuse large B-cell lymphoma (DLBCL) is a hematological malignancy representing one-third of non-Hodgkin's lymphoma cases. Notwithstanding immunotherapy in combination with chemotherapy (R-CHOP) is an effective therapeutic approach for DLBCL, a subset of patients encounters treatment resistance, leading to low survival rates. Thus, there is an urgent need to identify predictive biomarkers for DLBCL including the elderly population, which represents the fastest-growing segment of the population in Western countries. Methods: Gene expression profiles of n=414 DLBCL biopsies were retrieved from the public dataset GSE10846. Differentially expressed genes (DEGs) (fold change >1.4, p-value <0.05, n=387) have been clustered in responder and non-responder patient cohorts. An enrichment analysis has been performed on the top 30 up-regulated genes of responder and non-responder patients to identify the signatures involved in gene ontology (MSigDB). The more significantly up-regulated DEGs have been validated in our independent collection of formalin-fixed paraffin-embedded (FFPE) biopsy samples of elderly DLBCL patients, treated with R-CHOP as first-line therapy. Results: From the analysis of two independent cohorts of DLBCL patients emerged a gene signature able to predict the response to R-CHOP therapy. In detail, expression levels of EBF1, MYO6, CALR are associated with a significant worse overall survival. Conclusions: These results pave the way for a novel characterization of DLBCL biomarkers, aiding the stratification of responder versus non-responder patients.

Subtype-specific kinase dependency regulates growth and metastasis of poor-prognosis mesenchymal colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020). METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns. RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2. CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.

Recapitulating thyroid cancer histotypes through engineering embryonic stem cells.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.