Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

Proneural and mesenchymal glioma stem cells display major differences in splicing and lncRNA profiles.

Therapy resistance and recurrence in high-grade gliomas are driven by their populations of glioma stem cells (GSCs). Thus, detailed molecular characterization of GSCs is needed to develop more effective therapies. We conducted a study to identify differences in the splicing profile and expression of long non-coding RNAs in proneural and mesenchymal GSC cell lines. Genes related to cell cycle, DNA repair, cilium assembly, and splicing showed the most differences between GSC subgroups. We also identified genes distinctly associated with survival among patients of mesenchymal or proneural subgroups. We determined that multiple long non-coding RNAs with increased expression in mesenchymal GSCs are associated with poor survival of glioblastoma patients. In summary, our study established critical differences between proneural and mesenchymal GSCs in splicing profiles and expression of long non-coding RNA. These splicing isoforms and lncRNA signatures may contribute to the uniqueness of GSC subgroups, thus contributing to cancer phenotypes and explaining differences in therapeutic responses.

The Effects of Neoadjuvant Chemoradiation in Locally Advanced Rectal Cancer-The Impact in Intratumoral Heterogeneity.

Purpose: Intratumoral genetic heterogeneity (ITGH) is a common feature of solid tumors. However, little is known about the effect of neoadjuvant chemoradiation (nCRT) in ITGH of rectal tumors that exhibit poor response to nCRT. Here, we examined the impact of nCRT in the mutational profile and ITGH of rectal tumors and its adjacent irradiated normal mucosa in the setting of incomplete response to nCRT. Methods and Materials: To evaluate ITGH in rectal tumors, we analyzed whole-exome sequencing (WES) data from 79 tumors obtained from The Cancer Genome Atlas (TCGA). We also compared matched peripheral blood cells, irradiated normal rectal mucosa and pre and post-treatment tumor samples (PRE-T and POS-T) from one individual to examine the iatrogenic effects of nCRT. Finally, we performed WES of 7 PRE-T/POST-T matched samples to examine how nCRT affects ITGH. ITGH was assessed by quantifying subclonal mutations within individual tumors using the Mutant-Allele Tumor Heterogeneity score (MATH score). Results: Rectal tumors exhibit remarkable ITGH that is ultimately associated with disease stage (MATH score stage I/II 35.54 vs. stage III/IV 44.39, p = 0.047) and lymph node metastasis (MATH score N0 35.87 vs. N+ 45.79, p = 0.026). We also showed that nCRT does not seem to introduce detectable somatic mutations in the irradiated mucosa. Comparison of PRE-T and POST-T matched samples revealed a significant increase in ITGH in 5 out 7 patients and MATH scores were significantly higher after nCRT (median 41.7 vs. 28.8, p = 0.04). Finally, we were able to identify a subset of "enriched mutations" with significant changes in MAFs between PRE-T and POST-T samples. These "enriched mutations" were significantly more frequent in POST-T compared to PRE-T samples (92.9% vs. 7.1% p < 0.00001) and include mutations in genes associated with genetic instability and drug resistance in colorectal cancer, indicating the expansion of tumor cell subpopulations more prone to resist to nCRT. Conclusions: nCRT increases ITGH and may result in the expansion of resistant tumor cell populations in residual tumors. The risk of introducing relevant somatic mutations in the adjacent mucosa is minimal but non-responsive tumors may have potentially worse biological behavior when compared to their untreated counterparts. This was an exploratory study, and due to the limited number of samples analyzed, our results need to be validated in larger cohorts.

Antagonism between the RNA-binding protein Musashi1 and miR-137 and its potential impact on neurogenesis and glioblastoma development.

RNA-binding proteins (RBPs) and miRNAs are critical gene expression regulators that interact with one another in cooperative and antagonistic fashions. We identified Musashi1 (Msi1) and miR-137 as regulators of a molecular switch between self-renewal and differentiation. Msi1 and miR-137 have opposite expression patterns and functions, and Msi1 is repressed by miR-137. Msi1 is a stem-cell protein implicated in self-renewal while miR-137 functions as a proneuronal differentiation miRNA. In gliomas, miR-137 functions as a tumor suppressor while Msi1 is a prooncogenic factor. We suggest that the balance between Msi1 and miR-137 is a key determinant in cell fate decisions and disruption of this balance could contribute to neurodegenerative diseases and glioma development. Genomic analyses revealed that Msi1 and miR-137 share 141 target genes associated with differentiation, development, and morphogenesis. Initial results pointed out that these two regulators have an opposite impact on the expression of their target genes. Therefore, we propose an antagonistic model in which this network of shared targets could be either repressed by miR-137 or activated by Msi1, leading to different outcomes (self-renewal, proliferation, tumorigenesis).

Patient-derived conditionally reprogrammed cells maintain intra-tumor genetic heterogeneity.

Preclinical in vitro models provide an essential tool to study cancer cell biology as well as aid in translational research, including drug target identification and drug discovery efforts. For any model to be clinically relevant, it needs to recapitulate the biology and cell heterogeneity of the primary tumor. We recently developed and described a conditional reprogramming (CR) cell technology that addresses many of these needs and avoids the deficiencies of most current cancer cell lines, which are usually clonal in origin. Here, we used the CR cell method to generate a collection of patient-derived cell cultures from non-small cell lung cancers (NSCLC). Whole exome sequencing and copy number variations are used for the first time to address the capability of CR cells to keep their tumor-derived heterogeneity. Our results indicated that these primary cultures largely maintained the molecular characteristics of the original tumors. Using a mutant-allele tumor heterogeneity (MATH) score, we showed that CR cells are able to keep and maintain most of the intra-tumoral heterogeneity, suggesting oligoclonality of these cultures. CR cultures therefore represent a pre-clinical lung cancer model for future basic and translational studies.

High IL-1R8 expression in breast tumors promotes tumor growth and contributes to impaired antitumor immunity.

Tumors develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. The identification of mechanisms leading to local immune dysregulation is critical to improve cancer therapy. We here demonstrate that Interleukin-1 receptor 8 (IL-1R8 - previously known as SIGIRR/TIR8), a negative regulator of Toll-Like and Interleukin-1 Receptor family signaling, is up-regulated during breast epithelial cell transformation and in primary breast tumors. IL-1R8 expression in transformed breast epithelial cells reduced IL-1-dependent NF-κB activation and production of pro-inflammatory cytokines, inhibited NK cell activation and favored M2-like macrophage polarization. In a murine breast cancer model (MMTV-neu), IL-1R8-deficiency reduced tumor growth and metastasis and was associated with increased mobilization and activation of immune cells, such as NK cells and CD8+ T cells. Finally, immune-gene signature analysis in clinical specimens revealed that high IL-1R8 expression is associated with impaired innate immune sensing and T-cell exclusion from the tumor microenvironment. Our results indicate that high IL-1R8 expression acts as a novel immunomodulatory mechanism leading to dysregulated immunity with important implications for breast cancer immunotherapy.

Intratumoral Genetic Heterogeneity in Rectal Cancer: Are Single Biopsies representative of the entirety of the tumor?

OBJECTIVE: Demonstrate intratumoral genetic heterogeneity in rectal cancer. BACKGROUND: Several clinical management decisions in rectal cancer may be influenced by pretreatment biopsy information. However, in the setting of significant intratumoral heterogeneity, biopsies may not be representative of the entirety of the tumor and limit the reliability of the information provided from them for clinical decision management. METHODS: Three fragments from a single rectal adenocarcinoma were chosen for whole-exome sequencing followed by mutation detection analysis. About 25 Gb of unambiguously mapped sequences were generated for each sample resulting in a median fold-coverage of 35x. Captured sequences mapped to the reference human genome were then used for the detection of somatic point mutations. RESULTS: Overall, 193 unique somatic point mutations were identified. Only 53 (27%) of these were shared by all three fragments, including known genes involved in early phases of the adenoma-carcinoma sequence (such as, APC). Approximately, 115 (59%) mutations were exclusively present in only one of the fragments, including mutations in "driver" genes (DNAH12). Jaccard distances showed a median distance of 0.603 for pair-wise comparison of fragments indicating significant heterogeneity between them. CONCLUSIONS: Considerable intratumoral heterogeneity is present among naive rectal cancers. The majority of point mutations detected in different fragments from rectal cancers are frequently unique to a single fragment. These findings support that gene mutations found on single pretreatment biopsies will not necessarily be representative of mutations present in the entirety of the tumor and therefore may limit the utility of the biological information provided by single biopsy fragments for clinical management decisions.

Functional genomics analyses of RNA-binding proteins reveal the splicing regulator SNRPB as an oncogenic candidate in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive type of brain tumor. Currently, GBM has an extremely poor outcome and there is no effective treatment. In this context, genomic and transcriptomic analyses have become important tools to identify new avenues for therapies. RNA-binding proteins (RBPs) are master regulators of co- and post-transcriptional events; however, their role in GBM remains poorly understood. To further our knowledge of novel regulatory pathways that could contribute to gliomagenesis, we have conducted a systematic study of RBPs in GBM. RESULTS: By measuring expression levels of 1542 human RBPs in GBM samples and glioma stem cell samples, we identified 58 consistently upregulated RBPs. Survival analysis revealed that increased expression of 21 RBPs was also associated with a poor prognosis. To assess the functional impact of those RBPs, we modulated their expression in GBM cell lines and performed viability, proliferation, and apoptosis assays. Combined results revealed a prominent oncogenic candidate, SNRPB, which encodes core spliceosome machinery components. To reveal the impact of SNRPB on splicing and gene expression, we performed its knockdown in a GBM cell line followed by RNA sequencing. We found that the affected genes were involved in RNA processing, DNA repair, and chromatin remodeling. Additionally, genes and pathways already associated with gliomagenesis, as well as a set of general cancer genes, also presented with splicing and expression alterations. CONCLUSIONS: Our study provides new insights into how RBPs, and specifically SNRPB, regulate gene expression and directly impact GBM development.

miR-124, -128, and -137 Orchestrate Neural Differentiation by Acting on Overlapping Gene Sets Containing a Highly Connected Transcription Factor Network.

The ventricular-subventricular zone harbors neural stem cells (NSCs) that can differentiate into neurons, astrocytes, and oligodendrocytes. This process requires loss of stem cell properties and gain of characteristics associated with differentiated cells. miRNAs function as important drivers of this transition; miR-124, -128, and -137 are among the most relevant ones and have been shown to share commonalities and act as proneurogenic regulators. We conducted biological and genomic analyses to dissect their target repertoire during neurogenesis and tested the hypothesis that they act cooperatively to promote differentiation. To map their target genes, we transfected NSCs with antagomiRs and analyzed differences in their mRNA profile throughout differentiation with respect to controls. This strategy led to the identification of 910 targets for miR-124, 216 for miR-128, and 652 for miR-137. The target sets show extensive overlap. Inspection by gene ontology and network analysis indicated that transcription factors are a major component of these miRNAs target sets. Moreover, several of these transcription factors form a highly interconnected network. Sp1 was determined to be the main node of this network and was further investigated. Our data suggest that miR-124, -128, and -137 act synergistically to regulate Sp1 expression. Sp1 levels are dramatically reduced as cells differentiate and silencing of its expression reduced neuronal production and affected NSC viability and proliferation. In summary, our results show that miRNAs can act cooperatively and synergistically to regulate complex biological processes like neurogenesis and that transcription factors are heavily targeted to branch out their regulatory effect.

Comprehensive cancer-gene panels can be used to estimate mutational load and predict clinical benefit to PD-1 blockade in clinical practice.

Cancer gene panels (CGPs) are already used in clinical practice to match tumor's genetic profile with available targeted therapies. We aimed to determine if CGPs could also be applied to estimate tumor mutational load and predict clinical benefit to PD-1 and CTLA-4 checkpoint blockade therapy. Whole-exome sequencing (WES) mutation data obtained from melanoma and non-small cell lung cancer (NSCLC) patients published by Snyder et al. 2014 and Rizvi et al. 2015, respectively, were used to select nonsynonymous somatic mutations occurring in genes included in the Foundation Medicine Panel (FM-CGP) and in our own Institutional Panel (HSL-CGP). CGP-mutational load was calculated for each patient using both panels and was associated with clinical outcomes as defined and reported in the original articles. Higher CGP-mutational load was observed in NSCLC patients presenting durable clinical benefit (DCB) to PD-1 blockade (FM-CGP P=0.03, HSL-CGP P=0.01). We also observed that 69% of patients with high CGP-mutational load experienced DCB to PD-1 blockade, as compared to 20% of patients with low CGP-mutational load (FM-CGP and HSL-CGP P=0.01). Noteworthy, predictive accuracy of CGP-mutational load for DCB was not statistically different from that estimated by WES sequencing (P=0.73). Moreover, a high CGP-mutational load was significantly associated with progression-free survival (PFS) in patients treated with PD-1 blockade (FM-CGP P=0.005, HR 0.27, 95% IC 0.105 to 0.669; HSL-CGP P=0.008, HR 0.29, 95% IC 0.116 to 0.719). Similar associations between CGP-mutational load and clinical benefit to CTLA-4 blockade were not observed. In summary, our data reveals that CGPs can be used to estimate mutational load and to predict clinical benefit to PD-1 blockade, with similar accuracy to that reported using WES.

RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma.

The conserved RNA-binding protein Musashi1 (MSI1) has emerged as a key oncogenic factor in numerous solid tumors, including glioblastoma. However, its mechanism of action has not yet been established comprehensively. To identify its target genes comprehensively and determine the main routes by which it influences glioblastoma phenotypes, we conducted individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) experiments. We confirmed that MSI1 has a preference for UAG sequences contained in a particular structural context, especially in 3' untranslated regions. Although numerous binding sites were also identified in intronic sequences, our RNA transcriptome sequencing analysis does not favor the idea that MSI1 is a major regulator of splicing in glioblastoma cells. MSI1 target mRNAs encode proteins that function in multiple pathways of cell proliferation and cell adhesion. Since these associations indicate potentially new roles for MSI1, we investigated its impact on glioblastoma cell adhesion, morphology, migration, and invasion. These processes are known to underpin the spread and relapse of glioblastoma, in contrast to other tumors where metastasis is the main driver of recurrence and progression.

Mutational analysis of genes coding for cell surface proteins in colorectal cancer cell lines reveal novel altered pathways, druggable mutations and mutated epitopes for targeted therapy.

We carried out a mutational analysis of 3,594 genes coding for cell surface proteins (Surfaceome) in 23 colorectal cancer cell lines, searching for new altered pathways, druggable mutations and mutated epitopes for targeted therapy in colorectal cancer. A total of 3,944 somatic non-synonymous substitutions and 595 InDels, occurring in 2,061 (57%) Surfaceome genes were catalogued. We identified 48 genes not previously described as mutated in colorectal tumors in the TCGA database, including genes that are mutated and expressed in >10% of the cell lines (SEMA4C, FGFRL1, PKD1, FAM38A, WDR81, TMEM136, SLC36A1, SLC26A6, IGFLR1). Analysis of these genes uncovered important roles for FGF and SEMA4 signaling in colorectal cancer with possible therapeutic implications. We also found that cell lines express on average 11 druggable mutations, including frequent mutations (>20%) in the receptor tyrosine kinases AXL and EPHA2, which have not been previously considered as potential targets for colorectal cancer. Finally, we identified 82 cell surface mutated epitopes, however expression of only 30% of these epitopes was detected in our cell lines. Notwithstanding, 92% of these epitopes were expressed in cell lines with the mutator phenotype, opening new venues for the use of "general" immune checkpoint drugs in this subset of patients.

Recurrent somatic mutation in DROSHA induces microRNA profile changes in Wilms tumour.

Wilms tumour (WT) is an embryonal kidney neoplasia for which very few driver genes have been identified. Here we identify DROSHA mutations in 12% of WT samples (26/222) using whole-exome sequencing and targeted sequencing of 10 microRNA (miRNA)-processing genes. A recurrent mutation (E1147K) affecting a metal-binding residue of the RNase IIIb domain is detected in 81% of the DROSHA-mutated tumours. In addition, we identify non-recurrent mutations in other genes of this pathway (DGCR8, DICER1, XPO5 and TARBP2). By assessing the miRNA expression pattern of the DROSHA-E1147K-mutated tumours and cell lines expressing this mutation, we determine that this variant leads to a predominant downregulation of a subset of miRNAs. We confirm that the downregulation occurs exclusively in mature miRNAs and not in primary miRNA transcripts, suggesting that the DROSHA E1147K mutation affects processing of primary miRNAs. Our data underscore the pivotal role of the miRNA biogenesis pathway in WT tumorigenesis, particularly the major miRNA-processing gene DROSHA.

Gene Expression Profiling Reveals Regulation of ERK Phosphorylation by Androgen-Induced Tumor Suppressor U19/EAF2 in the Mouse Prostate.

U19/EAF2 is regulated by androgens in the prostate and capable of regulating transcriptional elongation of RNA Pol II via interaction with the ELL family proteins. Inactivation of U19/EAF2 induces tumorigenesis in multiple organs; however the mechanism of U19/EAF2 tumor suppression remains unclear. To elucidate potential mechanisms of U19/EAF2 action, we performed cDNA microarray analysis and identified 164 mRNA transcripts regulated by U19/EAF2 in the mouse ventral prostate. Bioinformatics analysis indicated that U19/EAF2 knockout activates the RAS-BRAF-ERK signaling pathway, which is known to play important roles in carcinogenesis. qPCR verified increased expression of BRAF mRNA, and immunostaining and Western blot analysis demonstrated increased expression of p-ERK at the protein level suggested U19/EAF2 knockout activates this important pathway. These findings indicate that loss of EAF2 up-regulates transcription of RAS cascade genes including Grb2, PI3K, and BRAF, leading to elevated p-ERK levels, which may represent a major functional role of U19/EAF2 in the prostate. Furthermore, these observations suggest that U19/EAF2 is a key player in crosstalk between androgen receptor and the RAS-BRAF-ERK signaling pathway.