Bruna R. Correa

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.

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 tumors 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.

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. Stem Cells 2016;34:220–232

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

ABSTRACT 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.

A Mouse Model of Targeted Musashi1 Expression in Whole Intestinal Epithelium Suggests Regulatory Roles in Cell Cycle and Stemness

The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA‐binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self‐renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell‐like features in medulloblastoma, glioblastoma, and breast cancer cells, and has pro‐proliferative and pro‐tumorigenic properties in intestinal epithelial progenitor cells in vitro. To undertake a detailed study of Musashi1s function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v‐Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium. Compared with wild type litters, v‐Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA‐seq revealed Musashi1s association with gut stem cell signature, cell cycle, DNA replication, and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6, and Sox4. In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle, and DNA synthesis and repair. Stem Cells 2015;33:3621–3634

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

BackgroundGlioblastoma (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.ResultsBy 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.ConclusionsOur study provides new insights into how RBPs, and specifically SNRPB, regulate gene expression and directly impact GBM development.

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.

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.

Abstract A133: Genetic heterogeneity in rectal cancer: Identification of subpopulations of tumor cells resistant to neoadjuvant CRT.

One of the benefits of neoadjuvant chemotherapy and radiation therapy (CRT) for the management of rectal cancer is tumor downstaging that can ultimately lead to complete tumor regression (known as complete pathological response - pCR). In a previous study using PET/CT imaging to assess tumor response to CRT, 50% of rectal cancers showed a continuous decrease in metabolic activity (estimated by standard uptake value measurements) between 6 and 12 weeks from CRT completion. However the remaining 50% of the cases showed increased metabolic activity within that period. We reasoned that the increased metabolic activity observed in these later patients could be determined by the clonal expansion of a genetically distinct subpopulation of tumor cells resistant to CRT. To address this question we performed exome sequencing and mutation detection analysis on normal tissue, primary tumor and residual tumor from 7 patients with rectal cancer that exhibited an increase in metabolic activity after CRT. Overall, about 20Gb of unambiguously mapped sequences were generated for each sample resulting in an average fold-coverage of 30X. Captured sequences mapped to the reference human genome were then used for the detection of SNPs and non-synonymous somatic point mutations in all three samples from each patient. Approximately 30,000 single nucleotide variants (SNVs) were identified in each sample and most of these SNVs were common to all samples. As expected, the majority of these common inherited variants (96%) have already been described in dbSNP. To identify non-synonymous somatic mutations occurring in primary and residual tumor samples, we excluded those present in the normal sample and those already described as a known SNP in dbSNP resulting in a mean of 243 SNVs per patient. Noteworthy, a significant number of non-synonymous somatic mutations were exclusively found in the primary and residual tumor samples of each patient, 30 and 32 SNVs on average respectively, revealing a high degree of tumor genetic heterogeneity. Furthermore, we were able to identify non-synonymous somatic mutations that were presented in both samples (mean of 107 SNVs), and for these mutations we determined the mutant allele frequency (number of reads representing the mutation/total number of reads covering the mutated base) in the primary and the residual tumor. We then searched for mutations with significantly different allele frequencies in the two samples, as these mutations would likely represent genetically distinct subpopulations of tumor cells selected during CRT. We were able to identify, on average, 4 SNVs enriched in primary tumor and 37 SNVs enriched in residual tumor per patient. In conclusion, based on exome sequencing of rectal tumors that exhibit incomplete response to CRT and have increased tumor metabolism we were able to identify non-synonymous mutations that may be associated with specific mechanisms of resistance or sensitivity to CRT. Functional analysis of these mutations and mutated genes will be presented, providing new insights into the molecular events influencing response to neoadjuvant CRT in rectal cancer. Citation Format: Fabiana Bettoni, Elisa R. Donnard, Bruna R. S. Correa, Paula F. Asprino, Fernanda C. Koyama, Natalia M. Felicio, Bruna H. Hessel, Pedro A. F. Galante, Anamaria A. Camargo, Angelita Habr-Gama, Rodrigo O. Perez. Genetic heterogeneity in rectal cancer - Identification of subpopulations of tumor cells resistant to neoadjuvant CRT. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 05.