Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the US. Despite multiple large-scale genetic sequencing studies, identification of predictors of patient survival ...remains challenging. We performed a comprehensive assessment and integrative analysis of large-scale gene expression datasets, across multiple platforms, to enable discovery of a prognostic gene signature for patient survival in pancreatic cancer. PDAC RNA-Sequencing data from The Cancer Genome Atlas was stratified into Survival+ (>2-year survival) and Survival-(<1-year survival) cohorts (n = 47). Comparisons of RNA expression profiles between survival groups and normal pancreatic tissue expression data from the Gene Expression Omnibus generated an initial PDAC specific prognostic differential expression gene list. The candidate prognostic gene list was then trained on the Australian pancreatic cancer dataset from the ICGC database (n = 103), using iterative sampling based algorithms, to derive a gene signature predictive of patient survival. The gene signature was validated in 2 independent patient cohorts and against existing PDAC subtype classifications. We identified 707 candidate prognostic genes exhibiting differential expression in tumor versus normal tissue. A substantial fraction of these genes was also found to be differentially methylated between survival groups. From the candidate gene list, a 5-gene signature (ADM, ASPM, DCBLD2, E2F7, and KRT6A) was identified. Our signature demonstrated significant power to predict patient survival in two distinct patient cohorts and was independent of AJCC TNM staging. Cross-validation of our gene signature reported a better ROC AUC (≥ 0.8) when compared to existing PDAC survival signatures. Furthermore, validation of our signature through immunohistochemical analysis of patient tumor tissue and existing gene expression subtyping data in PDAC, demonstrated a correlation to the presence of vascular invasion and the aggressive squamous tumor subtype. Assessment of these genes in patient biopsies could help further inform risk-stratification and treatment decisions in pancreatic cancer.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to ...hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.
We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.
Sensitivity to binimetinib and ribociclib was inversely related (
= -0.58,
= 0.009).
amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.
Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling.
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Despite breakthroughs achieved with cancer checkpoint blockade therapy (CBT), many patients do not respond to anti-programmed cell death-1 (PD-1) due to primary or acquired resistance. Human tumor ...profiling and preclinical studies in tumor models have recently uncovered transforming growth factor-β (TGFβ) signaling activity as a potential point of intervention to overcome primary resistance to CBT. However, the development of therapies targeting TGFβ signaling has been hindered by dose-limiting cardiotoxicities, possibly due to nonselective inhibition of multiple TGFβ isoforms. Analysis of mRNA expression data from The Cancer Genome Atlas revealed that
is the most prevalent TGFβ isoform expressed in many types of human tumors, suggesting that TGFβ1 may be a key contributor to primary CBT resistance. To test whether selective TGFβ1 inhibition is sufficient to overcome CBT resistance, we generated a high-affinity, fully human antibody, SRK-181, that selectively binds to latent TGFβ1 and inhibits its activation. Coadministration of SRK-181-mIgG1 and an anti-PD-1 antibody in mice harboring syngeneic tumors refractory to anti-PD-1 treatment induced profound antitumor responses and survival benefit. Specific targeting of TGFβ1 was also effective in tumors expressing more than one TGFβ isoform. Combined SRK-181-mIgG1 and anti-PD-1 treatment resulted in increased intratumoral CD8
T cells and decreased immunosuppressive myeloid cells. No cardiac valvulopathy was observed in a 4-week rat toxicology study with SRK-181, suggesting that selectively blocking TGFβ1 activation may avoid dose-limiting toxicities previously observed with pan-TGFβ inhibitors. These results establish a rationale for exploring selective TGFβ1 inhibition to overcome primary resistance to CBT.
The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% ...to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.
CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
Abstract Alternatively activated “M2” macrophages are believed to function during late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the ...pro-inflammatory response caused by “M1” macrophages. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL-4)-stimulated “M2a” macrophages and IL-10-stimulated “M2c” macrophages, are not well understood. M2a macrophages are characterized by their ability to inhibit inflammation and contribute to the stabilization of angiogenesis. However, the role and temporal profile of M2c macrophages in wound healing are not known. Therefore, we performed next generation sequencing (RNA-seq) to identify biological functions and gene expression signatures of macrophages polarized in vitro with IL-10 to the M2c phenotype in comparison to M1 and M2a macrophages and an unactivated control (M0). We then explored the expression of these gene signatures in a publicly available data set of human wound healing. RNA-seq analysis showed that hundreds of genes were upregulated in M2c macrophages compared to the M0 control, with thousands of alternative splicing events. Following validation by Nanostring, 39 genes were found to be upregulated by M2c macrophages compared to the M0 control, and 17 genes were significantly upregulated relative to the M0, M1, and M2a phenotypes (using an adjusted p-value cutoff of 0.05 and fold change cutoff of 1.5). Many of the identified M2c-specific genes are associated with angiogenesis, matrix remodeling, and phagocytosis, including CD163, MMP8, TIMP1, VCAN, SERPINA1, MARCO, PLOD2, PCOCLE2 and F5 . Analysis of the macrophage-conditioned media for secretion of matrix-remodeling proteins showed that M2c macrophages secreted higher levels of MMP7, MMP8, and TIMP1 compared to the other phenotypes. Interestingly, temporal gene expression analysis of a publicly available microarray data set of human wound healing showed that M2c-related genes were upregulated at early times after injury, similar to M1-related genes, while M2a-related genes appeared at later stages or were downregulated after injury. While further studies are required to confirm the timing and role of M2c macrophages in vivo , these results suggest that M2c macrophages may function at early stages of wound healing. Identification of markers of the M2c phenotype will allow more detailed investigations into the role of M2c macrophages in vivo.
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GEOZS, IJS, IMTLJ, KISLJ, NUK, PNG, SAZU, SBCE, SBJE, UL, UPCLJ, UPUK, ZRSKP
Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. ...However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1α) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.
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► Unbiased genomic and metabolomic approaches to reveal downstream functions of mTORC1 ► mTORC1 stimulates glucose uptake and glycolysis through HIF1 ► mTORC1 stimulates the pentose phosphate pathway and lipid biosynthesis through SREBP1 ► S6K1 regulates SREBP1 processing and transcriptional activation of target genes
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Neurodevelopmental disorders represent a frequent indication for clinical exome sequencing. Fifty percent of cases, however, remain undiagnosed even upon exome reanalysis. Here we show RNA sequencing ...(RNA-seq) on human B-lymphoblastoid cell lines (LCL) is highly suitable for neurodevelopmental Mendelian gene testing and demonstrate the utility of this approach in suspected cases of Cornelia de Lange syndrome (CdLS).
Genotype-Tissue Expression project transcriptome data for LCL, blood, and brain were assessed for neurodevelopmental Mendelian gene expression. Detection of abnormal splicing and pathogenic variants in these genes was performed with a novel RNA-seq diagnostic pipeline and using a validation CdLS-LCL cohort (n = 10) and test cohort of patients who carry a clinical diagnosis of CdLS but negative genetic testing (n = 5).
LCLs share isoform diversity of brain tissue for a large subset of neurodevelopmental genes and express 1.8-fold more of these genes compared with blood (LCL, n = 1706; whole blood, n = 917). This enables testing of more than 1000 genetic syndromes. The RNA-seq pipeline had 90% sensitivity for detecting pathogenic events and revealed novel diagnoses such as abnormal splice products in NIPBL and pathogenic coding variants in BRD4 and ANKRD11.
The LCL transcriptome enables robust frontline and/or reflexive diagnostic testing for neurodevelopmental disorders.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in ...tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. ...Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.
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•Neuroblastomas differentially express high levels of GPC2 versus normal tissues•MYCN and gain of chromosome 7q mediate neuroblastoma GPC2 differential expression•GPC2 is integral in neuroblastoma cell proliferation•A GPC2-directed antibody-drug conjugate is cytotoxic to neuroblastoma cells
Bosse et al. show that GPC2 is expressed at high levels on most neuroblastomas (NB) but not at appreciable levels in normal childhood tissues and that GPC2 is critical for NB maintenance. They develop a GPC2-directed antibody-drug conjugate that is cytotoxic to GPC2-expressing NB cells in vitro and in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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