Integrative Analysis Of RNA-Seq Data Research Articles

Blood transcriptomics analysis offers insights into variant-specific immune response to SARS-CoV-2

Bulk RNA sequencing (RNA-seq) of blood is typically used for gene expression analysis in biomedical research but is still rarely used in clinical practice. In this study, we propose that RNA-seq should be considered a diagnostic tool, as it offers not only insights into aberrant gene expression and splicing but also delivers additional readouts on immune cell type composition as well as B-cell and T-cell receptor (BCR/TCR) repertoires. We demonstrate that RNA-seq offers insights into a patient’s immune status via integrative analysis of RNA-seq data from patients infected with various SARS-CoV-2 variants (in total 196 samples with up to 200 million reads sequencing depth). We compare the results of computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) to complete blood count data, the current gold standard in clinical practice. We observe varying levels of lymphocyte depletion and significant differences in neutrophil levels between SARS-CoV-2 variants. Additionally, we identify B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that—combined with sequence alignments and BLASTp—they could be used to classify a patient's disease. Finally, we investigated the sequencing depth required for such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods using bulk RNA-seq analyses can supplement missing CBC data and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

Abstract 1131: Pharmacogenomic insight into targetable vulnerabilities and modifiers of response to MRTX1133 in KRASG12D-mutant models

Abstract The ability to effectively target mutated KRAS has remained elusive despite decades of research. Recently, the identification of selective KRASG12C inhibitors has provided an effective treatment option for patients harboring this particular mutation and has also provided insight toward targeting other KRAS mutants, including KRASG12D. MRTX1133 was identified via a structure-based drug design strategy as a potent, selective, and non-covalent KRASG12D inhibitor directed at the switch II binding pocket. MRTX1133 exhibited dose dependent KRAS pathway and tumor growth inhibition and demonstrated marked anti-tumor activity across a panel of models in vivo, including tumor regression >30% in 8 out of 11 pancreatic ductal adenocarcinoma (PDAC) models. While evaluation of MRTX1133 across a panel of cell and patient-derived xenograft models demonstrated strong single agent activity in the majority of models tested, a subset of models were less responsive. Focused sgRNA libraries targeting ~5,000 genes were tested in MRTX1133-anchored CRISPR/Cas9 screens in vitro and in vivo in PDAC and colorectal KRASG12D-mutant cell lines. Similar to what was seen in a targeted genetic screen with our selective KRASG12C inhibitor MRTX849, several genes that act either upstream or downstream of KRAS were depleted with MRTX1133 treatment which illuminate specific targetable vulnerabilities in the context of KRASG12D inhibition. sgRNAs targeting EGFR, PIK3CA, PTPN11, mTOR, and CDK2/4/6 were depleted in MRTX1133-treated (or vehicle-treated) cell lines or xenograft models, while hallmark tumor suppressor genes RB1, KEAP1, NF1 and PTEN were enriched. The enrichment of KEAP1 sgRNAs in PDAC models parallels findings with MRTX849 in preclinical models and is in line with emerging clinical data suggesting increased capacity to scavenge reactive oxygen species (ROS) may represent a mechanism of partial resistance to a non-covalent, non-electrophilic warhead-containing KRASG12D-selective inhibitor in PDAC. Treatment using small molecules targeting selected vulnerabilities in vitro and in vivo confirmed these genetic findings. Further evaluation of dependencies utilizing an integrated analysis of RNAseq data also revealed KRAS regulates and is critically dependent on pro-survival and cell cycle genes for cancer cell viability. These data lend further insight into tumor response to KRASG12D inhibition and provide key insight into the genes that mediate the mechanism of action of, as well as confer partial resistance to MRTX1133, and identify combination targets that can augment the anti-tumor effect of MRTX1133. Citation Format: Vickie Bowcut, Andrew Calinisan, David M. Briere, Lauren Hargis, Lars D. Engstrom, Jade Laguer, Darin Vanderpool, Xiaolun Wang, J David Lawson, Nicole Thomas, Robin Gunn, Christopher R. Smith, Julio Fernandez-Banet, Leo He, Adam Pavlicek, Lisa Rahbaek, Matthew A. Marx, Peter Olson, James G. Christensen, Jill Hallin. Pharmacogenomic insight into targetable vulnerabilities and modifiers of response to MRTX1133 in KRASG12D-mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1131.

Bortezomib Induces Anti-Multiple Myeloma Immune Response Mediated By Cgas/Sting Pathway Activation, Type I Interferon Secretion, and Immunogenic Cell Death: Clinical Application

Immune escape underlies progression of disease and resistance to therapy in MM, and dysfunction of both innate and adaptive immunity highlight the urgent need for scientifically-informed strategies to restore anti-MM immunity and improve patient outcome. Here we demonstrate that proteasome inhibitor bortezomib (BTZ) modulates the immunosuppressive bone marrow milieu by inducing immunogenic cell death (ICD) and a type-I interferon (IFN) response which contribute to its clinical efficacy. Moreover, we identified STING as a mediator of its anti-MM immune response, and provide the preclinical rationale for clinical trials of BTZ-STING agonist combination therapy. We first show that BTZ induces hallmarks of ICD in both human and murine MM cell lines, including exposure of endoplasmic reticulum protein calreticulin (CALR), that functions as an "eat me signal". Specifically, co-culture with BTZ-treated MM cells induced functional maturation of dendritic cells (DCs) and enhanced uptake of BTZ-treated MM cells, assessed by flow cytometry and confocal microscopy. Notably, these functional sequelae were abrogated when DCs were co-cultured with CALRKO MM cells; and add-back experiments by stable overexpression of CALR in KO clones confirmed the specific role of CALRin BTZ-induced immunogenicity. We next validated these findings in 2 in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent murine 5TGM1 tumor cell shrinkage in immunocompetent than immunodeficient hosts, an effect mediated by ICD induction since it was abrogated in immunocompetent mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to induce a protective anti-MM immune response: vaccination protected against tumor growth upon rechallenge with 5TGM1 cells; conversely, palpable tumors were detected in non-vaccinated mice by 1 week. Moreover, vaccination with BTZ-treated CALRKO 5TGM1 cells abrogated the protective effect of BTZ since only 50% mice were tumor free by 30 days. Consistently, ELISPOT assay on mice splenocytes confirmed that vaccination with BTZ-treated 5TGM1 cells triggered a MM-specific T cell response. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both CALRWT and CALRKO cells growing in immunocompetent mice; and then carried out an integrative analysis of RNAseq data from clinically-annotated MM patients (n=327) uniformly treated with BTZ-based regimens (IFM/DFCI 2009). We identified a specific ICD signature induced by BTZ only in CALRWT tumors in mice; and importantly, we found that increased expression of the human orthologs of this ICD signature was strongly and positively correlated with clinical outcome (overall survival (OS), p value=0.01). Moreover, the predictive value of this signature was confirmed in an independent dataset of BTZ-treated patients (GSE9782) (OS p value=0.024). Gene ontology analysis of the ICD signature genes revealed enrichment in inflammatory response pathways, including IFN stimulated genes (ISGs). Using RNAseq analysis and qRT-PCR validation, we showed that MM cells demonstrate a type-I IFN response after BTZ treatment which contributes to its in vivo efficacy, since neutralization of type-I IFNs signaling in both MM and host cells with a type-I IFNs receptor 1 (IFNAR) blocking antibody significantly reduced BTZ anti-MM activity. Finally, we showed that BTZ increases genomic instability/micronuclei formation in MM cells and activates innate cGAS/STING immune pathway, thereby stimulating a type-I IFN response. Conversely, STINGKO in MM cells abrogated this effect and decreased BTZ-triggered anti-MM T cell response. Moreover, expression of the ISGs included in ICD signature was positively correlated with STING expression in MM patients. Importantly, pharmacological activation of STING with STING agonist induced potent anti-MM activity in vivo; and combination of STING agonist with BTZ further potentiated this in vivo anti-MM response, with increased T cell infiltration into retrieved tumors, evidenced by IHC analysis. In conclusion, our studies delineate a novel mechanism whereby BTZ triggers anti-MM immune responses, and show that STING agonists can enhance this response. These findings provide the framework for clinical evaluation of STING agonists in combination with BTZ to induce potent anti-MM immune responses and thereby improve patient outcome. Disclosures Fulciniti: NIH: Research Funding. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Chauhan:Oncopeptide AB: Consultancy; consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.. Munshi:Karyopharm: Consultancy; Takeda: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract 5684: Drug-anchored in vitro and in vivo CRISPR screens to identify targetable vulnerabilities and modifiers of response to MRTX849 in KRASG12C-mutant models

Abstract MRTX849 is a recently described covalent KRASG12C inhibitor in active clinical development in non-small cell lung cancer (NSCLC) and other cancers harboring this mutation. Additional genetic alterations in hallmark pathways are co-incident with KRASG12C mutations in NSCLC and several of these alterations have been shown to contribute to the oncogenic phenotype. The impact of co-occurring genetic alterations on KRAS dependency or response to KRAS targeted therapy is unknown. Evaluation of MRTX849 across a panel of cell and patient-derived xenograft models demonstrated strong single agent activity in the majority of models tested, however, a subset of models were less responsive. A focused sgRNA library targeting ~1,000 genes was tested in MRTX849-anchored CRISPR/Cas9 screens in vitro and in vivo in multiple KRASG12C-mutant cell lines. Several genes that act either upstream or downstream of KRAS were depleted with MRTX849 treatment which illuminated specific targetable vulnerabilities in the context of KRASG12C inhibition. These data provided a catalogue of putative combination targets. sgRNAs targeting EGFR, PTPN11 (SHP2), SOS1, mTOR, CDK1/2/4/6 were all depleted in MRTX849-treated (or vehicle-treated) cell lines or xenograft models. Furthermore, treatment with small molecules targeting selected vulnerabilities confirmed these genetic data. Finally, targeting FGFR1 exhibited context-dependent dropout that may conditionally address receptor tyrosine kinase reactivation following MRTX849 treatment. This observation was further confirmed utilizing the FGFR family inhibitor BGJ398. Further evaluation of dependencies utilizing an integrated analysis of RNAseq data also revealed KRAS regulates and is critically dependent on specific pro-survival genes BCL2L1, MCL1 and BIRC5 (Survivin) for cancer cell viability. On the other hand, loss of tumor suppressors including KEAP1, NF1, CBL, RB1 and PTEN conferred a growth advantage and partial resistance to MRTX849 treatment in selected models. sgRNAs targeting the RAS pathway genes SPRY2 and NRAS were also enriched providing additional insight into the wiring of KRAS mutant tumors and adaptive response to KRASG12C inhibition. These data provide key insight into the genes that mediate the mechanism of action of, as well as confer partial resistance to, MRTX849, and identify combination targets that augment the anti-tumor effect of MRTX849. Citation Format: Lars D. Engstrom, Laura Waters, Sole Gatto, Julio Fernandez-Banet, Ruth Aranda, Adam Pavlicek, James G. Christensen, Peter Olson. Drug-anchored in vitro and in vivo CRISPR screens to identify targetable vulnerabilities and modifiers of response to MRTX849 in KRASG12C-mutant models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5684.

Integrated analysis of a compendium of RNA-Seq datasets for splicing factors

A vast amount of public RNA-sequencing datasets have been generated and used widely to study transcriptome mechanisms. These data offer precious opportunity for advancing biological research in transcriptome studies such as alternative splicing. We report the first large-scale integrated analysis of RNA-Seq data of splicing factors for systematically identifying key factors in diseases and biological processes. We analyzed 1,321 RNA-Seq libraries of various mouse tissues and cell lines, comprising more than 6.6 TB sequences from 75 independent studies that experimentally manipulated 56 splicing factors. Using these data, RNA splicing signatures and gene expression signatures were computed, and signature comparison analysis identified a list of key splicing factors in Rett syndrome and cold-induced thermogenesis. We show that cold-induced RNA-binding proteins rescue the neurite outgrowth defects in Rett syndrome using neuronal morphology analysis, and we also reveal that SRSF1 and PTBP1 are required for energy expenditure in adipocytes using metabolic flux analysis. Our study provides an integrated analysis for identifying key factors in diseases and biological processes and highlights the importance of public data resources for identifying hypotheses for experimental testing.

Epigenome and transcriptome study of moringa isothiocyanate in mouse kidney mesangial cells induced by high glucose, a potential model for diabetic-induced nephropathy.

Moringa isothiocyanate (MIC-1) is a bioactive constituent found abundantly in Moringa oleifera which possesses antioxidant and anti-inflammation properties. However, epigenome and transcriptome effects of MIC-1 in kidney mesangial cells challenged with high glucose (HG), a pre-condition for diabetic nephropathy (DN) remain unknown. Herein, we examined the transcriptome gene expression and epigenome DNA methylation in mouse kidney mesangial cells (MES13) using next-generation sequencing (NGS) technology. After HG treatment, epigenome and transcriptome were significantly altered. More importantly, MIC-1 exposure reversed some of the changes caused by HG. Integrative analysis of RNA-Seq data identified 20 canonical pathways showing inverse correlations between HG and MIC-1. These pathways included GNRH signaling, P2Y purigenic receptor signaling pathway, calcium signaling, LPS/IL-1-mediated inhibition of RXR function, and oxidative ethanol degradation III. In terms of alteration of DNA methylation patterns, 173 differentially methylation regions (DMRs) between the HG group and low glucose (LG) group and 149 DMRs between the MIC-1 group and the HG group were found. Several HG related DMRs could be reversed by MIC-1 treatment. Integrative analysis of RNA-Seq and Methyl-Seq data yielded a subset of genes associated with HG and MIC-1, and the gene expression changes may be driven by promoter CpG status. These genes include Col4a2, Tceal3, Ret, and Agt. In summary, our study provides novel insights related to transcriptomic and epigenomic/CpG methylomic alterations in MES13 upon challenged by HG but importantly, MIC-1 treatment reverses some of the transcriptome and epigenome/CpG methylome. These results may provide potential molecular targets and therapeutic strategies for DN.

Loss-of-Function of Gabarap Impairs Bortezomib-Induced Anti-Tumor Immunity in Multiple Myeloma: Clinical Application

Immune escape underlies progression of disease and resistance to therapy in multiple myeloma (MM). Conversely, restoration of anti-tumor immunity primed by immunogenic cancer cells and intratumoral dendritic cells (DCs) may be exploited to convert the immunologically "cold" MM into a "hot" MM; and may lead to long-term clinical benefit, even in patient subgroups with high-risk (HR) cytogenetics and poor survival. Here, we investigated the mechanisms, biologic sequelae, and clinical benefits of bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by DCs. We first show that BTZ can induce hallmarks of ICD in human and murine MM cell lines (n=5), including exposure of endoplasmic reticulum protein calreticulin (CALR) that functions as an "eat me signal". Specifically, our data show that co-culture with BTZ-treated MM cells can induce phenotypic and functional changes in immature DCs including higher expression of CD86/CD83 on cell surface and enhanced uptake of BTZ-treated MM cells, as assessed by flow cytometry- and confocal-based phagocytosis assay, respectively. Notably, we show that CALR has a key role in BTZ-induced immunogenicity, since these functional sequelae were abrogated in vitro when DCs were co-cultured with CALRKO MM cells. We then validated these findings in 2 different in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent 5TGM1 tumor cell shrinkage in immunocompetent hosts; and that this effect was directly linked to ICD induction, since it was abrogated in mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to enhance an anti-MM immune response: injection of live tumor cells resulted in palpable tumors in non-vaccinated mice by 1 week; conversely, injection of live tumor cells in vaccinated mice did not result in detectable tumor after 30 days. In contrast, in mice similarly vaccinated with BTZ-treated CALRKO 5TGM1 cells and challenged with injection of live WT cells, only 50% of vaccinated mice were tumor-free at day 30. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both immunodeficient or immunocompetent mice; and also carried out an integrative analysis of RNAseq data from newly diagnosed and clinically annotated MM patients (n=360) uniformly treated with BTZ-based regimes (IFM/DFCI 2009). Importantly, increased expression of the human orthologs of the immune genes induced in mice by BTZ was strongly and positively correlated with patient clinical outcome (OS p value=0.00089). The predictive value of this signature was confirmed in an independent dataset (GSE9782) (OS p value=0.024). Moreover, by interrogating the IFM/DFCI patient dataset, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) as a top differentially expressed gene among patients with longer survival rate (>5 years) as compared to those with poor survival (<1.5 years) after BTZ-based treatment. We found that low level of GABARAP, located on chr17p13.1, is associated with poor clinical outcome in MM patients (EFS, p= 0.0032); and that its prognostic value is still maintained even excluding HR patients with 17p deletion (EFS, p= 0.018). Interestingly, KMS11 cells that carry monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and stable overexpression of the gene in these cells restored the functional sequelae of ICD induction upon drug exposure. Moreover, GABARAPKO in 2 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; add-back experiments by pre-treatment with recombinant CALR (rCALR) or stable overexpression of GABARAP in KO clones confirmed the on-target effect GABARAPKO. Finally, mass Cytometry (CyTOF) after T cell culture with mature DCs primed by both WT and GABARAPKO AMO1 clones showed that treatment of MM cells with BTZ switched CD4+T cells towards an effector memory function; in contrast to treatment of GABARAPKO clones with BTZ, which led to T cell exhaustion. In conclusion, our studies demonstrate the clinical benefits of BTZ-induced ICD in MM; and that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, abrogates induction of antitumor immunity after drug exposure. These studies provide the framework for novel combination treatments to trigger anti-MM immunity and improve patient outcome in MM. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Munshi:Celgene: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Amgen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Discovery and characterization of long intergenic non-coding RNAs (lincRNA) module biomarkers in prostate cancer: an integrative analysis of RNA-Seq data.

BackgroundProstate cancer (PCa) is a leading cause of cancer-related death of men worldwide. There is an urgent need to develop novel biomarkers for PCa prognosis and diagnosis in the post prostate-specific antigen era. Long intergenic noncoding RNAs (lincRNAs) play essential roles in many physiological processes and can serve as alternative biomarkers for prostate cancer, but there has been no systematic investigation of lincRNAs in PCa yet.ResultsNine lincRNA co-expression modules were identified from PCa RNA-Seq data. The association between the principle component of each module and the PCa phenotype was examined by calculating the Pearson's correlation coefficients. Three modules (M1, M3, and M5) were found associated with PCa. Two modules (M3 and M5) were significantly enriched with lincRNAs, and one of them, M3, may be used as a lincRNA module-biomarker for PCa diagnosis. This module includes seven essential lincRNAs: TCONS_l2_00001418, TCONS_l2_00008237, TCONS_l2_00011130, TCONS_l2_00013175, TCONS_l2_00022611, TCONS_l2_00022670 and linc-PXN-1. The clustering analysis and microRNA enrichment analysis further confirmed our findings.ConclusionThe correlation between lincRNAs and protein-coding genes is helpful for further exploration of functional mechanisms of lincRNAs in PCa. This study provides some important insights into the roles of lincRNAs in PCa and suggests a few lincRNAs as candidate biomarkers for PCa diagnosis and prognosis.

Automated transcription start site prediction for comparative Transcriptomics using the SuperGenome

RNA deep-sequencing (RNA-seq) has been revolutionizing transcriptome analyses. Despite the high-throughput nature of this technology, very often transcriptome features such as transcription start sites (TSS) are still manually annotated. The problem is compounded for comparative transcriptomics of several species. We developed the SuperGenome algorithm, a novel general approach to comparative and integrative analysis of RNA-seq data. The SuperGenome can be utilized for comparative analyses of gene expression data, promoter sequences or SNPs in several species. Furthermore, it can be used for the comparative detection of TSS, for which we developed an automated TSS prediction method for differential RNA-seq experiments.