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Purpose: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. Experimental Design: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. Results: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. Conclusions: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment.

Cytogenetic aberrations at the single-cell level represent an important characteristic of cancer cells relevant to tumor evolution and prognosis. However, with the advent of The Cancer Genome Atlas (TCGA), there has been a major shift in cancer research to the use of data from aggregate cell populations. Given that tumor cells harbor hundreds to thousands of biologically relevant genetic alterations that manifest as intratumor heterogeneity, these aggregate analyses may miss alterations readily observable at single-cell resolution. Using the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, we developed an algorithm to parse International System for Cytogenetic Nomenclature notation for quantitative abnormalities. Comparison of the Mitelman database and TCGA demonstrated that the Mitelman database is a powerful resource, and that cytogenetic aberrations captured by traditional approaches used in Mitelman database are on par with population-based genomic analyses used in TCGA. This algorithm will help nonspecialists to overcome the challenges associated with the format and syntax of the Mitelman database.Significance:A novel in silico approach compares cytogenetic data between the Mitelman database and TCGA, highlighting the advantages and limitations of both datasets.

This file contains two figures and five tables which enhance the reader's comprehension by providing a pictorial representation of common cytogenetic aberrations mentioned in the text, as well as additional data such as statistics regarding the frequency of specific cytogenetic events. Supplementary table 5 also provides key information needed to interpret figure 5 of the main text.

Supplementary Figure 1 from The EphB6 Receptor Cooperates with c-Cbl to Regulate the Behavior of Breast Cancer Cells

Supplementary Figure 2 from The EphB6 Receptor Cooperates with c-Cbl to Regulate the Behavior of Breast Cancer Cells

Fig. S5. A. Heatmap of the BioID hits that are enriched in the top 15 GO processes based onGSEA software. B. Gene Set Enrichment Analysis plots of the top 15 GO process for the BioIDdata produced by GSEA software used to generate the heatmap in A.

Purpose:Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics.Experimental Design:Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models.Results:Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab.Conclusions:Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment.

Table S6: Hits from the EPHA2 BioID assay.

Supplementary Materials

Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility.To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells orin vitro, most notably, the iron transporters transferrin (TF), transferrin receptor 2 (TFR2), and mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria.We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that regulates iron homeostasis in cancer cells.

Loss of function screens using shRNA and CRISPR are routinely used to identify genes that modulate responses of tumor cells to anti-cancer drugs. Here, by integrating GSEA and CMAP analyses of multiple published shRNA screens, we identified a core set of pathways that affect responses to multiple drugs with diverse mechanisms of action. This suggests that these pathways represent “weak points” or “Achilles heels”, whose mild disturbance should make cancer cells vulnerable to a variety of treatments. These “weak points” include proteasome, protein synthesis, RNA splicing, RNA synthesis, cell cycle, Akt-mTOR, and tight junction-related pathways. Therefore, inhibitors of these pathways are expected to sensitize cancer cells to a variety of drugs. This hypothesis was tested by analyzing the diversity of drugs that synergize with FDA-approved inhibitors of the proteasome, RNA synthesis, and Akt-mTOR pathways. Indeed, the quantitative evaluation indicates that inhibitors of any of these signaling pathways can synergize with a more diverse set of pharmaceuticals, compared to compounds inhibiting targets distinct from the “weak points” pathways. Our findings described here imply that inhibitors of the “weak points” pathways should be considered as primary candidates in a search for synergistic drug combinations.

Large-scale genetic screens are becoming increasingly used as powerful tools to query the genome to identify therapeutic targets in cancer. The advent of the CRISPR technology has revolutionized the effectiveness of these screens and has made it possible to carry out loss-of-function screens to identify cancer-specific genetic interactions. Such loss-of-function screens can be performed in silico, in vitro, and in vivo, depending on the scale of the screen, as well as research questions to be answered. Performing screens in vivo has its challenges but also advantages, providing opportunities to study the tumor microenvironment and cancer immunity. In this chapter, we present a procedural framework and associated notes for conducting in vivo CRISPR knockout screens in cancer models to study cancer biology, anti-tumor immune responses, tumor microenvironment, and predicting treatment responses.

The human hepatocyte nuclear factor 1 homeobox A (HNF1A) gene loci express the protein-coding HNF1A transcript and a long non-coding RNA in the anti-sense (HNF1A-AS1) direction. HNF1A-AS1 is expressed in numerous types of cancers and poor clinical outcomes such as higher mortality rates, greater metastatic capacity, and poor prognosis of the disease are the results of this expression. In this study, we determined the epigenetic features of the HNF1A gene loci, and expression and cellular localization of HNF1A-AS1 RNA, HNF1A RNA, and HNF1A protein in colorectal cancer (HT-29, HTC116, RKO, and SW480) and normal colon epithelial (CCD841) cells. The HT-29 HNF1A gene had active histone marks (H3K4me3, H3K27ac) and DNase 1 accessible sites at the promoter regions of the HNF1A and HNF1A-AS1 genes. These epigenetic marks were not observed in the other colorectal cancer cells or in the normal colon epithelial cells. Consistent with the active gene epigenetic signature of the HNF1A gene in HT-29 cells, HNF1A protein, and HNF1A/HNF1A-AS1 transcripts were detected in HT-29 cells but poorly, if at all observed, in the other cell types. In HT-29 cells, HNF1A-AS1 localized to the nucleus and was found to bind to the enhancer of zeste homolog 2 (EZH2, a member of PRC2 complex) and potentially form RNA-DNA triplexes with DNase 1 accessible sites in the HT-29 genome. These activities of HNF1A-AS1 may contribute to the oncogenic properties of this long non-coding RNA.

Irreversible electroporation (IRE) is a new cancer ablation technology, but methods to improve IRE-induced therapeutic immunity are only beginning to be investigated. We developed a mouse model bearing large primary (300 mm(3)) and medium distant (100 mm(3)) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with Toll-like receptor (TLR)3/9 agonists (poly I:C/CpG) (IRE + pIC/CpG), PD-1 blockade (IRE + PD-1 blockade), or both (IRE + Combo) to investigate therapeutic effects on primary and distant EG7 tumors and conversion-promoting effects on the immunotolerant tumor microenvironment (TME). We demonstrated that IRE alone simulated very weak OVA-specific CD8(+) T cell responses and did not inhibit primary tumor growth. IRE + pIC/CpG synergistically stimulated more efficient OVA-specific CD8(+) T cell responses and primary tumor growth inhibition than IRE + PD-1 blockade. IRE + pIC/CpG played a major role in the modulation of immune cell profiles but a minor role in the downregulation of PD-L1 expression in the TME and vice versa for IRE + PD-1 blockade. IRE + Combo cooperatively induced potent OVA-specific CD8(+) T cell immunity and rescued exhausted intratumoral CD8(+) T cells, leading to eradication of not only primary tumors but also untreated concomitant distant tumors and lung metastases. IRE + Combo efficiently modulated immune cell profiles, as evidenced by reductions in immunotolerant type-2 (M2) macrophages, myeloid-derived suppressor-cells, plasmacytoid dendritic cells, and regulatory T cells and by increases in immunogenic M1 macrophages, CD169(+) macrophages, type-1 conventional dendritic cells, and CD8(+) T cells, leading to conversion of immunotolerance in not only primary TMEs but also untreated distant TMEs. IRE + Combo also showed effective therapeutic effects in two breast cancer models. Therefore, our results suggest that IRE + Combo is a promising strategy to improve IRE ablation therapy in cancer.

Cancer is one of the leading causes of death and chromosomal instability (CIN) is a hallmark feature of cancer. CIN, a source of genetic variation in either altered chromosome number or structure contributes to tumor heterogeneity and has become a hot topic in recent years prominently for its role in therapeutic responses. Synthetic lethality and synthetic rescue based approaches, for example, advancing CRISPR-Cas9 platform, are emerging as a powerful strategy to identify new potential targets to selectively eradicate cancer cells. Unfortunately, only few of them are further explored therapeutically due to the difficulty in linking these targets to small molecules for pharmacological intervention. This, however, can be alleviated by the efforts to bring chemical, bioactivity, and genomic data together, as well as established computational approaches. In this chapter, we will discuss some of these advances, including established databases and in silico target-ligand prediction, with the aim to navigate through the synthetically available chemical space to the biologically targetable landscape, and eventually, to the chemical modeling of synthetic lethality and synthetic rescue interactions, that are of great clinical and pharmaceutical relevance and significance.

Cytogenetic aberrations at the single-cell level represent an important characteristic of cancer cells relevant to tumor evolution and prognosis. However, with the advent of The Cancer Genome Atlas (TCGA), there has been a major shift in cancer research to the use of data from aggregate cell populations. Given that tumor cells harbor hundreds to thousands of biologically relevant genetic alterations that manifest as intratumor heterogeneity, these aggregate analyses may miss alterations readily observable at single-cell resolution. Using the Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, we developed an algorithm to parse International System for Cytogenetic Nomenclature notation for quantitative abnormalities. Comparison of the Mitelman database and TCGA demonstrated that the Mitelman database is a powerful resource, and that cytogenetic aberrations captured by traditional approaches used in Mitelman database are on par with population-based genomic analyses used in TCGA. This algorithm will help nonspecialists to overcome the challenges associated with the format and syntax of the Mitelman database. Significance: A novel in silico approach compares cytogenetic data between the Mitelman database and TCGA, highlighting the advantages and limitations of both datasets.

Genomic instability, and more specifically chromosomal instability (CIN), arises from a number of processes that are defective in cancer, such as aberrant mitotic cell division, replication stress, defective DNA damage repair, and ineffective telomere maintenance. CIN is an emerging hallmark of cancer that contributes to tumor heterogeneity through increased rates of genetic alterations. As genetic heterogeneity within a single tumor and between tumors is a key challenge leading to treatment failures, this brings to question, whether therapeutic approaches should aim at the genetic diversity or a specific mutation present within these tumors. Answering this question will determine the future of personalized targeted therapies. Here we discuss, how the genetic diversity associated with CIN in tumor cells can be used as a therapeutic advantage and targeted by exploiting the genetic concepts of synthetic lethality and synthetic dosage lethality. Given that a number of CIN-related pathways work together to fix the DNA damage within our genome and ensure proper segregation of chromosomes, we specifically focus on the genetic interactions amongst these pathways and their potential therapeutic applicability in cancer. We also discuss, how tumor genetic heterogeneity can be targeted in emerging immunotherapeutic approaches.

Background Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. Methods We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student’s t-test from at least four independent experiments. Results We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. Conclusions We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.

Gastro-esophageal (GE) cancers are one of the major causes of cancer-related death in the world. There is a need for novel biomarkers in the management of GE cancers, to yield predictive response to the available therapies. Our study aims to identify leading genes that are differentially regulated in patients with these cancers. We explored the expression data for those genes whose protein products can be detected in the plasma using the Cancer Genome Atlas to identify leading genes that are differentially regulated in patients with GE cancers. Our work predicted several candidates as potential biomarkers for distinct stages of GE cancers, including previously identified CST1, INHBA, STMN1, whose expression correlated with cancer recurrence, or resistance to adjuvant therapies or surgery. To define the predictive accuracy of these genes as possible biomarkers, we constructed a co-expression network and performed complex network analysis to measure the importance of the genes in terms of a ratio of closeness centrality (RCC). Furthermore, to measure the significance of these differentially regulated genes, we constructed an SVM classifier using machine learning approach and verified these genes by using receiver operator characteristic (ROC) curve as an evaluation metric. The area under the curve measure was > 0.9 for both the overexpressed and downregulated genes suggesting the potential use and reliability of these candidates as biomarkers. In summary, we identified leading differentially expressed genes in GE cancers that can be detected in the plasma proteome. These genes have potential to become diagnostic and therapeutic biomarkers for early detection of cancer, recurrence following surgery and for development of targeted treatment.

Cancer is one of the leading causes of death and chromosomal instability (CIN) is a hallmark feature of cancer. CIN, a source of genetic variation in either altered chromosome number or structure contributes to tumor heterogeneity and has become a hot topic in recent years prominently for its role in therapeutic responses. Synthetic lethality and synthetic rescue based approaches, for example, advancing CRISPR-Cas9 platform, are emerging as a powerful strategy to identify new potential targets to selectively eradicate cancer cells. Unfortunately, only few of them are further explored therapeutically due to the difficulty in linking these targets to small molecules for pharmacological intervention. This, however, can be alleviated by the efforts to bring chemical, bioactivity, and genomic data together, as well as established computational approaches. In this chapter, we will discuss some of these advances, including established databases and in silico target-ligand prediction, with the aim to navigate through the synthetically available chemical space to the biologically targetable landscape, and eventually, to the chemical modeling of synthetic lethality and synthetic rescue interactions, that are of great clinical and pharmaceutical relevance and significance.

Background: Gastro-esophageal cancers are one of the major causes of cancer-related death in the world. There is a need for novel biomarkers in the management of gastro-esophageal cancers to identify new therapeutic targets and to yield predictive response to the available therapies. Our study aims to identify leading genes that are dysregulated (upregulated or downregulated) in patients with gastro-esophageal cancers.Methods: We examined gene expression data for those genes whose protein products can be detected in the plasma in 600 independent tumor samples and 46 matching normal tissue samples using the Cancer Genome Atlas (TCGA) to identify leading genes that are dysregulated in patients with gastro-esophageal cancers. Non-parametric Mann-Whitney-U test was used to evaluate differential expression of genes using a cut-off of P< 0.05.Results: The comparison between tumors sample and healthy tissue showed BIRC5 (p=2.61 E-08), APOC2 (p=3.23E-08), CENPF (p=4.38E-08), STMN1 (p=5.74E-08), and HNRPC (p=8.21E-08) were the leading genes significantly overexpressed in esophageal cancer whereas CST1 (p=3.97 E-21), INHBA (p=9.22E-20), ACAN (p=1.08E-19), HSP90AB1 (p=2.62E-19), and HSPD1 (p=3.91E-19) were the leading genes that were overexpressed in stomach cancer. Conversely, C16orf89 (9.78E-08), AR (1.01E-07), CKB (1.17E-07), ADH1B (1.79E-07), and NCAM1 (2.15E-07) were the leading gene that were significantly downregulated in esophageal cancer whereas GPX3 (1.65E-19), CLEC3B (5.70E-19), CFD (5.68E-18), GSN (4.5IE-17), and CCL14 (1.12E-16) were significantly downregulated in stomach cancer. Furthermore, Stage-based examination showed stage-specific differential expression of various genes as well as stage-wise increasing or decreasing up-regulation or down-regulation of selected genes, respectively. Conclusions: The present study identified leading upregulated and downregulated genes in gastro-esophageal cancers that can be detected in the plasma proteome. These genes have potential to become diagnostic and therapeutic biomarkers for early detection of cancer, recurrence following surgery and for development of targeted-treatment.

Simple Summary Protein phosphorylation is a key regulatory mechanism that controls a wide variety of cellular responses. This process is catalysed by the members of the protein kinase superfamily that are classified into two main families based on their ability to phosphorylate either tyrosine or serine and threonine residues in their substrates. Massive research efforts have been invested in dissecting the functions of tyrosine kinases, revealing their importance in the initiation and progression of human malignancies. Based on these investigations, numerous tyrosine kinase inhibitors have been included in clinical protocols and proved to be effective in targeted therapies for various haematological malignancies. In this review, we provide insights into the role of tyrosine kinases in leukaemia and discuss their targeting for therapeutic purposes with the currently available inhibitory compounds. Abstract Protein kinases constitute a large group of enzymes catalysing protein phosphorylation and controlling multiple signalling events. The human protein kinase superfamily consists of 518 members and represents a complicated system with intricate internal and external interactions. Protein kinases are classified into two main families based on the ability to phosphorylate either tyrosine or serine and threonine residues. Among the 90 tyrosine kinase genes, 58 are receptor types classified into 20 groups and 32 are of the nonreceptor types distributed into 10 groups. Tyrosine kinases execute their biological functions by controlling a variety of cellular responses, such as cell division, metabolism, migration, cell–cell and cell matrix adhesion, cell survival and apoptosis. Over the last 30 years, a major focus of research has been directed towards cancer-associated tyrosine kinases owing to their critical contributions to the development and aggressiveness of human malignancies through the pathological effects on cell behaviour. Leukaemia represents a heterogeneous group of haematological malignancies, characterised by an uncontrolled proliferation of undifferentiated hematopoietic cells or leukaemia blasts, mostly derived from bone marrow. They are usually classified as chronic or acute, depending on the rates of their progression, as well as myeloid or lymphoblastic, according to the type of blood cells involved. Overall, these malignancies are relatively common amongst both children and adults. In malignant haematopoiesis, multiple tyrosine kinases of both receptor and nonreceptor types, including AXL receptor tyrosine kinase (AXL), Discoidin domain receptor 1 (DDR1), Vascular endothelial growth factor receptor (VEGFR), Fibroblast growth factor receptor (FGFR), Mesenchymal–epithelial transition factor (MET), proto-oncogene c-Src (SRC), Spleen tyrosine kinase (SYK) and pro-oncogenic Abelson tyrosine-protein kinase 1 (ABL1) mutants, are implicated in the pathogenesis and drug resistance of practically all types of leukaemia. The role of ABL1 kinase mutants and their therapeutic inhibitors have been extensively analysed in scientific literature, and therefore, in this review, we provide insights into the impact and mechanism of action of other tyrosine kinases involved in the development and progression of human leukaemia and discuss the currently available and emerging treatment options based on targeting these molecules.

CD8+ cytotoxic T lymphocyte (CTL) exhaustion is one of the major obstacles for the effectiveness of virus control in chronic infectious diseases. We previously generated novel ovalbumin (OVA)-specific 41BBL-expressing OVA-TEXO and human immunodeficiency virus (HIV-1) Gag-specific Gag-TEXO vaccines, inducing therapeutic immunity in wild-type C57BL/6 (B6) mice, and converting CTL exhaustion in recombinant OVA-specific adenovirus AdVOVA-infected B6 (AdVOVA-B6) mice with chronic infection. IL-21 cytokine plays an important role in controlling chronic infections. Therefore, in this study, we constructed recombinant transgene IL-21-expressing AdVIL-21, and generated IL-21-expressing OVA-TEXO/IL-21 and Gag-TEXO/IL21 vaccines, or control vaccines (OVA-TEXO/Null and Gag-TEXO/Null) by infecting OVA-TEXO and Gag-TEXO cells with AdVIL-21 or the control AdVNull, lacking transgene, and assessed their effects in B6 or AdVOVA-B6 mice. We demonstrate that both OVA-TEXO/IL-21 and control OVA-TEXO/Null vaccines are capable of converting CTL exhaustion in chronic infection. However, the OVA-TEXO/IL-21 vaccine more efficiently rescues exhausted CTLs by increasing stronger CTL proliferation and effector cytokine IFN-γ expression than the control OVA-TEXO/Null vaccine in AdVOVA-B6 mice with chronic infection, though both vaccines stimulated comparable OVA-specific CTL responses and protective immunity against OVA-expressing BL6-10OVA melanoma lung metastasis in wild-type B6 mice. In vivo, the OVA-TEXO/IL-21-stimulated CTLs more efficiently up-regulate phosphorylation of mTORC1-controlled EIF4E and expression of mTORC1- regulated T-bet molecule than the control OVA-TEXO/Null-stimulated ones. Importantly, the Gag-TEXO/IL21 vaccine induces stronger Gag-specific therapeutic immunity against established Gag-expressing BL6-10Gag melanoma lung metastases than the control Gag-TEXO/Null vaccine in chronic infection. Therefore, this study should have a strong impact on developing new therapeutic vaccines for patients with chronic infections.

Targeted therapies rely on the genetic and epigenetic status of the tumor cells and are seen as the most promising approach to treat cancer today. However, current targeted therapies focus on directly inhibiting those molecules that are altered in tumor cells. Unfortunately, targeting these molecules, even with specific inhibitors, is challenging as tumor cells rewire their genetic circuitry to eliminate genetic dependency on these targets. Here, we describe how synthetic lethality approaches can be used to identify genetic dependencies and develop personalized targeted therapies. We also discuss strategies to specifically target these genetic dependencies, using small molecule and biologic drugs.

Chromosomal rearrangements involving the mixed-lineage leukemia (MLL1) gene are common in a unique group of acute leukemias, with more than 100 fusion partners in this malignancy alone. However, do these fusions occur or have a role in solid tumors? We performed extensive network analyses of MLL1-fusion partners in patient datasets, revealing that multiple MLL1-fusion partners exhibited significant interactions with the androgen-receptor signaling pathway. Further exploration of tumor sequence data from TCGA predicts the presence of MLL1 fusions with truncated SET domain in prostate tumors. To investigate the physiological relevance of MLL1 fusions in solid tumors, we engineered a truncated version of MLL1 by fusing it with one of its known fusion partners, ZC3H13, to use as a model system. Functional characterization with cell-based assays revealed that MLL1-ZC3H13 fusion induced chromosomal instability, affected mitotic progression, and enhanced tumorsphere formation. The MLL1-ZC3H13 chimera consistently increased the expression of a cancer stem cell marker (CD44); in addition, we detected potential collateral lethality between DOT1L and MLL1 fusions. Our work reveals that MLL1 fusions are likely prevalent in solid tumors and exhibit a potential pro-tumorigenic role.

Triple-negative breast cancer (TNBC) tumours that lack expression of oestrogen, and progesterone receptors, and do not overexpress the HER2 receptor represent the most aggressive breast cancer subtype, which is characterised by the resistance to therapy in frequently relapsing tumours and a high rate of patient mortality. This is likely due to the resistance of slowly proliferating tumour-initiating cells (TICs), and understanding molecular mechanisms that control TICs behaviour is crucial for the development of effective therapeutic approaches. Here, we present our novel findings, indicating that an intrinsically catalytically inactive member of the Eph group of receptor tyrosine kinases, EPHB6, partially suppresses the epithelial–mesenchymal transition in TNBC cells, while also promoting expansion of TICs. Our work reveals that EPHB6 interacts with the GRB2 adapter protein and that its effect on enhancing cell proliferation is mediated by the activation of the RAS-ERK pathway, which allows it to elevate the expression of the TIC-related transcription factor, OCT4. Consistent with this, suppression of either ERK or OCT4 activities blocks EPHB6-induced pro-proliferative responses. In line with its ability to trigger propagation of TICs, EPHB6 accelerates tumour growth, potentiates tumour initiation and increases TIC populations in xenograft models of TNBC. Remarkably, EPHB6 also suppresses tumour drug resistance to DNA-damaging therapy, probably by forcing TICs into a more proliferative, drug-sensitive state. In agreement, patients with higher EPHB6 expression in their tumours have a better chance for recurrence-free survival. These observations describe an entirely new mechanism that governs TNBC and suggest that it may be beneficial to enhance EPHB6 action concurrent with applying a conventional DNA-damaging treatment, as it would decrease drug resistance and improve tumour elimination.

DNA vaccines composed of heterologous human HER2 and rat neu sequences induce stronger antibody response and protective antitumor immunity than either HER2 or neu DNA vaccines in transgenic mice. We previously developed HER2-specific exosome-targeted T-cell vaccine HER2-T EXO capable of stimulating HER2-specific CD8 + T-cell responses, but only leading to partial protective immunity in double-transgenic HLA-A2/HER2 mice with self-immune tolerance to HER2. Here, we constructed an adenoviral vector AdV HuRt expressing HuRt fusion protein composed of NH 2 -HER2 1-407 (Hu) and COOH-neu 408-690 (Rt) fragments, and developed a heterologous human/rat HER2-specific exosome-targeted T-cell vaccine HuRt-T EXO using polyclonal CD4 + T-cells uptaking exosomes released by AdV HuRt -transfected dendritic cells. We found that the HuRt-T EXO vaccine stimulates enhanced CD4 + T-cell responses leading to increased induction of HER2-specific antibody (∼70 µg/ml) compared to that (∼40 µg/ml) triggered by the homologous HER2-T EXO vaccine. By using PE-H-2K d /HER2 23-71 tetramer, we determined that HuRt-T EXO stimulates stronger HER2-specific CD8 + T-cell responses eradicating 90% of HER2-specific target cells, while HER2-T EXO -induced CD8 + T-cell responses only eliminating 53% targets. Furthermore, HuRt-T EXO , but not HER2-T EXO vaccination, is capable of suppressing early stage-established HER2-expressing 4T1 HER2 breast cancer in its lung metastasis or subcutaneous form in BALB/c mice, and of completely protecting transgenic HLA-A2/HER2 mice from growth of HLA-A2/HER2-expressing BL6-10 A2/HER2 melanoma. HuRt-T EXO -stimulated HER2-specific CD8 + T-cells not only are cytolytic to trastuzumab-resistant HLA-A2/HER2-expressing BT474/A2 breast tumor cells in vitro but also eradicates pre-established BT474/A2 tumors in athymic nude mice. Therefore, our novel heterologous human/rat HER2-specific T-cell vaccine HuRt-T EXO, circumventing HER2 tolerance, may provide a new therapeutic alternative for patients with trastuzumab-resistant HER2 + breast tumor.

Simulated microgravity (SMG) was reported to affect tumor cell proliferation and metastasis. However, the underlying mechanism is elusive. In this study, we demonstrate that clinostat-modelled SMG reduces BL6-10 melanoma cell proliferation, adhesion and invasiveness in vitro and decreases tumor lung metastasis in vivo. It down-regulates metastasis-related integrin α6β4, MMP9 and Met72 molecules. SMG significantly reduces formation of focal adhesions and activation of focal adhesion kinase (FAK) and Rho family proteins (RhoA, Rac1 and Cdc42) and of mTORC1 kinase, but activates AMPK and ULK1 kinases. We demonstrate that SMG inhibits NADH induction and glycolysis, but induces mitochondrial biogenesis. Interestingly, administration of a RhoA activator, the cytotoxic necrotizing factor-1 (CNF1) effectively converts SMG-triggered alterations and effects on mitochondria biogenesis or glycolysis. CNF1 also converts the SMG-altered cell proliferation and tumor metastasis. In contrast, mTORC inhibitor, rapamycin, produces opposite responses and mimics SMG-induced effects in cells at normal gravity. Taken together, our observations indicate that SMG inhibits focal adhesions, leading to inhibition of signaling FAK and RhoA, and the mTORC1 pathway, which results in activation of the AMPK pathway and reduced melanoma cell proliferation and metastasis. Overall, our findings shed a new light on effects of microgravity on cell biology and human health.

While impressive improvements have been achieved in T-ALL therapy, current treatment approaches fail in approximately 25% of patients and these patients have limited treatment options. Another significant group of patients is being overtreated, which causes long-lasting side effects. Identification of molecules controlling drug resistance in T-ALL is crucial for treatment optimisation in both scenarios. We report here the EphB6 receptor is frequently overexpressed in T-ALL. Remarkably, our observations indicate that EphB6 acts in T-ALL cells to enhance sensitivity to a DNA-damaging drug, doxorubicin, as interruption of EphB6 activity interferes with the efficiency of doxorubicin-induced eradication of T-ALL cells in cell culture and in xenograft animals. This effect relies on the protection of Akt kinase signaling, while Akt inhibition combined with doxorubicin application produces synergistic effects on the elimination of EphB6-deficient T-ALL cells. These data imply that EphB6 suppresses T-ALL resistance by interfering with Akt activity. Our observations highlight a novel role for EphB6 in reducing drug resistance of T-ALL and suggest that doxorubicin treatment should produce better results if personalised based on EphB6 levels. If successfully verified in clinical studies, this approach should improve outcomes for T-ALL patients resistant to current therapies and for patients, who are being overtreated.

Whether inflation of CD8+ memory T (mT) cells, which is often derived from repeated prime-boost vaccinations or chronic viral infections in the elderly, would affect late CD8+ T-cell immunity is a long-standing paradox. We have previously established an animal model with mT-cell inflation by transferring ConA-stimulated monoclonal CD8+ T cells derived from Ova-specific T-cell-receptor transgenic OTI mice into irradiation-induced lymphopenic B6 mice. In this study, we also established another two animal models with mT-cell inflation by transferring, 1) ConA-stimulated monoclonal CD8+ T cells derived from lymphocytic choriomeningitis virus glycoprotein-specific T-cell-receptor transgenic P14 mice, and 2) ConA-stimulated polyclonal CD8+ T cells derived from B6.1 mice into B6 mice with irradiation-induced lymphopenia. We vaccinated these mice with recombinant Ova-expressing Listeria monocytogenes and Ova-pulsed dendritic cells, which stimulated CD4+ T cell-independent and CD4+ T-cell-dependent CD8+ T-cell responses, respectively, and assessed Ova-specific CD8+ T-cell responses by flow cytometry. We found that Ova-specific CD8+ T-cell responses derived from the latter but not the former vaccination were significantly reduced in mice with CD8+ mT-cell inflation compared to wild-type B6 mice. We determined that naive CD8+ T cells purified from splenocytes of mice with mT-cell inflation had defects in cell proliferation upon stimulation in vitro and in vivo and upregulated T-cell anergy-associated Itch and GRAIL molecules. Taken together, our data reveal that CD8+ mT-cell inflation renders compromised CD4+ T-cell-dependent CD8+ T-cell immunity via naive T-cell anergy, and thus show promise for the design of efficient vaccines for elderly patients with CD8+ mT-cell inflation.

Expansion of PD-1-expressing CD8+ cytotoxic T lymphocytes (CTLs) and associated CTL exhaustion are chief issues for ineffective virus-elimination in chronic infectious diseases. PD-1 blockade using antagonistic anti-PD-L1 antibodies results in a moderate conversion of CTL exhaustion. We previously demonstrated that CD40L signaling of ovalbumin (OVA)-specific vaccine, OVA-Texo, converts CTL exhaustion via the activation of the mTORC1 pathway in OVA-expressing adenovirus (AdVova)-infected B6 mice showing CTL inflation and exhaustion. Here, we developed AdVova-infected B6 and transgenic CD11c-DTR (termed AdVova-B6 and AdVova-CD11c-DTR) mice with chronic infection, and assessed a potential effect of CD40 agonist on the conversion of CTL exhaustion and on a potential enhancement of PD-1 antagonist action in rescuing exhausted CTLs in our chronic infection models. We demonstrate that a single dose of anti-CD40 alone can effectively convert CTL exhaustion by activating the mTORC1 pathway, leading to CTL proliferation, up-regulation of an effector-cytokine IFN-γ and the cytolytic effect in AdVova-B6 mice. Using anti-CD4 antibody and diphtheria toxin (DT) to deplete CD4+ T-cells and dendritic cells (DCs), we discovered that the CD40 agonist-induced conversion in AdVova-B6 and AdVova-CD11c-DTR mice is dependent upon host CD4+ T-cell and DC involvements. Moreover, CD40 agonist significantly enhances PD-1 antagonist effectiveness in rescuing exhausted CTLs in chronic infection. Taken together, our data demonstrate the importance of CD40 signaling in the conversion of CTL exhaustion and its ability to enhance PD-1 antagonist action in rescuing exhausted CTLs in chronic infection. Therefore, our findings may positively impact the design of new therapeutic strategies for chronic infectious diseases.

Compared with CD4+25+ regulatory T cells (Tregs), the mechanisms for natural, polyclonal CD8+25+ Treg immune suppression have been significantly less studied. We previously showed that polyclonal T cells can acquire antigen-specific targeting activity through arming with exosomal peptide-MHC (pMHC). In this study, we assessed the suppressive effect of CD8+25+ Tregs or CD8+25+ Tregs armed with ovalbumin (OVA)-specific exosomes on other immune cells and OVA-specific dendritic cell (DCOVA)-stimulated antitumor immunity. We demonstrate that CD8+25+ Tregs inhibit T cell proliferation in vitro in a cell contact-dependent fashion but independent of the expression of immunosuppressive IL-10, TGF-β, and CTLA-4. CD8+25+ Tregs anergize naïve T cells upon stimulation by up-regulating T cell anergy-associated Egr2 and down-regulating IL-2 production. Tregs also anergize DCs by preventing DC maturation through the down-regulation of Iab, CD80, CD86, and inflammatory cytokines, leading to defects in T cell stimulation. Moreover, CD8+25+ Tregs inhibit CTLs through inducing CTL death via perforin-mediated apoptosis and through reducing effector CTL cytotoxic activity via down-regulating CTL perforin-production and degranulation. In addition, we show that CD8+25+ Tregs suppress DCOVA-stimulated CTL responses in priming and effector phases and inhibit immunity against OVA-expressing CCLOVA lung cancer. Remarkably, polyclonal CD8+25+ Tregs armed with OVA-specific exosomal pMHC class-II (pMHC-II), or pMHC class-I (pMHC-I) complexes exert their enhanced inhibition of CTL responses in the priming and the effector phases, respectively. Taken together, our investigation reveals that assigning antigen specificity to nonspecific polyclonal CD8+25+ Tregs for enhanced immune suppression can be achieved through exosomal pMHC arming. This principle may have a great effect on Treg-mediated immunotherapy of autoimmune diseases.