Soluble CTLA-4 attenuates T cell activation and modulates anti-tumor immunity.

CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumor control. Antibodies targeting CTLA-4 have been promising treatments for numerous cancers, but the mechanistic basis of their anti-tumoral immune-boosting effects is poorly understood. Although the ctla4 gene also encodes an alternatively spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in a murine cancer model. We show that expression of sCTLA-4 by tumor cells suppresses CD8+ T cells in vitro and accelerates growth and experimental metastasis of murine tumors in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8+ T cells in a non-cytotoxic state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoral CD8+ T cell activation and cytolytic potential, correlating with therapeutic efficacy and tumor control. This previously unappreciated role of sCTLA-4 suggests that the biology and function of multi-gene products of immune checkpoint receptors need to be fully elucidated for improved mechanistic understanding of cancer immunotherapies.

Differential Regulation of the STING Pathway in Human Papillomavirus-Positive and -Negative Head and Neck Cancers.

Squamous cell carcinomas, which arise from the cells that line the mucosal surfaces of the head and neck, represent the most common type of head and neck cancers (HNSCC). Human papillomavirus (HPV) infection has been strongly associated with the development of oropharyngeal cancers, which are cancers that occur in the back of the throat, including the tonsils and base of the tongue. HNSCCs with and without HPV infection have distinct pathology, with HPV-positive patients having higher levels of immune infiltration, activation in the tumor microenvironment and better response to radiation and chemotherapy. It is, however, unclear whether HPV infection in HNSCCs has the potential to activate innate-immune sensing pathways and if these cancers possess intrinsic immunogenicity associated with HPV infection. Here we investigate the innate immune stimulator of interferon genes (STING) pathway and immune responses to STING activation in HNSCCs and uncover fundamental differences in the regulation of this pathway in cell lines versus primary human clinical specimens. We show that while STING is differentially expressed in HPV-positive and -negative HNSCC cell lines, they exhibit a gross functional defect in signaling through this pathway. However, STING activation in immune cell populations generated immune signatures predicted to elicit useful tumoricidal mechanisms. In contrast, IHC analysis of human tissue microarrays revealed enhanced STING expression in HPV-related tumors and high intratumoral expression of STING correlated with increased survival. SIGNIFICANCE: STING is an important innate immune sensor of cytosolic DNA, inducing essential antiviral and antitumoral responses. This research shows that STING expression is enhanced in HPV-positive HNSCC patient tissue, with high intratumoral STING expression correlating with increased survival. In addition, STING activation in immune cell populations augmented antitumoral effects against HNSCCs, suggesting patients may benefit from the use of STING agonists in combination with traditional therapies.

Neuroblastoma: an ongoing cold front for cancer immunotherapy.

Neuroblastoma is the most frequent extracranial childhood tumour but effective treatment with current immunotherapies is challenging due to its immunosuppressive microenvironment. Efforts to date have focused on using immunotherapy to increase tumour immunogenicity and enhance anticancer immune responses, including anti-GD2 antibodies; immune checkpoint inhibitors; drugs which enhance macrophage and natural killer T (NKT) cell function; modulation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway; and engineering neuroblastoma-targeting chimeric-antigen receptor-T cells. Some of these strategies have strong preclinical foundation and are being tested clinically, although none have demonstrated notable success in treating paediatric neuroblastoma to date. Recently, approaches to overcome heterogeneity of neuroblastoma tumours and treatment resistance are being explored. These include rational combination strategies with the aim of achieving synergy, such as dual targeting of GD2 and tumour-associated macrophages or natural killer cells; GD2 and the B7-H3 immune checkpoint; GD2 and enhancer of zeste-2 methyltransferase inhibitors. Such combination strategies provide opportunities to overcome primary resistance to and maximize the benefits of immunotherapy in neuroblastoma.

Worked to the bone: antibody-based conditioning as the future of transplant biology.

Conditioning of the bone marrow prior to haematopoietic stem cell transplant is essential in eradicating the primary cause of disease, facilitating donor cell engraftment and avoiding transplant rejection via immunosuppression. Standard conditioning regimens, typically comprising chemotherapy and/or radiotherapy, have proven successful in bone marrow clearance but are also associated with severe toxicities and high incidence of treatment-related mortality. Antibody-based conditioning is a developing field which, thus far, has largely shown an improved toxicity profile in experimental models and improved transplant outcomes, compared to traditional conditioning. Most antibody-based conditioning therapies involve monoclonal/naked antibodies, such as alemtuzumab for graft-versus-host disease prophylaxis and rituximab for Epstein-Barr virus prophylaxis, which are both in Phase II trials for inclusion in conditioning regimens. Nevertheless, alternative immune-based therapies, including antibody-drug conjugates, radio-labelled antibodies and CAR-T cells, are showing promise in a conditioning setting. Here, we analyse the current status of antibody-based drugs in pre-transplant conditioning regimens and assess their potential in the future of transplant biology.

Recent Progress in Interferon Therapy for Myeloid Malignancies.

Myeloid malignancies are a heterogeneous group of clonal haematopoietic disorders, caused by abnormalities in haematopoietic stem cells (HSCs) and myeloid progenitor cells that originate in the bone marrow niche. Each of these disorders are unique and present their own challenges with regards to treatment. Acute myeloid leukaemia (AML) is considered the most aggressive myeloid malignancy, only potentially curable with intensive cytotoxic chemotherapy with or without allogeneic haematopoietic stem cell transplantation. In comparison, patients diagnosed with chronic myeloid leukaemia (CML) and treated with tyrosine kinase inhibitors (TKIs) have a high rate of long-term survival. However, drug resistance and relapse are major issues in both these diseases. A growing body of evidence suggests that Interferons (IFNs) may be a useful therapy for myeloid malignancies, particularly in circumstances where patients are resistant to existing front-line therapies and have risk of relapse following haematopoietic stem cell transplant. IFNs are a major class of cytokines which are known to play an integral role in the non-specific immune response. IFN therapy has potential as a combination therapy in AML patients to reduce the impact of minimal residual disease on relapse. Alongside this, IFNs can potentially sensitize leukaemic cells to TKIs in resistant CML patients. There is evidence also that IFNs have a therapeutic role in myeloproliferative neoplasms (MPNs) such as polycythaemia vera (PV) and primary myelofibrosis (PMF), where they can restore polyclonality in patients. Novel formulations have improved the clinical effectiveness of IFNs. Low dose pegylated IFN formulations improve pharmacokinetics and improve patient tolerance to therapies, thereby minimizing the risk of haematological toxicities. Herein, we will discuss recent developments and the current understanding of the molecular and clinical implications of Type I IFNs for the treatment of myeloid malignancies.

STINGing Viral Tumors: What We Know from Head and Neck Cancers.

It has now become increasingly clear that viruses, which may not be directly oncogenic, can affect the biology of tumors as well as immune behavior against tumors. This has led to a fundamental question: Should tumors associated with viral infection be considered distinct from those without? Typically, viruses activate the host innate immune responses by stimulating pathogen recognition receptors and DNA-sensing pathways, including the stimulator of interferon genes (STING) pathway. However, regulation of the STING pathway in a virus-associated tumor microenvironment is poorly understood. Human papillomavirus (HPV) infection within a subset of head and neck squamous cell carcinomas (HNSCC) promotes a unique etiology and clinical outcome. For reasons currently not well understood, patients with HPV+ tumors have a better outcome in terms of both overall survival and reduced risk of recurrence compared with HPV- HNSCC. This observation may reflect a greater intrinsic immunogenicity associated with HPV infection, pertaining to innate immune system pathways activated following recognition of viral nucleotides. Here we discuss how HNSCC provides a unique model to study the STING pathway in the context of viral-induced tumor type as well as recent advances in our understanding of this pathway in HSNCC.

Old Dog, New Trick: Type I IFN-Based Treatment for Acute Myeloid Leukemia.

Despite strong biological rationale for the use of type-I IFNs for the treatment of acute myeloid leukemia (AML), their usage is limited to few hematologic malignancies. Here, we propose that innate immune sensing machinery, particularly the stimulator of IFN genes pathway, may be exploited to deliver antileukemic effects in AML.

DNA Damage/Repair Management in Cancers.

DNA damage is well recognized as a critical factor in cancer development and progression. DNA lesions create an abnormal nucleotide or nucleotide fragment, causing a break in one or both chains of the DNA strand. When DNA damage occurs, the possibility of generated mutations increases. Genomic instability is one of the most important factors that lead to cancer development. DNA repair pathways perform the essential role of correcting the DNA lesions that occur from DNA damaging agents or carcinogens, thus maintaining genomic stability. Inefficient DNA repair is a critical driving force behind cancer establishment, progression and evolution. A thorough understanding of DNA repair mechanisms in cancer will allow for better therapeutic intervention. In this review we will discuss the relationship between DNA damage/repair mechanisms and cancer, and how we can target these pathways.

SubID, a non-median dichotomization tool for heterogeneous populations, reveals the pan-cancer significance of INPP4B and its regulation by EVI1 in AML.

Our previous studies demonstrated that INPP4B, a member of the PI3K/Akt signaling pathway, is overexpressed in a subset of AML patients and is associated with lower response to chemotherapy and shorter survival. INPP4B expression analysis in AML revealed a right skewed frequency distribution with 25% of patients expressing significantly higher levels than the majority. The 75% low/25% high cut-off revealed the prognostic power of INPP4B expression status in AML, which would not have been apparent with a standard median cut-off approach. Our identification of a clinically relevant non-median cut-off for INPP4B indicated a need for a generalizable non-median dichotomization approach to optimally study clinically relevant genes. To address this need, we developed Subgroup Identifier (SubID), a tool which examines the relationship between a continuous variable (e.g. gene expression), and a test parameter (e.g. CoxPH or Fisher's exact P values). In our study, Fisher's exact SubID was used to reveal EVI1 as a transcriptional regulator of INPP4B in AML; a finding which was validated in vitro. Next, we used CoxPH SubID to conduct a pan-cancer analysis of INPP4B's prognostic significance. Our analysis revealed that INPP4Blow is associated with shorter survival in kidney clear cell, liver hepatocellular, and bladder urothelial carcinomas. Conversely, INPP4Blow was shown to be associated with increased survival in pancreatic adenocarcinoma in three independent datasets. Overall, our study describes the development and application of a novel subgroup identification tool used to identify prognostically significant rare subgroups based upon gene expression, and for investigating the association between a gene with skewed frequency distribution and potentially important upstream and downstream genes that relate to the index gene.

Measurement of PTEN by Flow Cytometry.

Recent advancements have driven the development of smaller footprint, less expensive, and user-friendly flow cytometers introducing the technology to more users.Flow cytometry is an established tool for multiparametric analysis of various important cellular characteristics. Fluorescent dyes or fluorophore-conjugated antibodies allow for measurement of protein expression, identification of cell populations, or DNA content analysis. This is combined with analysis of light-scattering detection to determine cell size and complexity to allow for the study of complex cell samples, such as whole blood. Through antibody staining for a variety of surface markers as well as intracellular proteins we can also elucidate intracellular signaling, and phosphor-signaling, on a single-cell basis.Here we describe the application of flow cytometry analysis to the tumor suppressor PTEN in various cancer cell lines and a mouse model.

Phosphoinositide signaling in cancer: INPP4B Akt(s) out.

INPP4B acts as a tumor suppressor in various epithelial cancers by inhibiting PI3K/Akt signaling. Unexpectedly, tumor-promoting features of INPP4B in leukemia and breast cancer have been recently uncovered. In this spotlight, we discuss the seemingly paradoxical nature of INPP4B-mediated signaling in cancer.

NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells.

Internal tandem duplication of the FMS-like tyrosine kinase (FLT3-ITD) receptor is present in 20% of acute myeloid leukemia (AML) patients and it has been associated with an aggressive AML phenotype. FLT3-ITD expressing cell lines have been shown to generate increased levels of reactive oxygen species (ROS) and DNA double strand breaks (DSBs). However, the molecular basis of how FLT3-ITD-driven ROS leads to the aggressive form of AML is not clearly understood. Our group has previously reported that inhibition of FLT3-ITD signaling results in post-translational down-regulation of p22(phox), a small membrane-bound subunit of the NADPH oxidase (NOX) complex. Here we demonstrated that 32D cells, a myeloblast-like cell line transfected with FLT3-ITD, have a higher protein level of p22(phox) and p22(phox)-interacting NOX isoforms than 32D cells transfected with the wild type FLT3 receptor (FLT3-WT). The inhibition of NOX proteins, p22(phox), and NOX protein knockdowns caused a reduction in ROS, as measured with a hydrogen peroxide (H2O2)-specific dye, peroxy orange 1 (PO1), and nuclear H2O2, as measured with nuclear peroxy emerald 1 (NucPE1). These reductions in the level of H2O2 following the NOX knockdowns were accompanied by a decrease in the number of DNA DSBs. We showed that 32D cells that express FLT3-ITD have a higher level of both oxidized DNA and DNA DSBs than their wild type counterparts. We also observed that NOX4 and p22(phox) localize to the nuclear membrane in MV4-11 cells expressing FLT3-ITD. Taken together these data indicate that NOX and p22(phox) mediate the ROS production from FLT3-ITD that signal to the nucleus causing genomic instability.

Imatinib and Nilotinib inhibit Bcr-Abl-induced ROS through targeted degradation of the NADPH oxidase subunit p22phox.

Constitutive expression of the Bcr-Abl kinase in Chronic Myelogenous Leukaemia (CML) is known to produce elevated levels of Reactive Oxygen Species (ROS) which can enhance cell survival as well as generate genomic instability. Our laboratory has previously demonstrated that NADPH oxidase (Nox) activity contributes to intracellular-ROS levels in Bcr-Abl-positive cells, while inducing increased pro-survival signalling through the PI3K/Akt pathway. How Bcr-Abl signalling regulates Nox activity still remains to be elucidated. In this study, using the K562 CML cell line we showed that inhibition of Bcr-Abl signalling, by either Imatinib or Nilotinib, led to a significant reduction in ROS levels which was concurrent with the GSK-3ß dependent, post-translational down-regulation of the small membrane-bound protein p22phox, an essential component of the Nox complex. Furthermore, siRNA knockdown of p22phox in these cells established its importance in ROS production and proliferation. Taken together we believe our results provide a possible link between Bcr-Abl signalling and ROS production through Nox activity and demonstrate a novel mechanism of action associated with Imatinib and Nilotinib treatment in CML.

Redox-regulated growth factor survival signaling.

SIGNIFICANCE: Once the thought of as unwanted byproducts of cellular respiration in eukaryotes, reactive oxygen species (ROS) have been shown to facilitate essential physiological roles. It is now understood that ROS are critical mediators of intracellular signaling. Control of signal transduction downstream of growth factor receptors by ROS is a complex process whose details are only recently coming to light. RECENT ADVANCES: Indeed, recent evidence points to control of signal propagation by ROS at multiple levels in the typical cascade. Growth factor stimulation activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs) at the membrane, producing superoxide in the extracellular matrix, which is catalyzed to the membrane-permeable hydrogen peroxide (H2O2) that mediates intracellular signaling events. CRITICAL ISSUES: The potential for H2O2, however, to disrupt cellular functions by damaging proteins and nucleic acids demands that its levels are kept in check by receptor-associated peroxiredoxins. This interplay of Nox and peroxiredoxin activity moderates levels of H2O2 sufficiently to modify signaling partners locally. Among the best studied of these partners are redox-controlled phosphatases that are inactivated by H2O2. Phosphatases regulate signal propagation downstream of receptors, and thus their inactivation allows a further level of control. Transmission of information further downstream to targets such as transcription factors, themselves regulated by ROS, completes this pathway. FUTURE DIRECTIONS: Thus, signal propagation or attenuation can be dictated by ROS at multiple points. Given the complex nature of these processes, we envisage the emerging trends in the field of redox signaling in the context of growth factor stimulation.

TLR-4 signalling accelerates colon cancer cell adhesion via NF-κB mediated transcriptional up-regulation of Nox-1.

Surgery induced inflammation is a potent promoter of tumour recurrence and metastasis in colorectal cancer. The recently discovered family of Nox enzymes represent a major source of endogenous reactive oxygen species (ROS) and are now heavily implicated in tumour cell metastasis. Interestingly, Nox enzymes can be 'purposefully' activated by inflammatory cytokines and growth factors which are present in abundance in the peri-operative window. As colon cancer cells express Nox enzymes and Toll-like receptor 4 (TLR-4), we hypothesised that LPS may potentiate the ability of colon cancer cells to metastasise via Nox enzyme mediated redox signalling. In support of this hypothesis, this paper demonstrates that LPS induces a significant, transient increase of endogenous ROS in SW480, SW620 and CT-26 colon cancer cells. This increase in LPS-induced ROS activity is completely abrogated by a Nox inhibitor, diphenyleneiodonium (DPI), Nox1 siRNA and an NF-κB inhibitor, Dihydrochloride. A significant increase in Nox1 and Nox2 protein expression occurs following LPS treatment. Inhibition of NF-κB also attenuates the increase of Nox1 and Nox2 protein expression. The sub-cellular location of LPS-induced ROS generation lies mainly in the endoplasmic reticulum. LPS activates the PI3K/Akt pathway via Nox generated ROS and this signal is inhibited by DPI. This LPS activated Nox mechanism facilitates a significant increase in SW480 colon cancer cell adhesion to collagen I, which is inhibited by DPI, Nox1 siRNA and a PI3K inhibitor. Altogether, these data suggest that the LPS-Nox1 redox signalling axis plays a crucial role in facilitation of colon cancer cell adhesion, thus increasing the metastatic potential of colon cancer cells. Nox1 may represent a valuable target in which to prevent colon cancer metastasis.

H2O2 production downstream of FLT3 is mediated by p22phox in the endoplasmic reticulum and is required for STAT5 signalling.

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H(2)O(2)-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H(2)O(2) after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H(2)O(2) in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-ß inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H(2)O(2) production in AML cells is mediated by p22phox and is critical for STAT5 signalling.

The isolation and identification of a secreted biomarker associated with cell stress in serum-free CHO cell culture.

One approach to improving mammalian culture productivity has been to reduce cell stress and cell death in the bioreactor, thus enhancing productivity through a longer phase of viability. Here we describe the isolation and identification of a biomarker for stress and viability loss in CHO culture. Using SELDI-TOF mass spectrometry to profile the protein component of supernatant culture media we have identified a peak at 7.7 kDa that was associated with loss of viability toward the end of the culture and simulated stress from both toxic metabolite accumulation and nutrient depletion. The relative intensity (signal/noise ratio) of the peak increased rapidly at the onset of dropping viability toward the end of the growth phase. Also, the peak height was seen to increase significantly when cells were grown under conditions emulating ammonia accumulation and glutamine deprivation. The species has been identified as a fragment of Galectin-1 (Gal-1) via MS/MS fingerprinting. We propose that this peak could be utilized as a marker for early onset of stress in cell culture. This work demonstrates the efficacy of SELDI technology to identify biomarkers in mammalian cell culture and highlights its value as a tool for the monitoring and improvement of culture processes.

The application of SELDI-TOF mass spectrometry to mammalian cell culture.

Surface Enhanced Laser Desorption/Ionisation Time-of-Fight Mass Spectrometry (SELDI-TOF MS) is a technique by which protein profiles can be rapidly produced from a wide variety of biological samples. By employing chromatographic surfaces combined with the specificity and reproducibility of mass spectrometry it has allowed for profiles from complex biological samples to be analysed. Profiling and biomarker identification have been employed widely throughout the biological sciences. To date, however, the benefits of SELDI-TOF MS have not been realised in the area of mammalian cell culture. The advantages in identifying markers for cell stresses, apoptosis and other culture parameters mean that these tools could help greatly to enhance monitoring and control of bioreaction process and improve the production of therapeutics. Better characterisation of culture systems through proteome analysis will allow for improved productivity and better yields.