The potential and controversy of targeting STAT family members in cancer.

The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors that play a complex and essential role in the regulation of physiologic cell processes, such as proliferation, differentiation, apoptosis and angiogenesis, and serves to organize the epigenetic landscape of immune cells. To date, seven STAT genes have been identified in the human genome; STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6. They all account for diverse effects in response to extracellular signaling proteins, mainly by altering gene transcription in the effector cells. Members of the STAT family have been implicated in human cancer development, progression, metastasis, survival and resistance to treatment. Particularly STAT3 and STAT5 are of interest in cancer biology. They are currently considered as oncogenes, but their signaling is embedded into a complex and delicate balance between different (counteracting) transcription factors, and thus, in some contexts they can have a tumor suppressive role. Assessing STAT signaling mutations as well as screening for aberrant STAT pathway activation may have a role to predict sensitivity to immunotherapy and targeted STAT inhibition. In the present comprehensive review of the literature, we discuss in-depth the role of each STAT family member in cancer, assemble cutting-edge information on the use of these molecules as potential biomarkers and targets for treatment, and address why their clinical implementation is controversy.

Targeting the PD-1 Axis with Pembrolizumab for Recurrent or Metastatic Cancer of the Uterine Cervix: A Brief Update.

Even though cervical cancer is partly preventable, it still poses a great public health problem throughout the world. Current therapies have vastly improved the clinical outcomes of cervical cancer patients, but progress in new systemic treatment modalities has been slow in the last years. Especially for patients with advanced disease this is discouraging, as their prognosis remains very poor. The pathogen-induced nature, the considerable mutational load, the involvement of genes regulating the immune response, and the high grade of immune infiltration, suggest that immunotherapy might be a promising strategy to treat cervical cancer. In this literature review, we focus on the use of PD-1 blocking therapy in cervical cancer, pembrolizumab in particular, as it is the only approved immunotherapy for this disease. We discuss why it has great clinical potential, how it opens doors for personalized treatment in cervical cancer, and which trials are aiming to expand its clinical use.

Dendritic cell vaccination as postremission treatment to prevent or delay relapse in acute myeloid leukemia.

Relapse is a major problem in acute myeloid leukemia (AML) and adversely affects survival. In this phase 2 study, we investigated the effect of vaccination with dendritic cells (DCs) electroporated with Wilms' tumor 1 (WT1) messenger RNA (mRNA) as postremission treatment in 30 patients with AML at very high risk of relapse. There was a demonstrable antileukemic response in 13 patients. Nine patients achieved molecular remission as demonstrated by normalization of WT1 transcript levels, 5 of which were sustained after a median follow-up of 109.4 months. Disease stabilization was achieved in 4 other patients. Five-year overall survival (OS) was higher in responders than in nonresponders (53.8% vs 25.0%; P = .01). In patients receiving DCs in first complete remission (CR1), there was a vaccine-induced relapse reduction rate of 25%, and 5-year relapse-free survival was higher in responders than in nonresponders (50% vs 7.7%; P < .0001). In patients age ≤65 and >65 years who received DCs in CR1, 5-year OS was 69.2% and 30.8% respectively, as compared with 51.7% and 18% in the Swedish Acute Leukemia Registry. Long-term clinical response was correlated with increased circulating frequencies of polyepitope WT1-specific CD8+ T cells. Long-term OS was correlated with interferon-γ+ and tumor necrosis factor-α+ WT1-specific responses in delayed-type hypersensitivity-infiltrating CD8+ T lymphocytes. In conclusion, vaccination of patients with AML with WT1 mRNA-electroporated DCs can be an effective strategy to prevent or delay relapse after standard chemotherapy, translating into improved OS rates, which are correlated with the induction of WT1-specific CD8+ T-cell response. This trial was registered at www.clinicaltrials.gov as #NCT00965224.

Dendritic Cells as Pharmacological Tools for Cancer Immunotherapy.

Although the earliest­rudimentary­attempts at exploiting the immune system for cancer therapy can be traced back to the late 18th Century, it was not until the past decade that cancer immunotherapeutics have truly entered mainstream clinical practice. Given their potential to stimulate both adaptive and innate antitumor immune responses, dendritic cells (DCs) have come under intense scrutiny in recent years as pharmacological tools for cancer immunotherapy. Conceptually, the clinical effectiveness of this form of active immunotherapy relies on the completion of three critical steps: 1) the DCs used as immunotherapeutic vehicles must properly activate the antitumor immune effector cells of the host, 2) these immune effector cells must be receptive to stimulation by the DCs and be competent to mediate their antitumor effects, which 3) requires overcoming the various immune-inhibitory mechanisms used by the tumor cells. In this review, following a brief overview of the pivotal milestones in the history of cancer immunotherapy, we will introduce the reader to the basic immunobiological and pharmacological principles of active cancer immunotherapy using DCs. We will then discuss how current research is trying to define the optimal parameters for each of the above steps to realize the full clinical potential of DC therapeutics. Given its high suitability for immune interventions, acute myeloid leukemia was chosen here to showcase the latest research trends driving the field of DC-based cancer immunotherapy.

Tumoricidal activity of human dendritic cells.

Dendritic cells (DCs) are a family of professional antigen-presenting cells (APCs) that are able to initiate innate and adaptive immune responses against pathogens and tumor cells. The DC family is heterogeneous and is classically divided into two main subsets, each with its unique phenotypic and functional characteristics: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). Recent results have provided intriguing evidence that both DC subsets can also function as direct cytotoxic effector cells; in particular, against cancer cells. In this review, we delve into this understudied function of human DCs and discuss why these so-called killer DCs might become important tools in future cancer immunotherapies.

Engineering monocyte-derived dendritic cells to secrete interferon-α enhances their ability to promote adaptive and innate anti-tumor immune effector functions.

Dendritic cell (DC) vaccination has demonstrated potential in clinical trials as a new effective cancer treatment, but objective and durable clinical responses are confined to a minority of patients. Interferon (IFN)-α, a type-I IFN, can bolster anti-tumor immunity by restoring or increasing the function of DCs, T cells and natural killer (NK) cells. Moreover, type-I IFN signaling on DCs was found to be essential in mice for tumor rejection by the innate and adaptive immune system. Targeted delivery of IFN-α by DCs to immune cells could boost the generation of anti-tumor immunity, while avoiding the side effects frequently associated with systemic administration. Naturally circulating plasmacytoid DCs, major producers of type-I IFN, were already shown capable of inducing tumor antigen-specific T cell responses in cancer patients without severe toxicity, but their limited number complicates their use in cancer vaccination. In the present work, we hypothesized that engineering easily generated human monocyte-derived mature DCs to secrete IFN-α using mRNA electroporation enhances their ability to promote adaptive and innate anti-tumor immunity. Our results show that IFN-α mRNA electroporation of DCs significantly increases the stimulation of tumor antigen-specific cytotoxic T cell as well as anti-tumor NK cell effector functions in vitro through high levels of IFN-α secretion. Altogether, our findings mark IFN-α mRNA-electroporated DCs as potent inducers of both adaptive and innate anti-tumor immunity and pave the way for clinical trial evaluation in cancer patients.

Clinical use of dendritic cells for cancer therapy.

Since the mid-1990s, dendritic cells have been used in clinical trials as cellular mediators for therapeutic vaccination of patients with cancer. Dendritic cell-based immunotherapy is safe and can induce antitumour immunity, even in patients with advanced disease. However, clinical responses have been disappointing, with classic objective tumour response rates rarely exceeding 15%. Paradoxically, findings from emerging research indicate that dendritic cell-based vaccination might improve survival, advocating implementation of alternative endpoints to assess the true clinical potency of dendritic cell-based vaccination. We review the clinical effectiveness of dendritic cell-based vaccine therapy in melanoma, prostate cancer, malignant glioma, and renal cell carcinoma, and summarise the most important lessons from almost two decades of clinical studies of dendritic cell-based immunotherapy in these malignant disorders. We also address how the specialty is evolving, and which new therapeutic concepts are being translated into clinical trials to leverage the clinical effectiveness of dendritic cell-based cancer immunotherapy. Specifically, we discuss two main trends: the implementation of the next-generation dendritic cell vaccines that have improved immunogenicity, and the emerging paradigm of combination of dendritic cell vaccination with other cancer therapies.

HPV vaccine stimulates cytotoxic activity of killer dendritic cells and natural killer cells against HPV-positive tumour cells.

Cervarix™ is approved as a preventive vaccine against infection with the human papillomavirus (HPV) strains 16 and 18, which are causally related to the development of cervical cancer. We are the first to investigate in vitro the effects of this HPV vaccine on interleukin (IL)-15 dendritic cells (DC) as proxy of a naturally occurring subset of blood DC, and natural killer (NK) cells, two innate immune cell types that play an important role in antitumour immunity. Our results show that exposure of IL-15 DC to the HPV vaccine results in increased expression of phenotypic maturation markers, pro-inflammatory cytokine production and cytotoxic activity against HPV-positive tumour cells. These effects are mediated by the vaccine adjuvant, partly through Toll-like receptor 4 activation. Next, we demonstrate that vaccine-exposed IL-15 DC in turn induce phenotypic activation of NK cells, resulting in a synergistic cytotoxic action against HPV-infected tumour cells. Our study thus identifies a novel mode of action of the HPV vaccine in boosting innate immunity, including killing of HPV-infected cells by DC and NK cells.

Human plasmacytoid dendritic cells are equipped with antigen-presenting and tumoricidal capacities.

Human plasmacytoid dendritic cells (pDCs) represent a highly specialized naturally occurring dendritic-cell subset and are the main producers of type I interferons (IFNs) in response to viral infections. We show that human pDCs activated by the preventive vaccine FSME specifically up-regulate CD56 on their surface, a marker that was thought to be specific for NK cells and associated with cytolytic effector functions. We observed that FSME-activated pDCs specifically lysed NK target cells and expressed cytotoxic molecules, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and granzyme B. Elevated levels of these molecules coincided with the expression of CD56, indicative for skewing human pDCs toward an interferon-producing killer DC subset. Detailed phenotypical and functional analysis revealed that pDCs attained a mature phenotype, secreted proinflammatory cytokines, and had the capacity to present antigens and stimulate T cells. Here, we report on the generation of CD56(+) human interferon producing killer pDCs with the capacity to present antigens. These findings aid in deciphering the role for pDCs in antitumor immunity and present a promising prospect of developing antitumor therapy using pDCs.

Induction of complete and molecular remissions in acute myeloid leukemia by Wilms' tumor 1 antigen-targeted dendritic cell vaccination.

Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far, most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads. Here, in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms' tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two patients in partial remission after chemotherapy were brought into complete remission after intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these two patients and three other patients who were in complete remission, the AML-associated tumor marker returned to normal after dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8+ T cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer cells postvaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-gamma-producing CD8+ T cells and features of general immune activation. These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a postremission treatment to prevent full relapse in AML patients.

Auranofin Synergizes with the PARP Inhibitor Olaparib to Induce ROS-Mediated Cell Death in Mutant p53 Cancers.

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as 'aurola'). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF's anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

Humoral and Cellular Immune Responses against SARS-CoV-2 after Third Dose BNT162b2 following Double-Dose Vaccination with BNT162b2 versus ChAdOx1 in Patients with Cancer.

PURPOSE: Patients with cancer display reduced humoral responses after double-dose COVID-19 vaccination, whereas their cellular response is more comparable with that in healthy individuals. Recent studies demonstrated that a third vaccination dose boosts these immune responses, both in healthy people and patients with cancer. Because of the availability of many different COVID-19 vaccines, many people have been boosted with a different vaccine from the one used for double-dose vaccination. Data on such alternative vaccination schedules are scarce. This prospective study compares a third dose of BNT162b2 after double-dose BNT162b2 (homologous) versus ChAdOx1 (heterologous) vaccination in patients with cancer. EXPERIMENTAL DESIGN: A total of 442 subjects (315 patients and 127 healthy) received a third dose of BNT162b2 (230 homologous vs. 212 heterologous). Vaccine-induced adverse events (AE) were captured up to 7 days after vaccination. Humoral immunity was assessed by SARS-CoV-2 anti-S1 IgG antibody levels and SARS-CoV-2 50% neutralization titers (NT50) against Wuhan and BA.1 Omicron strains. Cellular immunity was examined by analyzing CD4+ and CD8+ T-cell responses against SARS-CoV-2-specific S1 and S2 peptides. RESULTS: Local AEs were more common after heterologous boosting. SARS-CoV-2 anti-S1 IgG antibody levels did not differ significantly between homologous and heterologous boosted subjects [GMT 1,755.90 BAU/mL (95% CI, 1,276.95-2,414.48) vs. 1,495.82 BAU/mL (95% CI, 1,131.48-1,977.46)]. However, homologous-boosted subjects show significantly higher NT50 values against BA.1 Omicron. Subjects receiving heterologous boosting demonstrated increased spike-specific CD8+ T cells, including higher IFNγ and TNFα levels. CONCLUSIONS: In patients with cancer who received double-dose ChAdOx1, a third heterologous dose of BNT162b2 was able to close the gap in antibody response.

NK cells: key to success of DC-based cancer vaccines?

The cytotoxic and regulatory antitumor functions of natural killer (NK) cells have become attractive targets for immunotherapy. Manipulation of specific NK cell functions and their reciprocal interactions with dendritic cells (DCs) might hold therapeutic promise. In this review, we focus on the engagement of NK cells in DC-based cancer vaccination strategies, providing a comprehensive overview of current in vivo experimental and clinical DC vaccination studies encompassing the monitoring of NK cells. From these studies, it is clear that NK cells play a key regulatory role in the generation of DC-induced antitumor immunity, favoring the concept that targeting both innate and adaptive immune mechanisms may synergistically promote clinical outcome. However, to date, DC vaccination trials are only infrequently accompanied by NK cell monitoring. Here, we discuss different strategies to improve DC vaccine preparations via exploitation of NK cells and provide a summary of relevant NK cell parameters for immune monitoring. We underscore that the design of DC-based cancer vaccines should include the evaluation of their NK cell stimulating potency both in the preclinical phase and in clinical trials.

Dendritic cell vaccination in acute myeloid leukemia.

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, with a 5-year overall survival rate of only 5.2% for the continuously growing subgroup of AML patients older than 65 years. These patients are generally not considered eligible for intensive chemotherapy and/or allogeneic hematopoietic stem cell transplantation because of high treatment-related morbidity and mortality, emphasizing the need for novel, less toxic, treatment alternatives. It is within this context that immunotherapy has gained attention in recent years. In this review, we focus on the use of dendritic cell (DC) vaccines for immunotherapy of AML. DC are central orchestrators of the immune system, bridging innate and adaptive immunity and critical to the induction of anti-leukemic immunity. We discuss the rationale and basic principles of DC-based therapy for AML and review the clinical experience that has been obtained so far with this form of immunotherapy for patients with AML.

Targeting hedgehog signaling in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer related death. The urgent need for effective therapies is highlighted by the lack of adequate targeting. In PDAC, hedgehog (Hh) signaling is known to be aberrantly activated, which prompted the pathway as a possible target for effective treatment for PDAC patients. Unfortunately, specific targeting of upstream molecules within the Hh signaling pathway failed to bring clinical benefit. This led to the ongoing debate on Hh targeting as a therapeutic treatment for PDAC patients. Additionally, concurrent non-canonical activation routes also result in translocation of Gli transcription factors into the nucleus. Therefore, different downstream targets of the Hh signaling pathway were identified and evaluated in preclinical and clinical research. In this review we summarize the variety of Hh signaling antagonists in different preclinical models of PDAC. Furthermore, we discuss published and ongoing clinical trials that evaluated Hh antagonists and point out the current hurdles and future perspectives in the light of redesigning Hh-targeting therapies for the treatment of PDAC patients.

Expression of SARS-CoV-2-Related Surface Proteins in Non-Small-Cell Lung Cancer Patients and the Influence of Standard of Care Therapy.

In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.

Optimization of the Solvent and In Vivo Administration Route of Auranofin in a Syngeneic Non-Small Cell Lung Cancer and Glioblastoma Mouse Model.

The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10-15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF's optimal administration route and solvent in two syngeneic cancer mouse models.

Clinical evaluation of cellular immunotherapy in acute myeloid leukaemia.

Immunotherapy is currently under active investigation as an adjuvant therapy to improve the overall survival of patients with acute myeloid leukaemia (AML) by eliminating residual leukaemic cells following standard therapy. The graft-versus-leukaemia effect observed following allogeneic haematopoietic stem cell transplantation has already demonstrated the significant role of immune cells in controlling AML, paving the way to further exploitation of this effect in optimized immunotherapy protocols. In this review, we discuss the current state of cellular immunotherapy as adjuvant therapy for AML, with a particular focus on new strategies and recently published results of preclinical and clinical studies. Therapeutic vaccines that are being tested in AML include whole tumour cells as an autologous source of multiple leukaemia-associated antigens (LAA) and autologous dendritic cells loaded with LAA as effective antigen-presenting cells. Furthermore, adoptive transfer of cytotoxic T cells or natural killer cells is under active investigation. Results from phase I and II trials are promising and support further investigation into the potential of cellular immunotherapeutic strategies to prevent or fight relapse in AML patients.