Identification of Novel Isatin Derivative Bearing a Nitrofuran Moiety as Potent Multi-Isoform Aldehyde Dehydrogenase Inhibitor.

Aldehyde dehydrogenases (ALDHs) are a family of enzymes that aid in detoxification and are overexpressed in several different malignancies. There is a correlation between increased expression of ALDH and a poor prognosis, stemness, and resistance to several drugs. Several ALDH inhibitors have been generated due to the crucial role that ALDH plays in cancer stem cells. All of these inhibitors, however, are either ineffective, very toxic, or have yet to be subjected to rigorous testing on their effectiveness. Although various drug-like compounds targeting ALDH have been reported in the literature, none have made it to routine use in the oncology clinic. As a result, new potent, non-toxic, bioavailable, and therapeutically effective ALDH inhibitors are still needed. In this study, we designed and synthesized potent multi-ALDH isoform inhibitors based on the isatin and indazole pharmacophore. Molecular docking studies and enzymatic tests revealed that among all of the synthesized analogs, compound 3 is the most potent inhibitor of ALDH1A1, ALDH3A1, and ALDH1A3, exhibiting 51.32%, 51.87%, and 36.65% inhibition, respectively. The ALDEFLUOR assay further revealed that compound 3 acts as an ALDH broad spectrum inhibitor at 500 nM. Compound 3 was also the most cytotoxic to cancer cells, with an IC50 in the range of 2.1 to 3.8 µM for ovarian, colon, and pancreatic cancer cells, compared to normal and embryonic kidney cells (IC50 7.1 to 8.7 µM). Mechanistically, compound 3 increased ROS activity due to potent multi-ALDH isoform inhibition, which increased apoptosis. Taken together, this study identified a potent multi-isoform ALDH inhibitor that could be further developed as a cancer therapeutic.

Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition.

Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic.

Pharmacological agents targeting drug-tolerant persister cells in cancer.

Current cancer therapy can be effective, but the development of drug resistant disease is the usual outcome. These drugs can eliminate most of the tumor burden but often fail to eliminate the rare, "Drug Tolerant Persister" (DTP) cell subpopulations in residual tumors, which can be referred to as "Persister" cells. Therefore, novel therapeutic agents specifically targeting or preventing the development of drug-resistant tumors mediated by the remaining persister cells subpopulations are needed. Since approximately ninety percent of cancer-related deaths occur because of the eventual development of drug resistance, identifying, and dissecting the biology of the persister cells is essential for the creation of drugs to target them. While there remains uncertainty surrounding all the markers identifying DTP cells in the literature, this review summarizes the drugs and therapeutic approaches that are available to target the persister cell subpopulations expressing the cellular markers ATP-binding cassette sub-family B member 5 (ABCB5), CD133, CD271, Lysine-specific histone demethylase 5 (KDM5), and aldehyde dehydrogenase (ALDH). Persister cells expressing these markers were selected as the focus of this review because they have been found on cells surviving following drug treatments that promote recurrent drug resistant cancer and are associated with stem cell-like properties, including self-renewal, differentiation, and resistance to therapy. The limitations and obstacles facing the development of agents targeting these DTP cell subpopulations are detailed, with discussion of potential solutions and current research areas needing further exploration.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

Complementarity between Microbiome and Immunity May Account for the Potentiating Effect of Quercetin on the Antitumor Action of Cyclophosphamide in a Triple-Negative Breast Cancer Model.

Immunotherapy targeting program cell death protein 1 (PD-1) in addition to chemotherapy has improved the survival of triple-negative breast cancer (TNBC) patients. However, the development of resistance and toxicity remain significant problems. Using the translationally relevant 4T1 mouse model of TNBC, we report here that dietary administration of the phytochemical quercetin enhanced the antitumor action of Cyclophosphamide, a cytotoxic drug with significant immunogenic effects that is part of the combination chemotherapy used in TNBC. We observed that quercetin favorably modified the host fecal microbiome by enriching species such as Akkermansia muciniphilia, which has been shown to improve response to anti-PD-1 therapy. We also show that quercetin and, to a greater extent, Cyclophosphamide increased the systemic frequency of T cells and NK cells. In addition, Cyclophosphamide alone and in combination with quercetin reduced the frequency of Treg, which is consistent with an antitumor immune response. On the other hand, Cyclophosphamide did not significantly alter the host microbiome, suggesting complementarity between microbiome- and immune-mediated mechanisms in potentiating the antitumor action of Cyclophosphamide by quercetin. Overall, these results support the potential for microbiota-centered dietary intervention to overcome resistance to chemoimmunotherapy in TNBC.

Acute Myeloid Leukemia Causes T Cell Exhaustion and Depletion in a Humanized Graft-versus-Leukemia Model.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is, in many clinical settings, the only curative treatment for acute myeloid leukemia (AML). The clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. However, AML relapse remains the top cause of posttransplant death; this highlights the urgent need to enhance GVL. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. In this article, we report, the successful establishment of a novel (to our knowledge) humanized GVL model system by transplanting clinically paired donor PBMCs and patient AML into MHC class I/II knockout NSG mice. We observed significantly reduced leukemia growth in humanized mice compared with mice that received AML alone, demonstrating a functional GVL effect. Using this model system, we studied human GVL responses against human AML cells in vivo and discovered that AML induced T cell depletion, likely because of increased T cell apoptosis. In addition, AML caused T cell exhaustion manifested by upregulation of inhibitory receptors, increased expression of exhaustion-related transcription factors, and decreased T cell function. Importantly, combined blockade of human T cell-inhibitory pathways effectively reduced leukemia burden and reinvigorated CD8 T cell function in this model system. These data, generated in a highly clinically relevant humanized GVL model, not only demonstrate AML-induced inhibition of alloreactive T cells but also identify promising therapeutic strategies targeting T cell depletion and exhaustion for overcoming GVL failure and treating AML relapse after alloSCT.

IGF2BP family of RNA-binding proteins regulate innate and adaptive immune responses in cancer cells and tumor microenvironment.

Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP1, IGF2BP2, and IGF2BP3) are a family of RNA-binding proteins that play an essential role in the development and disease by regulating mRNA stability and translation of critical regulators of cell division and metabolism. Genetic and chemical inhibition of these proteins slows down cancer cell proliferation, decreases invasiveness, and prolongs life span in a variety of animal models. The role of RNA-binding proteins in the induction of tissues' immunogenicity is increasingly recognized, but, the impact of the IGF2BPs family of proteins on the induction of innate and adaptive immune responses in cancer is not fully understood. Here we report that downregulation of IGF2BP1, 2, and 3 expression facilitates the expression of interferon beta-stimulated genes. IGF2BP1 has a greater effect on interferon beta and gamma signaling compared to IGF2BP2 and IGF2BP3 paralogs. We demonstrate that knockdown or knockout of IGF2BP1, 2, and 3 significantly potentiates inhibition of cell growth induced by IFNß and IFNγ. Mouse melanoma cells with Igf2bp knockouts demonstrate increased expression of MHC I (H-2) and induce intracellular Ifn-γ expression in syngeneic T-lymphocytes in vitro. Increased immunogenicity, associated with Igf2bp1 inhibition, "inflames" mouse melanoma tumors microenvironment in SM1/C57BL/6 and SW1/C3H mouse models measured by a two-fold increase of NK cells and tumor-associated myeloid cells. Finally, we demonstrate that the efficiency of anti-PD1 immunotherapy in the mouse melanoma model is significantly more efficient in tumors that lack Igf2bp1 expression. Our retrospective data analysis of immunotherapies in human melanoma patients indicates that high levels of IGF2BP1 and IGF2BP3 are associated with resistance to immunotherapies and poor prognosis. In summary, our study provides evidence of the role of IGF2BP proteins in regulating tumor immunogenicity and establishes those RBPs as immunotherapeutic targets in cancer.

Angelica gigas: Signature Compounds, In Vivo Anticancer, Analgesic, Neuroprotective and Other Activities, and the Clinical Translation Challenges.

Angelica gigas Nakai (AGN) root is a medicinal herbal widely used in traditional medicine in Korea. AGN root ethanolic extract dietary supplements are marketed in the United States for memory health and pain management. We comprehensively reviewed the anticancer, analgesic, pro-memory and other bio-activities of AGN extract and its signature phytochemicals decursin, decursinol angelate, and decursinol a decade ago in 2012 and updated their anticancer activities in 2015. In the last decade, significant progress has been made for understanding the pharmacokinetics (PK) and metabolism of these compounds in animal models and single dose human PK studies have been published by us and others. In addition to increased knowledge of the known bioactivities, new bioactivities with potential novel health benefits have been reported in animal models of cerebral ischemia/stroke, anxiety, sleep disorder, epilepsy, inflammatory bowel disease, sepsis, metabolic disorders, osteoporosis, osteoarthritis, and even male infertility. Herein, we will update PK and metabolism of pyranocoumarins, review in vivo bioactivities from animal models and human studies, and critically appraise the relevant active compounds, the cellular and molecular pharmacodynamic targets, and pertinent mechanisms of action. Knowledge gaps include whether human pyranocoumarin PK metrics are AGN dose dependent and subjected to metabolic ceiling, or metabolic adaptation after repeated use. Critical clinical translation challenges include sourcing of AGN extracts, product consistency and quality control, and AGN dose optimization for different health conditions and disease indications. Future research directions are articulated to fill knowledge gaps and address these challenges.

Drug-Tolerant Persister Cells in Cancer Therapy Resistance.

One of the current stumbling blocks in our fight against cancer is the development of acquired resistance to therapy, which is attributable to approximately 90% of cancer-related deaths. Undercutting this process during treatment could significantly improve cancer management. In many cases, drug resistance is mediated by a drug-tolerant persister (DTP) cell subpopulation present in tumors, often referred to as persister cells. This review provides a summary of currently known persister cell subpopulations and approaches to target them. A specific DTP cell subpopulation with elevated levels of aldehyde dehydrogenase (ALDH) activity has stem cell-like characteristics and a high level of plasticity, enabling them to switch rapidly between high and low ALDH activity. Further studies are required to fully elucidate the functions of ALDH-high DTP cells, how they withstand drug concentrations that kill other cells, and how they rapidly adapt under levels of high cellular stress and eventually lead to more aggressive, recurrent, and drug-resistant cancer. Furthermore, this review addresses the processes used by the ALDH-high persister cell subpopulation to enable cancer progression, the ALDH isoforms important in these processes, interactions of ALDH-high DTPs with the tumor microenvironment, and approaches to therapeutically modulate this subpopulation in order to more effectively manage cancer.

A novel clinically relevant graft-versus-leukemia model in humanized mice.

The prognosis for acute myeloid leukemia (AML) relapse post allogeneic hematopoietic stem cell transplantation (alloSCT) is dismal. Novel effective treatment is urgently needed. Clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. The mechanisms that mediate immune escape of leukemia (thus causing GVL failure) remain poorly understood. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. Here, using our large, longitudinal clinical tissue bank that include AML cells and G-CSF mobilized donor hematopoietic stem cells (HSCs), we successfully established a novel GVL model in humanized mice. Donor HSCs were injected into immune-deficient NOD-Cg-Prkdcscid IL2rgtm1Wjl /SzJ (NSG) mice to build humanized mice. Immune reconstitution in these mice recapitulated some clinical scenario in the patient who received the corresponding HSCs. Allogeneic but HLA partially matched patient-derived AML cells were successfully engrafted in these humanized mice. Importantly, we observed a significantly reduced (yet incomplete elimination of) leukemia growth in humanized mice compared with that in control NSG mice, demonstrating a functional (but defective) GVL effect. Thus, for the first time, we established a novel humanized mouse model that can be used for studying human GVL responses against human AML cells in vivo. This novel clinically relevant model provides a valuable platform for investigating the mechanisms of human GVL and development of effective leukemia treatments.

Multi-dimensional analysis identifies an immune signature predicting response to decitabine treatment in elderly patients with AML.

Decitabine is a DNA-hypomethylating agent that has been widely applied for the treatment of acute myeloid leukaemia (AML) patients who are elderly or unfit for intensive therapy. Although effective, the complete response rate to decitabine is only around 30% and the overall survival remains poor. Emerging data support that regulation of DNA methylation is critical to control immune cell development, differentiation and activation. We hypothesize that defining how decitabine influences the immune responses in AML will facilitate the development of novel immune-based leukaemia therapeutics. Here, we performed phenotypic and functional immune analysis on clinical samples from AML patients receiving decitabine treatment and demonstrated a significant impact of decitabine on the immune system. T-cell expression of inhibitory molecules was upregulated and the ability of CD8 T cells to produce cytokines was decreased upon decitabine treatment. Importantly, in an unbiased comprehensive analysis, we identified a unique immune signature containing a cluster of key immune markers that clearly separate patients who achieved complete remission after decitabine from those who failed to do so. Therefore, this immune signature has a strong predictive value for clinical response. Collectively, our study suggests that immune-based analyses may predict clinical response to decitabine and provide a therapeutic strategy to improve the treatment of AML.

Schweinfurthin natural products induce regression of murine melanoma and pair with anti-PD-1 therapy to facilitate durable tumor immunity.

Metastatic melanoma is a significant clinical problem with a 5-year survival rate of only 15-20%. Recent approval of new immunotherapies and targeted inhibitors have provided much needed options for these patients, in some cases promoting dramatic disease regressions. In particular, antibody-based therapies that block the PD-1/PD-L1 checkpoint inhibitory pathway have achieved an increased overall response rate in metastatic melanoma, yet durable response rates are reported only around 15%. To improve the overall and durable response rates for advanced-stage melanoma, combined targeted and immune-based therapies are under investigation. Here, we investigated how the natural products called schweinfurthins, which have selective anti-proliferative activity against many cancer types, impact anti-(α)PD-1-mediated immunotherapy of murine melanomas. Two different compounds efficiently reduced the growth of human and murine melanoma cells in vitro and induced plasma membrane surface localization of the ER-resident protein calreticulin in B16.F10 melanoma cells, an indicator of immunogenic cell death. In addition, both compounds improved αPD-1-mediated immunotherapy of established tumors in immunocompetent C57BL/6 mice either by delaying tumor progression or resulting in complete tumor regression. Improved immunotherapy was accomplished following only a 5-day course of schweinfurthin, which was associated with initial tumor regression even in the absence of αPD-1. Schweinfurthin-induced tumor regression required an intact immune system as tumors were unaffected in NOD scid gamma (NSG) mice. These results indicate that schweinfurthins improve αPD-1 therapy, leading to enhanced and durable anti-tumor immunity and support the translation of this novel approach to further improve response rates for metastatic melanoma.

Eomes+T-betlow CD8+ T Cells Are Functionally Impaired and Are Associated with Poor Clinical Outcome in Patients with Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a devastating blood cancer with poor prognosis. Immunotherapy targeting inhibitory pathways to unleash the antileukemia T-cell response is a promising strategy for the treatment of leukemia, but we must first understand the underlying molecular mechanisms. Eomesodermin (Eomes) and T-bet are both T-box transcription factors that regulate CD8+ T-cell responses in a context-specific manner. Here, we examined the role of these transcription factors in CD8+ T-cell immunity in AML patients. We report that the frequency of Eomes+T-betlow CD8+ T cells increased in newly diagnosed AML. This cell subset produced fewer cytokines and displayed reduced killing capacity, whereas depletion of Eomes by siRNA reversed these functional defects. Furthermore, Eomes bound the promoter of T-cell immunoglobulin and ITIM domain (TIGIT) and positively regulated the expression of this inhibitory receptor on patient-derived T cells. A high frequency of Eomes+T-betlow CD8+ T cells was associated with poor response to induction chemotherapy and shorter overall survival in AML patients. These findings have significant clinical implications as they not only identify a predictive and prognostic biomarker for AML, but they also provide an important target for effective leukemia therapeutics. SIGNIFICANCE: These findings reveal that a high frequency of Eomes+T-betlow CD8+ T cells predicts poor clinical outcome in AML and that targeting Eomes may provide a therapeutic benefit against AML.

Aldehyde dehydrogenase in regulatory T-cell development, immunity and cancer.

The role of aldehyde dehydrogenase (ALDH) in carcinogenesis and resistance to cancer therapies is well known. Mounting evidence also suggests a potentially important role for ALDH in the induction and function of regulatory T (Treg) cells. Treg cells are important cells of the immune system involved in promoting immune tolerance and preventing aberrant immune responses to beneficial or non-harmful antigens. However, Treg cells also impair tumor immunity, leading to the progression of various carcinomas. ALDH expression and the subsequent production of retinoic acid by numerous cells, including dendritic cells, macrophages, eosinophils and epithelial cells, seems important in Treg induction and function in multiple organ systems. This is particularly evident in the gastrointestinal tract, pulmonary tract and skin, which are exposed to a myriad of environmental antigens and represent interfaces between the human body and the outside world. Expression of ALDH in Treg cells themselves may also be involved in the proliferation of these cells and resistance to certain cytotoxic therapies. Hence, inhibition of ALDH expression may be useful to treat cancer. Besides the direct effect of ALDH inhibition on carcinogenesis and resistance to cancer therapies, inhibition of ALDH could potentially augment the immune response to tumor antigens by inhibiting Treg induction, function and ability to promote immune tolerance to tumor cells in multiple cancer types.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

KEY POINTS: Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. ABSTRACT: In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexin V, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.

PD-1/PD-L1 co-inhibition shapes anticancer T cell immunodominance: facing the consequences of an immunological ménage à trois.

PD-1- and PD-L1-blocking monoclonal antibodies have shown significant promise in clinical settings and rekindled the hope for successful cancer immunotherapy. We recently demonstrated that interfering with PD-1/PD-L1 signaling selectively augments CD8+ T cell (TCD8) responses to subdominant determinants (SDDs) of a model tumor antigen. This was likely due to decreased lysis of SDD-specific TCD8 by neighboring immunodominant clones co-engaging the same antigen-presenting cells (APCs). We therefore proposed that PD-1-based checkpoint inhibitors widen the range of tumor determinants that can be effectively targeted by TCD8. Subsequently and using different tumor models, Chen et al. reported, in Proceedings of the National Academy of Sciences of the United States of America, that PD-L1 protects APCs from the lytic function of immunodominant TCD8 and that PD-L1 blockade narrows, rather than broadens, the overall anticancer T cell response. Here, we briefly compare and contrast the experimental systems employed by the two groups, which may account, at least partially, for the opposing conclusions drawn. We argue that the pathway(s) of tumor antigen presentation, direct presentation versus cross-presentation, and the intensity of PD-1 expression by immunodominant and subdominant TCD8 must be taken into consideration in rational design of anti-PD-1/PD-L1-adjuvanted tumor vaccines and therapies.

Beta blocker use correlates with better overall survival in metastatic melanoma patients and improves the efficacy of immunotherapies in mice.

Immunotherapy has expanded treatment options for cancers with historically poor outcomes, yet a significant proportion of patients still fail to achieve durable clinical benefit. We defined the contribution of ß-adrenergic receptor (ßAR) signaling, a component of the stress response, on success of immunotherapy for melanoma since the use of antagonists (ß-blockers) is associated with improved clinical outcomes in some cancers. We show that metastatic melanoma patients who received immunotherapy had improved overall survival if they also received pan ß-blockers. This retrospective analysis is reinforced by results showing that ßAR blockade enhances the control of murine melanoma growth by anti-(α)PD-1 checkpoint blockade. However, this effect was most significant when ß-blocker was combined with dual αPD-1 + high dose interleukin-2 therapy and was reproduced by selective blockade of ß2ARs. These results identify a novel strategy that can be quickly introduced to potentially increase the number of patients who benefit from immune-based therapies.

Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice.

BACKGROUND & AIMS: Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. METHODS: Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. RESULTS: Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. CONCLUSIONS: In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Combined sublethal irradiation and agonist anti-CD40 enhance donor T cell accumulation and control of autochthonous murine pancreatic tumors.

Tumor-reactive T lymphocytes can promote the regression of established tumors. However, their efficacy is often limited by immunosuppressive mechanisms that block T cell accumulation or function. ACT provides the opportunity to ameliorate immune suppression prior to transfer of tumor-reactive T cells to improve the therapeutic benefit. We evaluated the combination of lymphodepleting whole body irradiation (WBI) and agonist anti-CD40 (αCD40) antibody on control of established autochthonous murine neuroendocrine pancreatic tumors following the transfer of naïve tumor-specific CD8 T cells. Sublethal WBI had little impact on disease outcome but did promote T cell persistence in the lymphoid organs. Host conditioning with αCD40, an approach known to enhance APC function and T cell expansion, transiently increased donor T cell accumulation in the lymphoid organs and pancreas, but failed to control tumor progression. In contrast, combined WBI and αCD40 prolonged T cell proliferation and dramatically enhanced accumulation of donor T cells in both the lymphoid organs and pancreas. This dual conditioning approach also promoted high levels of inflammation in the pancreas and tumor, induced histological regression of established tumors, and extended the lifespan of treated mice. Prolonged survival was entirely dependent upon adoptive transfer, but only partially dependent upon IFNγ production by donor T cells. Our results identify the novel combination of two clinically relevant host conditioning approaches that synergize to overcome immune suppression and drive strong tumor-specific T cell accumulation within well-established tumors.