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Despite the clinical success of T-cell checkpoint blockade, most patients with cancer still fail to have durable responses to immunotherapy. The molecular mechanisms driving checkpoint blockade resistance, whether preexisting or evolved, remain unclear. To address this critical knowledge gap, we treated B16 melanoma with the combination of CTLA-4, PD-1, and PD-L1 blockade and a Flt3 ligand vaccine (≥75% curative), isolated tumors resistant to therapy, and serially passaged them in vivo with the same treatment regimen until they developed complete resistance. Using gene expression analysis and immunogenomics, we determined the adaptations associated with this resistance phenotype. Checkpoint resistance coincided with acquisition of a “hypermetabolic” phenotype characterized by coordinated upregulation of the glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways. These resistant tumors flourished under hypoxic conditions, whereas metabolically starved T cells lost glycolytic potential, effector function, and the ability to expand in response to immunotherapy. Furthermore, we found that checkpoint-resistant versus -sensitive tumors could be separated by noninvasive MRI imaging based solely on their metabolic state. In a cohort of patients with melanoma resistant to both CTLA-4 and PD-1 blockade, we observed upregulation of pathways indicative of a similar hypermetabolic state. Together, these data indicated that melanoma can evade T-cell checkpoint blockade immunotherapy by adapting a hypermetabolic phenotype.

Despite the success of immune checkpoint blockade against melanoma, many "cold" tumors like prostate cancer remain unresponsive. We found that hypoxic zones were prevalent across preclinical prostate cancer and resisted T cell infiltration even in the context of CTLA-4 and PD-1 blockade. We demonstrated that the hypoxia-activated prodrug TH-302 reduces or eliminates hypoxia in these tumors. Combination therapy with this hypoxia-prodrug and checkpoint blockade cooperated to cure more than 80% of tumors in the transgenic adenocarcinoma of the mouse prostate-derived (TRAMP-derived) TRAMP-C2 model. Immunofluorescence imaging showed that TH-302 drives an influx of T cells into hypoxic zones, which were expanded by checkpoint blockade. Further, combination therapy reduced myeloid-derived suppressor cell density by more than 50%, and durably reduced the capacity of the tumor to replenish the granulocytic subset. Spontaneous prostate tumors in TRAMP transgenic mice, which completely resist checkpoint blockade, showed minimal adenocarcinoma tumor burden at 36 weeks of age and no evidence of neuroendocrine tumors with combination therapy. Survival of Pb-Cre4, Ptenpc-/-Smad4pc-/- mice with aggressive prostate adenocarcinoma was also significantly extended by this combination of hypoxia-prodrug and checkpoint blockade. Hypoxia disruption and T cell checkpoint blockade may sensitize some of the most therapeutically resistant cancers to immunotherapy.

Purpose: Agonist antibodies targeting the T-cell costimulatory receptor 4-1BB (CD137) are among the most effective immunotherapeutic agents across preclinical cancer models. In the clinic, however, development of these agents has been hampered by dose-limiting liver toxicity. Lack of knowledge of the mechanisms underlying this toxicity has limited the potential to separate 4-1BB agonist–driven tumor immunity from hepatotoxicity. Experimental Design: The capacity of 4-1BB agonist antibodies to induce liver toxicity was investigated in immunocompetent mice, with or without coadministration of checkpoint blockade, via (i) measurement of serum transaminase levels, (ii) imaging of liver immune infiltrates, and (iii) qualitative and quantitative assessment of liver myeloid and T cells via flow cytometry. Knockout mice were used to clarify the contribution of specific cell subsets, cytokines, and chemokines. Results: We find that activation of 4-1BB on liver myeloid cells is essential to initiate hepatitis. Once activated, these cells produce interleukin-27 that is required for liver toxicity. CD8 T cells infiltrate the liver in response to this myeloid activation and mediate tissue damage, triggering transaminase elevation. FoxP3+ regulatory T cells limit liver damage, and their removal dramatically exacerbates 4-1BB agonist–induced hepatitis. Coadministration of CTLA-4 blockade ameliorates transaminase elevation, whereas PD-1 blockade exacerbates it. Loss of the chemokine receptor CCR2 blocks 4-1BB agonist hepatitis without diminishing tumor-specific immunity against B16 melanoma. Conclusions: 4-1BB agonist antibodies trigger hepatitis via activation and expansion of interleukin-27–producing liver Kupffer cells and monocytes. Coadministration of CTLA-4 and/or CCR2 blockade may minimize hepatitis, but yield equal or greater antitumor immunity. Clin Cancer Res; 24(5); 1138–51. ©2018 AACR.

Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder, is currently conceptualized as a disease of synaptic failure. Synaptic impairments are robust within the AD brain and better correlate with dementia severity when compared with other pathological features of the disease. Nevertheless, the series of events that promote synaptic failure still remain under debate, as potential triggers such as β-amyloid (Aβ) can vary in size, configuration and cellular location, challenging data interpretation in causation studies. Here we present data obtained using adeno-associated viral (AAV) constructs that drive the expression of oligomeric Aβ either intra or extracellularly. We observed that expression of Aβ in both cellular compartments affect learning and memory, reduce the number of synapses and the expression of synaptic-related proteins, and disrupt chemical long-term potentiation (cLTP). Together, these findings indicate that during the progression AD the early accumulation of Aβ inside neurons is sufficient to promote morphological and functional cellular toxicity, a phenomenon that can be exacerbated by the buildup of Aβ in the brain parenchyma. Moreover, our AAV constructs represent a valuable tool in the investigation of the pathological properties of Aβ oligomers both in vivo and in vitro.

Diabetes mellitus (DM) is one of the most devastating diseases that currently affects the aging population. Recent evidence indicates that DM is a risk factor for many brain disorders, due to its direct effects on cognition. New findings have shown that the microtubule‐associated protein tau is pathologically processed in DM; however, it remains unknown whether pathological tau modifications play a central role in the cognitive deficits associated with DM. To address this question, we used a gain‐of‐function and loss‐of‐function approach to modulate tau levels in type 1 diabetes (T1DM) and type 2 diabetes (T2DM) mouse models. Our study demonstrates that tau differentially contributes to cognitive and synaptic deficits induced by DM. On one hand, overexpressing wild‐type human tau further exacerbates cognitive and synaptic impairments induced by T1DM, as human tau mice treated under T1DM conditions show robust deficits in learning and memory processes. On the other hand, neither a reduction nor increase in tau levels affects cognition in T2DM mice. Together, these results shine new light onto the different molecular mechanisms that underlie the cognitive and synaptic impairments associated with T1DM and T2DM.

Despite intensive research efforts over the past few decades, the mechanisms underlying the etiology of sporadic Alzheimer's disease (AD) remain unknown. This fact is of major concern because the number of patients affected by this medical condition is increasing exponentially and the existing treatments are only palliative in nature and offer no disease modifying affects. Interestingly, recent epidemiological studies indicate that diabetes significantly increases the risk of developing AD, suggesting that diabetes may play a causative role in the development of AD pathogenesis. Therefore, elucidating the molecular interactions between diabetes and AD is of critical significance because it might offer a novel approach to identifying mechanisms that may modulate the onset and progression of sporadic AD cases. This review highlights the involvement of several novels pathological molecular mechanisms induced by diabetes that increase AD pathogenesis. Furthermore, we discuss novel findings in animal model and clinical studies involving the use of anti-diabetic compounds as promising therapeutics for AD.

Advances in our understanding of tumor immune biology and development of cancer immunotherapies have led to improved outcomes for patients who suffer from aggressive cancers such as melanoma. Despite the clinical success of immune checkpoint blockade, a majority of patients still fail to respond, and the underlying mechanisms that drive resistance remain unclear. To understand why a subset of tumors fail to respond to immunotherapy, we established a novel murine model of melanoma that is fully resistant to immune checkpoint blockade. By in vivo passaging nonresponding B16 melanoma tumor cells, we selected for a resistant variant that fails to respond to the combination of CTLA-4, PD-1, and PD-L1 blockade. In comparing gene expression of parental versus resistant tumor cells and analyzing the corresponding immune infiltrate, we determined the adaptations associated with resistance to therapy. We found that evasion of immunotherapy was associated with a “hypermetabolic” phenotype, characterized by an upregulation of glycolytic, oxidoreductase, and mitochondrial oxidative phosphorylation pathways to establish a hypoxic, metabolically hostile microenvironment. Enforced expression of two key genes associated with these pathways in parental tumor cells was sufficient to mediate resistance to triple checkpoint blockade. Flow cytometry assays determined that T cells infiltrating resistant tumors had diminished glycolytic capacity and effector function, indicating a metabolic disadvantage. Consistent with our findings, melanoma patients who failed dual checkpoint blockade exhibited similar metabolic alterations as seen in our resistant variant. Using a novel MRI-based imaging approach, we observed distinct metabolic changes that stratified responding versus nonresponding tumors in live mice. Applying this method to patients could provide insight into predicting response rates to checkpoint modulation. Overall, our data indicate that resistant melanoma tumor cells acquire a “hypermetabolic” phenotype to establish a hostile microenvironment that is capable of inhibiting the antitumor immune response. This abstract is also being presented as Poster A24. Citation Format: Ashvin R. Jaiswal, Arthur J. Liu, Shivanand Pudakalakatti, Prasanta Dutta, Priyamvada Jayaprakash, Todd Bartkowiak, Casey Ager, Zhiqiang Wang, Alex Reuben, Zachary Cooper, Cristina Ivan, Zhenlin Ju, Felix Nwajei, Jing Wang, Michael A. Davies, R. Eric Davis, Jennifer A. Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Melanoma evolves complete immunotherapy resistance through acquisition of a hypermetabolic phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR02.

Activation of the stimulator of interferon genes (STING) pathway by both exogenous and endogenous cytosolic DNA results in the production of interferon beta (IFN-β) and is required for the generation of cytotoxic T-cell priming against tumor antigens. In the clinical setting, pharmacological stimulation of the STING pathway has the potential to synergize with immunotherapy antibodies by boosting anti-tumor immune responses. We report the discovery of two highly potent cyclic dinucleotide STING agonists, IACS-8803 and IACS-8779, which show robust activation of the STING pathway in vitro and a superior systemic anti-tumor response in the B16 murine model of melanoma when compared to one of the clinical benchmark compounds.

Immunosuppressive myeloid populations including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are abundant within pancreatic adenocarcinoma (PDAC) tumors and play critical roles in constraining cytotoxic T-cell function in the tumor microenvironment. We hypothesized that intratumoral engagement of innate pathogen recognition receptors such as Stimulator of Interferon Genes (STING) could induce proinflammatory polarization of the myeloid stroma and liberate the antitumor T-cell response to regress refractory PDAC tumors in the presence of checkpoint blockade.We developed and characterized a novel cyclic dinucleotide (CDN) STING agonist IACS-8803, and found that 8803 activates downstream STING signaling in both human (THP-1) and murine (J774) myeloid reporter cells with over 10-fold greater potency than ML-RR-S2-CDA, the first-in-class CDN currently undergoing clinical evaluation (NCT02675439, NCT03172936). Intratumoral delivery of 8803 into subcutaneous B16 melanoma and PDAC tumors additionally revealed a greater capacity to induce tumor regression relative to ML-RR-S2-CDA. In order to evaluate the specific effects of 8803 on the phenotype and function of suppressive myeloid populations, we generated in vitro polarized human M2 macrophages and murine bone marrow-derived MDSC. Upon exposure to 8803, we observe downregulation of M2 markers CD163, LAP/TGF-β, and Arginase on human M2 macrophages, concomitant with upregulation of M1 markers CD86, CD80, and IL-6. Additionally, 8803-stimulated murine MDSC exhibit reduced T-cell suppressive capacity compared to unstimulated MDSC. In these studies, we consistently observe that the magnitude of phenotypic and functional repolarization by 8803 is superior to that of ML-RR-S2-CDA as well as other known CDN, 2’3’-cGAMP and c-di-GMP. Therefore, we describe IACS-8803 as a novel, highly potent STING agonist with the capacity to induce inflammatory repolarization in suppressive myeloid cells of both human and murine origin. We next investigated the capacity for intratumoral delivery of IACS-8803 to sensitize murine pancreatic cancer to checkpoint blockade and to mobilize systemic immunity against disseminated lesions. We utilized mT4-2D, a novel pancreatic cancer cell line from Kras+/LSL-G12D Tp53+/LSL-R172H Pdx1-Cre tumor organoids. We isolateda single cell clone of mT4-2D with reduced in vivo growth kinetics (termed mT4-LS), as well as a clone that maintains the aggressive nature of the parental line (termed mT4-LA). Mice bearing 10-day established orthotopic and subcutaneous mT4-LS tumors received standard regimens of αCTLA-4, αPD-1, or combined αCTLA-4/αPD-1 in the presence or absence of 8803 CDN injected into the orthotopic pancreatic tumor. We find single-agent treatment with 8803, αCTLA-4, αPD-1, or αCTLA-4/αPD-1 can cure 40-60% of mice of both orthotopic and subcutaneous tumors in this system; however, combining 8803 with checkpoint blockade completely eradicates both injected and distal mT4-LS tumors in all mice. We replicated these studies using the highly aggressive and refractory mT4-LA model, and found that combination therapy with intra-pancreatic 8803 and systemic αCTLA-4/αPD-1 significantly extends survival compared to 8803 or αCTLA-4/αPD-1 alone (p=0.001, p=0.0086, respectively). Analysis of treated mT4-LA tumors by flow cytometry reveals that combination therapy enhances the cytotoxic potential of CD8 T-cells at both injected and uninjected lesions, and promotes dendritic cell proliferation within the pancreatic milieu. These studies provide a preclinical rationale for pursuing the use of STING-activating CDN as a localized approach to sensitize refractory PDAC tumors to checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Maria E. Di Francesco, Philip Jones, Michael A. Curran. Intratumoral delivery of a novel STING agonist synergizes with checkpoint blockade to regress multifocal pancreatic cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A050.

Background: Resistance of pancreatic ductal adenocarcinoma (PDAC) to checkpoint blockade therapy is commonly attributed to its status as an immunologically cold tumor. However, it remains to be determined whether the lack of endogenous immunity to PDAC is predominantly due to a paucity of targetable neoantigens or to the unique tolerogenic environment of malignant pancreatic tissue. Using an implantable model derived from the Kras+/LSL-G12DTrp53+/LSL-R172HPdx1-Cre (KPC) mouse, we investigated how tissue implantation site and exogenous antigen presentation influenced immune surveillance of KPC tumors and response to combination immunotherapy. Methods: The KPC organoid-derived cell line mT4-2D was stably transduced with various expression constructs including luciferase (mT4-Luc), ovalbumin (mT4-OVA), or mCherry (mT4-Cherry). Each line was implanted subcutaneously (2.5x105 cells) or orthotopically at indicated doses to assess effects of exogenous antigen expression on engraftment efficiency. To evaluate how tissue environment governs response to therapy, mice were co-implanted with mT4-Luc at both orthotopic and subcutaneous sites and were exposed to combination therapy consisting of intratumoral STING agonist ML-RR-CDA (10ug/mouse) and checkpoint antagonists αCTLA-4 and αPD-1 beginning on day 14 post-implantation. Tumor growth at both sites was monitored weekly by IVIS bioluminescent imaging and mice were followed for survival, or tumors were harvested on day 25 for analysis of immune infiltrates by flow cytometry. Results: mT4 tumors grew aggressively at both subcutaneous and orthotopic sites, with doses as low as 3.5x104 causing mortality within 3 weeks post-implantation. However, expression of either OVA or mCherry resulted in spontaneous clearance in 100% of mice, regardless of implantation site. Luciferase expression yielded progressing tumors, and ex vivo peptide restimulation failed to identify Luciferase-specific T cells within mT4-Luc challenged mice; suggesting that Luciferase is a minimally immunogenic protein in C57BL/6 mice. Despite the poor immunogenicity of the mT4 model, we found that intratumoral injections of ML-RR-CDA delayed growth of subcutaneously-implanted mT4, and that concomitant administration of αCTLA-4/αPD-1 induced complete clearance in ~40% of mice. In contrast, intratumoral delivery of ML-RR-CDA was ineffective against orthotopic mT4 tumors, and combination therapy with αCTLA-4/αPD-1 achieved only modest delays in tumor progression. Flow cytometric analysis of tumor infiltrating immune populations in mice with co-implanted orthotopic and subcutaneous lesions revealed dichotomies in the density of CD8 T cells, CD4 effector T cells, and dendritic cell populations between tissue sites in response to local therapy. Conclusions: Although implantable cell lines isolated from KPC tumors are highly aggressive and resistant models of PDAC, we found that engraftment and growth of mT4-2D was completely negated upon forced expression of a single foreign protein antigen. This suggests that the lack of targetable mutanome products may explain the resistance of KPC and potentially human PDAC tumors to immunotherapy. However, when implanted subcutaneously, mT4-2D was sensitive to in situ STING activation in combination with checkpoint blockade, which indicates that immune responses against tissue-specific antigens are potentially sufficient to control growth of KPC tumors when removed from the suppressive pancreatic environment. Flow analysis revealed a greater capacity of CD8 T cells, CD4 T cells, and dendritic cells to infiltrate subcutaneous vs orthotopic tumors following intratumoral therapy, reinforcing the theory that therapies which enhance infiltration of select immune populations are needed in order to sensitize PDAC tumors to therapy. Citation Format: Casey R. Ager, Michael A. Curran. Effects of tissue site and antigenicity on KPC-derived pancreatic tumor growth and response to combination immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B52.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~80% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF analysis, flow cytometry, confocal microscopy, and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by coordinate upregulation of the glycolytic and aldose reductase pathways to create a metabolically hostile microenvironment in which T-cell function is profoundly suppressed. When reintroduced into the parental tumor, the genes most closely associated with these metabolic adaptations confer enhanced immunotherapy resistance. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed MRI imaging to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patients. Conclusion: Upregulation of glycolytic metabolism and the aldose reductase pathway by melanoma tumor cells cripples T cells in the microenvironment and confers resistance to checkpoint blockade. This abstract is also being presented as Poster A71. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Prasanta Dutta, Arthur Liu, Todd Bartkowiak, Casey Ager, Cristina Ivan, Richard Eric Davis, Michael A. Davies, Jennifer Wargo, James P. Allison, Pratip K. Bhattacharya, David Hong, Michael A. Curran. Metabolic adaptations establish immunotherapy resistance in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR08.

Background: Despite the success of T-cell checkpoint blockade antibodies in treating an array of cancers, the majority of patients still fail to respond to these therapies, or respond transiently and then relapse. The molecular mechanisms that drive lack of response to checkpoint blockade, whether pre-existing or evolved on therapy, remain unclear. Materials and Methods: To address this critical gap in clinical knowledge, we established a mouse model of melanoma designed to elucidate the molecular mechanisms underlying immunotherapy resistance. Through multiple in vivo passages, we selected a B16 melanoma tumor line that evolved complete resistance to combination blockade of CTLA-4, PD-1, and PD-L1, which cures ~75% of mice of the parental tumor. Using gene expression analysis, proteomics, and immunogenomics, we determined the adaptations engaged by this melanoma to become completely immunotherapy resistant. NMR spectroscopy, Seahorse XF Analysis, flow cytometry, confocal microscopy and Western blot analysis provided further insight into the mechanisms driving checkpoint blockade resistance. Results: Acquisition of immunotherapy resistance by these melanomas was driven by acquisition of a hypermetabolic state through coordinate upregulation of both glycolysis and oxidative phosphorylation (OxPhos). Depletion of essential nutrients from the tumor microenvironment by these resistant tumors functionally compromises infiltrating T cells to an extent that cannot be rescued by even triple checkpoint blockade. The glycolysis and OxPhos-associated genes most highly induced by these resistant melanomas were capable of conferring enhanced resistance to checkpoint blockade when reintroduced monogenically into the parental tumor. We have validated upregulation of these pathways in a unique cohort of melanoma patients who failed dual checkpoint blockade. Additionally, we employed a novel MRI imaging technique to visualize metabolic changes acquired by resistant tumors in live mice. Clinical application of this technique could provide a much-needed noninvasive tool to predict immunotherapeutic sensitivity of patient cancers. Conclusion: Acquisition of a hypermetabolic state by melanoma tumor cells cripples T cells in the microenvironment and confers complete resistance to checkpoint blockade. Citation Format: Ashvin R. Jaiswal, Shivanand Pudakalakatti, Dutta Prasanta, Arthur Liu, Todd Bartkowiak, Casey Ager, Michael A. Davies, Richard E. Davis, Jennifer Wargo, Pratip K. Bhattacharya, David S. Hong, Michael A. Curran. Metabolic adaptations confer immunotherapy resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1700.

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by the accumulation of amyloid-beta protein and neuronal loss, is the leading cause of age-related dementia in the world today. The disease is also associated with neuroinflammation, robust activation of astrocytes and microglia, and evidence of activation of the complement system, localized with both fibrillar amyloid-beta (fAbeta) plaques and tangles. The observations are consistent with a complement-dependent component of AD progression. We have previously shown that inhibition of the major complement receptor for C5a (CD88) with the antagonist PMX205 results in a significant reduction in pathology in two mouse models of AD. To further characterize the role of complement in AD-related neuroinflammation, we examined the age- and disease-associated expression of CD88 in brain of transgenic mouse models of AD and the influence of PMX205 on the presence of various complement activation products using flow cytometry, western blot, and immunohistochemistry. CD88 was found to be up-regulated in microglia, in the immediate vicinity of amyloid plaques. While thioflavine plaque load and glial recruitment is significantly reduced after treatment with PMX205, C1q remains co-localized with fAbeta plaques and C3 is still expressed by the recruited astrocytes. Thus, with PMX205, potentially beneficial activities of these early complement components may remain intact, while detrimental activities resulting from C5a-CD88 interaction are inhibited. This further supports the targeted inhibition of specific complement mediated activities as an approach for AD therapy.

Alzheimer’s disease (AD) is an age-related dementia, characterized by amyloid plaques, neurofibrillary tangles, neuroinflammation, and neuronal loss in the brain. Components of the complement system, known to produce a local inflammatory reaction, are associated with the plaques and tangles in AD brain, and thus a role for complement-mediated inflammation in the acceleration or progression of disease has been proposed. A complement activation product, C5a, is known to recruit and activate microglia and astrocytes in vitro by activation of a G protein-coupled cell-surface C5aR. Here, oral delivery of a cyclic hexapeptide C5a receptor antagonist (PMX205) for 2–3 mo resulted in substantial reduction of pathological markers such as fibrillar amyloid deposits (49–62%) and activated glia (42–68%) in two mouse models of AD. The reduction in pathology was correlated with improvements in a passive avoidance behavioral task in Tg2576 mice. In 3xTg mice, PMX205 also significantly reduced hyperphosphorylated tau (69%). These data provide the first evidence that inhibition of a proinflammatory receptor-mediated function of the complement cascade (i.e., C5aR) can interfere with neuroinflammation and neurodegeneration in AD rodent models, suggesting a novel therapeutic target for reducing pathology and improving cognitive function in human AD patients.

Alzheimer's disease (AD) is a progressive neurological disorder that impairs memory and other cognitive functions in the elderly. The social and financial impacts of AD are overwhelming and are escalating exponentially as a result of population aging. Therefore, identifying AD-related risk factors and the development of more efficacious therapeutic approaches are critical to cure this neurological disorder. Current epidemiological evidence indicates that life experiences, including chronic stress, are a risk for AD. However, it is unknown if short-term stress, lasting for hours, influences the onset or progression of AD. Here, we determined the effect of short-term, multi-modal 'modern life-like' stress on AD pathogenesis and synaptic plasticity in mice bearing three AD mutations (the 3xTg-AD mouse model). We found that combined emotional and physical stress lasting 5 h severely impaired memory in wild-type mice and tended to impact it in already low-performing 3xTg-AD mice. This stress reduced the number of synapse-bearing dendritic spines in 3xTg-AD mice and increased Aβ levels by augmenting AβPP processing. Thus, short-term stress simulating modern-life conditions may exacerbate cognitive deficits in preclinical AD by accelerating amyloid pathology and reducing synapse numbers. Epidemiological evidence indicates that life experiences, including chronic stress, are a risk for Alzheimer disease (AD). However, it is unknown if short stress in the range of hours influences the onset or progression of AD. Here, we determined the effect of short, multi-modal 'modern-lifelike'stress on AD pathogenesis and synaptic plasticity in mice bearing three AD mutations (the 3xTg-AD mouse model). We found that combined emotional and physical stress lasting 5 h severely impaired memory in wild-type mice and tended to impact it in already low-performing 3xTg-AD mice. This stress reduced the number of synapse-bearing dendritic spines in 3xTg-AD mice and increased Aβ levels by augmenting AβPP processing. Thus, short stress simulating modern-life conditions may exacerbate cognitive deficits in preclinical AD by accelerating amyloid pathology and reducing synapse numbers.

The initiation of an inflammatory response is critical to the survival of an organism. However, when inflammation fails to reach resolution, a chronic inflammatory state may occur, potentially leading to bystander tissue damage. Accumulating evidence suggests that chronic inflammation contributes to the progression of Alzheimer's disease (AD), and identifying mechanisms to resolve the pro-inflammatory environment stimulated by AD pathology remains an area of active investigation. Previously, we found that treatment with the pro-resolving mediator aspirin-triggered lipoxin A4 (ATL), improved cognition, reduced Aβ levels, and enhanced microglia phagocytic activity in Tg2576 transgenic AD mice. Here, we evaluated the effect of aging on brain lipoxin A4 (LXA4) levels using non-transgenic and 3xTg-AD mice. Additionally, we investigated the effect of ATL treatment on tau pathology in 3xTg-AD mice. We found that LXA4 levels are reduced with age, a pattern significantly more impacted in 3xTg-AD mice. Moreover, ATL delivery enhanced the cognitive performance of 3xTg-AD mice and reduced Aβ levels, as well as decreased the levels of phosphorylated-tau (p-tau). The decrease in p-tau was due in part to an inhibition of the tau kinases GSK-3β and p38 MAPK. In addition, microglial and astrocyte reactivity was inhibited by ATL treatment. Our results suggest that the inability to resolve the immune response during aging might be an important feature that contributes to AD pathology and cognitive deficits. Furthermore, we demonstrate that activation of LXA4 signaling could serve as a potential therapeutic target for AD-related inflammation and cognitive dysfunction.

Alzheimer's disease (AD) is the most prevalent age‐related neurodegenerative disorder, affecting over 35 million people worldwide. Pathologically, AD is characterized by the progressive accumulation of β‐amyloid (Aβ) plaques and neurofibrillary tangles within the brain. Together, these pathologies lead to marked neuronal and synaptic loss and corresponding impairments in cognition. Current treatments, and recent clinical trials, have failed to modify the clinical course of AD; thus, the development of novel and innovative therapies is urgently needed. Over the last decade, the potential use of stem cells to treat cognitive impairment has received growing attention. Specifically, neural stem cell transplantation as a treatment for AD offers a novel approach with tremendous therapeutic potential. We previously reported that intrahippocampal transplantation of murine neural stem cells (mNSCs) can enhance synaptogenesis and improve cognition in 3xTg‐AD mice and the CaM/Tet‐DTA model of hippocampal neuronal loss. These promising findings prompted us to examine a human neural stem cell population, HuCNS‐SC, which has already been clinically tested for other neurodegenerative disorders. In this study, we provide the first evidence that transplantation of research grade HuCNS‐SCs can improve cognition in two complementary models of neurodegeneration. We also demonstrate that HuCNS‐SC cells can migrate and differentiate into immature neurons and glia and significantly increase synaptic and growth‐associated markers in both 3xTg‐AD and CaM/Tet‐DTA mice. Interestingly, improvements in aged 3xTg‐AD mice were not associated with altered Aβ or tau pathology. Rather, our findings suggest that human NSC transplantation improves cognition by enhancing endogenous synaptogenesis. Taken together, our data provide the first preclinical evidence that human NSC transplantation could be a safe and effective therapeutic approach for treating AD. © 2014 The Authors. Hippocampus Published by Wiley Periodicals, Inc.

Frequency of alcohol use indicates the extent to which drinking is integrated into individuals' and groups' day to day social life and has potential public health implications. We used data from 25 longitudinal general population studies, disaggregated by gender and age group, to estimate the magnitude of changes in frequency of alcohol use over time, if any. Meta-analytic results indicated a reasonably consistent increase of about. 08 standard deviation across studies. We assessed the associations of characteristics of individuals (gender, age, birth cohort, initial abstinence rate, initial frequency), social contexts (country, historical period), and studies (sampling frames, measures of frequency, attrition rate) with findings of changes in frequency. Frequency of alcohol use appeared to stabilize by the third or fourth decade of life, with inconsistent rates of change across studies of younger persons. The data suggested simultaneous age, period, and cohort effects on changes in drinking frequency. St...

Tumor hypoxia contributes to chemotherapy and radiotherapy resistance, and helps foster metastasis. Hypoxia likely also supports tumor immune resistance by supporting development of suppressive myeloid and T cell populations by activating immunosuppressive signaling pathways in the tumor and stroma, and by creating a metabolically hostile environment for immune effector cells. TH-302 is a hypoxia-specific chemotherapeutic drug which is activated only in the hypoxic cores of tumors and remains harmless in the periphery. We hypothesized that concurrent combination therapy with TH-302 and T cell checkpoint blockade would promote inside-out tumor destruction with the drug killing at the core, releasing antigen, and diminishing suppression, while the antibodies helped expand and protect the T cells activated as a result. In the TRAMP-C2 prostatic adenocarcinoma model, we found that antibody blockade of CTLA-4 and PD-1, used in conjunction with TH-302 administration, promoted tumor rejection in a significantly larger percentage of mice than either therapy alone. The combination therapy promoted uniquely advantageous ratios of effector T cells to MDSC within the prostate cancer microenvironment. This novel combination of immunotherapy and hypoxia-specific chemotherapy has the potential to offer profound anti-tumor responses to a much greater percentage of patients than either chemotherapy or immunotherapy alone with the same or fewer side effects. Citation Format: Midan Ai, Todd Bartkowiak, Ashvin R. Jaiswal, Krishna Shah, Casey Ager, Beatrisa Lerman, Michael A. Curran. Combination hypoxia-specific chemotherapy and immunotherapy of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 629. doi:10.1158/1538-7445.AM2014-629

Tumor hypoxia predicts poor outcomes across all cancers and has long been recognized as a critical source of resistance to both chemotherapy and radiotherapy. Despite the success of T cell immune checkpoint blockade in treating melanoma, aggressive adenocarcinomas of the prostate and pancreas are largely resistant to CTLA-4 and PD-1 antibody therapy in the mouse and in man. We find that hypoxic zones of these tumors resist infiltration by T cells even in the context of robust infiltration of T cells in normoxic areas of the same tumor (e.g. in the context of T cell checkpoint blockade). Beyond this lack of accessibility to tumor-specific T cells, hypoxia drives the establishment of a highly interdependent network of immunosuppressive stromal cells. Among these, we find myeloid-derived suppressor cells and myofibroblasts to be the critical populations which act together to suppress T cell responses and mediate immunotherapy resistance. Evofosfamide is a hypoxia-specific chemotherapeutic pro-drug which is activated only in the hypoxic cores of tumors and thus can be co-administered with immunotherapy. We find that Evofosfamide-driven disruption of hypoxia zones sensitizes prostate cancer to antibody blockade of CTLA-4 and PD-1 in both transplantable and genetically-engineered murine models of prostate cancer. Co-administration of Evofosfamide and α-CTLA-4/α-PD-1 promotes tumor rejection in a significantly larger percentage of mice than either therapy alone. Mechanistic studies reveals that loss of immune resistance is a consequence of re-oxygenation of hypoxia zones which results in 1) loss of active myeloid suppressor cells, 2) reduced suppressive capacity of new myeloid immigrants, 3) loss of suppressive activation of myofibroblasts, and 4) enhanced infiltration of effector T cells. Therefore, this combination of hypoxia disruption and T cell checkpoint blockade has immense potential to render some of the most therapeutically resistant cancers sensitive to immunotherapy.