APR-246 increases tumor antigenicity independent of p53.

We previously reported that activation of p53 by APR-246 reprograms tumor-associated macrophages to overcome immune checkpoint blockade resistance. Here, we demonstrate that APR-246 and its active moiety, methylene quinuclidinone (MQ) can enhance the immunogenicity of tumor cells directly. MQ treatment of murine B16F10 melanoma cells promoted activation of melanoma-specific CD8+ T cells and increased the efficacy of a tumor cell vaccine using MQ-treated cells even when the B16F10 cells lacked p53. We then designed a novel combination of APR-246 with the TLR-4 agonist, monophosphoryl lipid A, and a CD40 agonist to further enhance these immunogenic effects and demonstrated a significant antitumor response. We propose that the immunogenic effect of MQ can be linked to its thiol-reactive alkylating ability as we observed similar immunogenic effects with the broad-spectrum cysteine-reactive compound, iodoacetamide. Our results thus indicate that combination of APR-246 with immunomodulatory agents may elicit effective antitumor immune response irrespective of the tumor's p53 mutation status.

Increased p53 expression induced by APR-246 reprograms tumor-associated macrophages to augment immune checkpoint blockade.

In addition to playing a major role in tumor cell biology, p53 generates a microenvironment that promotes antitumor immune surveillance via tumor-associated macrophages. We examined whether increasing p53 signaling in the tumor microenvironment influences antitumor T cell immunity. Our findings indicate that increased p53 signaling induced either pharmacologically with APR-246 (eprenetapopt) or in p53-overexpressing transgenic mice can disinhibit antitumor T cell immunity and augment the efficacy of immune checkpoint blockade. We demonstrated that increased p53 expression in tumor-associated macrophages induces canonical p53-associated functions such as senescence and activation of a p53-dependent senescence-associated secretory phenotype. This was linked with decreased expression of proteins associated with M2 polarization by tumor-associated macrophages. Our preclinical data led to the development of a clinical trial in patients with solid tumors combining APR-246 with pembrolizumab. Biospecimens from select patients participating in this ongoing trial showed that there was a suppression of M2-polarized myeloid cells and increase in T cell proliferation with therapy in those who responded to the therapy. Our findings, based on both genetic and a small molecule-based pharmacological approach, suggest that increasing p53 expression in tumor-associated macrophages reprograms the tumor microenvironment to augment the response to immune checkpoint blockade.

Isoform specific anti-TGFß therapy enhances antitumor efficacy in mouse models of cancer.

TGFß is a potential target in cancer treatment due to its dual role in tumorigenesis and homeostasis. However, the expression of TGFß and its inhibition within the tumor microenvironment has mainly been investigated in stroma-heavy tumors. Using B16 mouse melanoma and CT26 colon carcinoma as models of stroma-poor tumors, we demonstrate that myeloid/dendritic cells are the main sources of TGFß1 and TGFß3. Depending on local expression of TGFß isoforms, isoform specific inhibition of either TGFß1 or TGFß3 may be effective. The TGFß signature of CT26 colon carcinoma is defined by TGFß1 and TGFß1 inhibition results in tumor delay; B16 melanoma has equal expression of both isoforms and inhibition of either TGFß1 or TGFß3 controls tumor growth. Using T cell functional assays, we show that the mechanism of tumor delay is through and dependent on enhanced CD8+ T cell function. To overcome the local immunosuppressive environment, we found that combining TGFß inhibition with immune checkpoint blockade results in improved tumor control. Our data suggest that TGFß inhibition in stroma poor tumors shifts the local immune environment to favor tumor suppression.

Targeting Phosphatidylserine Enhances the Anti-tumor Response to Tumor-Directed Radiation Therapy in a Preclinical Model of Melanoma.

Phosphatidylserine (PS) is exposed on the surface of apoptotic cells and is known to promote immunosuppressive signals in the tumor microenvironment (TME). Antibodies that block PS interaction with its receptors have been shown to repolarize the TME into a proinflammatory state. Radiation therapy (RT) is an effective focal treatment of isolated solid tumors but is less effective at controlling metastatic cancers. We found that tumor-directed RT caused an increase in expression of PS on the surface of viable immune infiltrates in mouse B16 melanoma. We hypothesize that PS expression on immune cells may provide negative feedback to immune cells in the TME. Treatment with an antibody that targets PS (mch1N11) enhanced the anti-tumor efficacy of tumor-directed RT and improved overall survival. This combination led to an increase in proinflammatory tumor-associated macrophages. The addition of anti-PD-1 to RT and mch1N11 led to even greater anti-tumor efficacy and overall survival. We found increased PS expression on several immune subsets in the blood of patients with metastatic melanoma after receiving tumor-directed RT. These findings highlight the potential of combining PS targeting with RT and PD-1 pathway blockade to improve outcomes in patients with advanced-stage cancers.

Blockade of the AHR restricts a Treg-macrophage suppressive axis induced by L-Kynurenine.

Tryptophan catabolism by the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase 2 (IDO/TDO) promotes immunosuppression across different cancer types. The tryptophan metabolite L-Kynurenine (Kyn) interacts with the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) to drive the generation of Tregs and tolerogenic myeloid cells and PD-1 up-regulation in CD8+ T cells. Here, we show that the AHR pathway is selectively active in IDO/TDO-overexpressing tumors and is associated with resistance to immune checkpoint inhibitors. We demonstrate that IDO-Kyn-AHR-mediated immunosuppression depends on an interplay between Tregs and tumor-associated macrophages, which can be reversed by AHR inhibition. Selective AHR blockade delays progression in IDO/TDO-overexpressing tumors, and its efficacy is improved in combination with PD-1 blockade. Our findings suggest that blocking the AHR pathway in IDO/TDO expressing tumors would overcome the limitation of single IDO or TDO targeting agents and constitutes a personalized approach to immunotherapy, particularly in combination with immune checkpoint inhibitors.

iNOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiotherapy, chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated fibroblasts (CAF) are abundant in PDAC tumors, the effects of radiotherapy on CAFs and the response of PDAC cells to radiotherapy are unknown. Using patient samples and orthotopic PDAC biological models, we showed that radiotherapy increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in radiotherapy-activated tumor cells, iNOS expression and nitric oxide (NO) secretion were significantly increased in CAFs secretome following radiotherapy. Culture of PDAC cells with conditioned media from radiotherapy-activated CAFs increased iNOS/NO signaling in tumor cells through NF-κB, which, in turn, elevated the release of inflammatory cytokines by the tumor cells. Increased NO after radiotherapy in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP). In murine orthotopic PDAC models, pancreatic tumor growth was delayed when iNOS inhibition was combined with radiotherapy. These data show the important role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors. SIGNIFICANCE: A radiolabeled pH-targeted peptide can be used as a PET imaging tool to assess therapy response within PDAC and blocking iNOS/NO signaling may improve radiotherapy outcomes.

In situ vaccination with defined factors overcomes T cell exhaustion in distant tumors.

Irreversible T cell exhaustion limits the efficacy of programmed cell death 1 (PD-1) blockade. We observed that dual CD40-TLR4 stimulation within a single tumor restored PD-1 sensitivity and that this regimen triggered a systemic tumor-specific CD8+ T cell response. This approach effectively treated established tumors in diverse syngeneic cancer models, and the systemic effect was dependent on the injected tumor, indicating that treated tumors were converted into necessary components of this therapy. Strikingly, this approach was associated with the absence of exhausted PD-1hi T cells in treated and distant tumors, while sparing the intervening draining lymph node and spleen. Furthermore, patients with transcription changes like those induced by this therapy experienced improved progression-free survival with anti-PD-1 treatment. Dual CD40-TLR4 activation within a single tumor is thus an approach for overcoming resistance to PD-1 blockade that is unique in its ability to cause the loss of exhausted T cells within tumors while sparing nonmalignant tissues.

Targeted APC Activation in Cancer Immunotherapy to Enhance the Abscopal Effect.

In oncology, the "abscopal effect" refers to the therapeutic effect on a distant tumor resulting from the treatment of local tumor (e. g., ablation, injection, or radiation). Typically associated with radiation, the abscopal effect is thought to be mediated by a systemic antitumor immune response that is induced by two concurrent changes at the treated tumor: (1) the release of tumor antigens and (2) the exposure of damage-associated molecular patterns. Therapies that produce these changes are associated with immunogenic cell death (ICD). Some interventions have been shown to cause an abscopal effect without inducing the release of tumor antigens, suggesting that release of tumor antigens at baseline plays a significant role in mediating the abscopal effect. With tumor antigens already present, therapies that target activation of APCs alone may be sufficient to enhance the abscopal effect. Here, we discuss two therapies targeted at APC activation, TLR9 and CD40 agonists, and their use in the clinic to enhance the abscopal effect.

High IL-1R8 expression in breast tumors promotes tumor growth and contributes to impaired antitumor immunity.

Tumors develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. The identification of mechanisms leading to local immune dysregulation is critical to improve cancer therapy. We here demonstrate that Interleukin-1 receptor 8 (IL-1R8 - previously known as SIGIRR/TIR8), a negative regulator of Toll-Like and Interleukin-1 Receptor family signaling, is up-regulated during breast epithelial cell transformation and in primary breast tumors. IL-1R8 expression in transformed breast epithelial cells reduced IL-1-dependent NF-κB activation and production of pro-inflammatory cytokines, inhibited NK cell activation and favored M2-like macrophage polarization. In a murine breast cancer model (MMTV-neu), IL-1R8-deficiency reduced tumor growth and metastasis and was associated with increased mobilization and activation of immune cells, such as NK cells and CD8+ T cells. Finally, immune-gene signature analysis in clinical specimens revealed that high IL-1R8 expression is associated with impaired innate immune sensing and T-cell exclusion from the tumor microenvironment. Our results indicate that high IL-1R8 expression acts as a novel immunomodulatory mechanism leading to dysregulated immunity with important implications for breast cancer immunotherapy.

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Comprehensive cancer-gene panels can be used to estimate mutational load and predict clinical benefit to PD-1 blockade in clinical practice.

Cancer gene panels (CGPs) are already used in clinical practice to match tumor's genetic profile with available targeted therapies. We aimed to determine if CGPs could also be applied to estimate tumor mutational load and predict clinical benefit to PD-1 and CTLA-4 checkpoint blockade therapy. Whole-exome sequencing (WES) mutation data obtained from melanoma and non-small cell lung cancer (NSCLC) patients published by Snyder et al. 2014 and Rizvi et al. 2015, respectively, were used to select nonsynonymous somatic mutations occurring in genes included in the Foundation Medicine Panel (FM-CGP) and in our own Institutional Panel (HSL-CGP). CGP-mutational load was calculated for each patient using both panels and was associated with clinical outcomes as defined and reported in the original articles. Higher CGP-mutational load was observed in NSCLC patients presenting durable clinical benefit (DCB) to PD-1 blockade (FM-CGP P=0.03, HSL-CGP P=0.01). We also observed that 69% of patients with high CGP-mutational load experienced DCB to PD-1 blockade, as compared to 20% of patients with low CGP-mutational load (FM-CGP and HSL-CGP P=0.01). Noteworthy, predictive accuracy of CGP-mutational load for DCB was not statistically different from that estimated by WES sequencing (P=0.73). Moreover, a high CGP-mutational load was significantly associated with progression-free survival (PFS) in patients treated with PD-1 blockade (FM-CGP P=0.005, HR 0.27, 95% IC 0.105 to 0.669; HSL-CGP P=0.008, HR 0.29, 95% IC 0.116 to 0.719). Similar associations between CGP-mutational load and clinical benefit to CTLA-4 blockade were not observed. In summary, our data reveals that CGPs can be used to estimate mutational load and to predict clinical benefit to PD-1 blockade, with similar accuracy to that reported using WES.

Investigação do papel de SIGIRR/IL-1R8 no crosstalk entre células tumorais e o infiltrado leucocitário / Investigating the role of SIGIRR/IL-1R8 in the crosstalk between tumor cells and the immune system

Células tumorais desenvolvem diversas estratégias para escapar da identificação e eliminação pelo sistema imune. Dessa forma, a investigação dos mecanismos envolvidos na comunicação celular no microambiente tumoral e na desregulação local do sistema imune é crítica para uma melhor compreensão da progressão da doença e para o desenvolvimento de alternativas terapêuticas mais eficazes. Nós aqui demonstramos que SIGIRR/IL-1R8, um importante regulador negativo de receptores de Interleucina-1 (ILRs) e receptores do tipo Toll (TLRs), apresenta expressão aumentada em uma linhagem celular epitelial mamária transformada pela superexpressão do oncogene HER2 e em tumores primários de mama, e promove o crescimento tumoral e metástase através da modulação da inflamação associada ao câncer e da atenuação da resposta imune antitumoral. Observamos que IL-1R8 tem sua expressão correlacionada com HER2 em tecidos mamários e sua alta expressão é fator de pior prognóstico em câncer de mama de baixo grau. Notavelmente, níveis aumentados de IL-1R8 foram observados especialmente nos subtipos HER2+ e Luminais de tumores de mama, e sua expressão aumentada em células epiteliais de mama transformadas por HER2 diminui a ativação da via de NF-κB e a expressão de diferentes citocinas pro-inflamatórias (IL-6, IL-8, TNF, CSF2, CSF3 e IFN-ß1). Meio condicionado de células transformadas por HER2, mas não de variantes celulares com o gene IL-1R8 silenciado, induz a polarização de macrófagos para o fenótipo M2 e inibe a ativação de células NK. Em um modelo murino transgênico de tumorigênese espontânea mediada por HER2, MMTV-neu, verificamos que a deficiência de IL-1R8 (IL-1R8-/-neu) retardou o aparecimento de tumores e reduziu a incidência, a carga tumoral e a disseminação metastática. Contudo, não foram observadas diferenças significativas no crescimento tumoral quando animais IL-1R8-/-neu receberam medula óssea de animais IL-1R8+/+, confirmando um papel importante da expressão de IL-1R8 em células não hematopoiéticas na tumorigênese da mama. Tumores IL-1R8+/+neu apresentaram maiores níveis de citocinas pró-inflamatórias como IL-1ß e VEGF, e menores níveis da citocina imunomodulatória IFN-γ. Além disso, tumores que expressavam IL-1R8 apresentaram menor infiltrado de células NK maduras, células dendríticas (DCs) e linfócitos T-CD8+ e um maior infiltrado de macrófagos M2 e linfócitos T-CD4+. Coletivamente, esses resultados indicam que a expressão de IL-1R8 em tumores de mama pode representar um novo mecanismo de escape da resposta imune e suportam IL-1R8 como potencial alvo terapêutico

Tumor cells develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. Identification of new players that regulate the cellular crosstalk within the tumor microenvironment and promote local immune dysregulation is critical to understand disease progression and to improve therapeutic strategies. Here, we demonstrate that SIGIRR/IL-1R8, a negative regulator of IL-1R and TLRs, is up-regulated in a HER2-transformed epithelial mammary cell line and in primary breast tumors and promotes tumor growth and metastasis by modulating cancer-related inflammation and impairing anti-tumor immunity. IL-1R8 expression is correlated with HER2 in mammary tissue, and higher tumor IL-1R8 expression is a poor prognostic factor in lower grade breast tumors. Notably, higher levels of IL-1R8 expression were observed in HER2+ and Luminal breast tumor subtypes and IL-1R8 up-regulation in HER2-transformed mammary epithelial cells inhibited NF-κB activation and the expression of pro-inflammatory cytokines (IL-6, IL-8, TNFα, CSF2, CSF3, IFN-ß1). Conditioned medium from HER2-transformed cells, but not from IL-1R8 knockdown variants, induced M2-macrophage polarization and inhibited natural-killer (NK) cell activation. IL-1R8 deficiency in a transgenic mouse model of breast tumorigenesis (MMTV-neu) significantly delayed tumor onset and reduced tumor incidence, burden and metastasis. No significant differences in tumor growth were observed when IL-1R8-/-neu mice were transplanted with bone marrow from IL-1R8+/+ animals, confirming an important role for IL-1R8 expression in non-hematopoietic cells during breast tumorigenesis. IL-1R8+/+neu mammary tumors presented higher levels of pro-inflammatory cytokines such as IL-1ß and VEGF, but lower levels of IFN-γ. Besides, a lower infiltrate of mature NK cells, dendritic cells (DCs) and CD8+ T cells but higher infiltrate of M2-macrophages and CD4+ T cells were present in IL-1R8 expressing tumors. Collectively, our results support IL-1R8 expression as a novel tumor immune escape mechanism in breast cancer and putative target for immunotherapy

Differential macrophage activation alters the expression profile of NTPDase and ecto-5'-nucleotidase.

Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-5'-nucleotidase/CD73 (ecto-5'-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6-8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-5'-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set.

Homocysteine decreases extracellular nucleotide hydrolysis in rat platelets.

Hyperhomocysteinemia is an independent risk factor for atherothrombotic disease. Platelets play an important role in cardiovascular disease and release pro-aggregates mediators when activated, such as ADP, a physiological agonist involved in normal hemostasis and thrombosis. NTPDases and 5'-nucleotidase are ecto-enzymes that hydrolyze ATP, ADP and AMP to adenosine playing an important role on blood flow and thrombogenesis by regulating ADP catabolism. The aim of the present study was evaluate extracellular adenine nucleotide hydrolysis of rat platelets exposed to homocysteine in vitro and in vivo. In vitro homocysteine (Hcy) in the concentration range of 20 to 500 microM caused a significant decrease on ATP (around 30%) and ADP (around 45%) hydrolysis, respectively, while AMP hydrolysis was not altered. Hcy was not able to inhibit the hydrolysis of ATP and ADP catalyzed by purified apyrase at the same concentrations tested in vitro on platelets, suggesting an indirect effect. The inhibitory effect of Hcy on platelets was prevented by antioxidants agents in vitro and in vivo. Furthermore homocysteine treatment increased platelet aggregation induced by ADP. Based on the results presented herein, we propose that inhibition of extracellular ATP and ADP hydrolysis caused by homocysteine was probably due oxidative stress, since antioxidants prevented such effects. These findings may contribute to an increase platelet response to ADP and consequence development of thrombotic risk attributed to hyperhomocysteinemia.