Growth Of Established Tumors Research Articles

Targeting Autophagy in Cancer: Recent Advances and Future Directions

Autophagy, a multistep lysosomal degradation pathway that supports nutrient recycling and metabolic adaptation, has been implicated as a process that regulates cancer. Although autophagy induction may limit the development of tumors, evidence in mouse models demonstrates that autophagy inhibition can limit the growth of established tumors and improve response to cancer therapeutics. Certain cancer genotypes may be especially prone to autophagy inhibition. Different strategies for autophagy modulation may be needed depending on the cancer context. Here, we review new advances in the molecular control of autophagy, the role of selective autophagy in cancer, and the role of autophagy within the tumor microenvironment and tumor immunity. We also highlight clinical efforts to repurpose lysosomal inhibitors, such as hydroxychloroquine, as anticancer agents that block autophagy, as well as the development of more potent and specific autophagy inhibitors for cancer treatment, and review future directions for autophagy research. SIGNIFICANCE: Autophagy plays a complex role in cancer, but autophagy inhibition may be an effective therapeutic strategy in advanced cancer. A deeper understanding of autophagy within the tumor microenvironment has enabled the development of novel inhibitors and clinical trial strategies. Challenges and opportunities remain to identify patients most likely to benefit from this approach.

1011 - THE ROLE OF MUTANT P53 IN TUMOR DEVELOPMENT AND THE RESPONSE OF MALIGNANT CELLS TO ANTI-CANCER THERAPEUTICS

p53 is a highly potent tumour suppressor that acts through activation of several biological processes, including apoptotic cell death, cell growth arrest and cell senescence. In accordance with its critical role in tumour suppression, the p53 gene is the most frequently mutated gene in human cancers. Mutant p53 cancers are usually relatively resistant to chemotherapeutic drugs and such patients therefore often have a poor prognosis. Despite several decades of research into the functions of wild-type (wt) p53 in normal cells and mutant p53 in tumour cells, we still do not have a clear understanding of how mutations in p53 contribute to the development and sustained growth of cancers. There is evidence to support the notion that mutant p53 acts only to blocks wt p53 tumour suppressor functions (dominant negative effect; DNE) but there are also reports that mutant p53 has de novogain-of-function properties that additionally promote tumour growth. Of critical importance is the question of whether mutant p53 contributes to the sustained growth of established tumours, thereby validating whether abrogating its expression would be a promising therapeutic approach. Clarification of this point has profound implications for the future development and clinical success of cancer drugs that aim to directly target and degrade mutant p53. We have investigated the role of mutant p53 in aggressive MYC-driven lymphomas, focusing on how it functions during tumour development and also the response to conventional and targeted cancer therapeutics.

Oral administration of lipoteichoic acid from Lactobacillus rhamnosus GG overcomes UVB‐induced immunosuppression and impairs skin tumor growth in mice

There is increasing evidence of the relevant connection and regulation between the gut and skin immune axis. In fact, oral administration of lipoteichoic acid (LTA) from Lactobacillus rhamnosus GG (LGG) prevents the development of UV-induced skin tumors in chronically exposed mice. Here we aim to evaluate whether this LTA is able to revert UV-induced immunosuppression as a mechanism involved in its anti-tumor effect and whether it has an immunotherapeutic effect against cutaneous squamous cell carcinoma. Using a mouse model of contact hypersensitivity, we demonstrate that LTA overcomes UV-induced skin immunosuppression. This effect was in part achieved by modulating the phenotype of lymph node resident dendritic cells (DC) and the homing of skin migratory DC. Importantly, oral LTA reduced significantly the growth of established skin tumors once UV radiation was discontinued, demonstrating that it has a therapeutic, besides the already demonstrated preventive antitumor effect. The data presented here strongly indicates that oral administration of LTA represents a promising immunotherapeutic approach for different conditions in which the skin immune system is compromised.

Abstract 14: A new orally available drug for the treatment of metastatic cancers

Abstract Metastatic cancers account for the great majority of cancer deaths. We conducted a screen to indetify known drugs that selectively inhibit the viability of metastatic tumor cells. Results of the screen identified members of the FDA-approved benzimidazole methylcarbamate family (e.g. mebendazole (MBZ) and albendazole (ALB)) as potential therapies for metastatic disease. Earlier work also supports a role for this chemical family in the potential treatment of activity in multiple cancers, but progress has been stalled by their poor water solubility and poor bioavailability for systemic delivery to disseminated tumors. We therefore synthesized a novel compound (OBD9) containing the scaffold of MBZ coupled to an oxetane group to enhance aqueous solubility to 361 μM. OBD9 demonstrates significant cytotoxicity toward a variety of cancer cell types including colon, lung, and prostate cancers (IC50: 0.9-2 μM). In a mouse xenograft model of human A549 lung cancer cell line OBD9 dramatically inhibited the growth of established tumors at 30 or 90 μM without noticeable toxicity. In a mouse xenograft model using highly aggressive PCMLN3 prostate cancer cells, oral administration of OBD9 at 30 mg/kg also significantly repressed growth of established tumors with no visible toxicity. We have studied the working mechanisms behind OBD9 and found that the Wnt signaling pathway is suppressed by OBD9 treatment. Both qPCR and Western blot data suggest that the beta-catenin/TCF complex in the Wnt signaling pathway are inhibited by OBD9. Among the downstream targets of Wnt signaling pathway, the oncogene cMyc is also significantly decreased in different cancers including colon, lung and prostate cancer. Overall, our in vitro and in vivo data suggest that OBD9 potentially represents a novel therapeutic option for multiple cancers especially metastatic cancer through targeting Wnt signaling pathway and the downstream oncogene cMyc. Citation Format: Kun Zhou, Jae Eun Cheong, Michaela Zaffagni, Yingjie Xu, Lijun Sun, Bruce Zetter. A new orally available drug for the treatment of metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 14.

Abstract 3220: Enhancing breast cancer immunotherapy via IONP-mediated photothermal therapy

Abstract Despite immunotherapy by harnessing host’s own immune system to fight against cancer has elicited promising durable responses in metastatic melanoma and lung cancer patients, its efficacy in treating breast cancer remains to be low. In this work, we demonstrate that sequential photothermal therapy (PTT), mediated with systemically administered stealthy iron oxide nanoparticles (IONPs), can significantly enhance checkpoint blockade-based immunotherapy for breast cancer. Our data suggests that the combination therapy when combining IONP-mediated PTT with anti-CTLA-4 therapy results in a synergistic antitumor effect to completely inhibit established 4T1 murine breast tumor growth in BALB/c mice, while control treatments, including the antibody alone or IONP-mediated PTT alone, fail to do so. Our data further reveal that the enhanced antitumor effect may be attributed by the effective regulation of the suppressive tumor immunity by IONP-mediated PTT. Our flow cytometry data suggests that CD4+FoxP3+ regulatory T-cells (Tregs) are effectively depleted via IONP-mediated PTT, with a significant reduction of the Treg percentage from 42.2 ± 15.8% (control) to 11.9 ± 6.5% (p < 0.05). Our data further suggest that IONP-mediated PTT significantly reduces suppressor-cell-attractive cytokine granulocyte-colony stimulating factor (G-CSF) secretion, which is the driving force for Treg recruitment. Upon effective regulation of suppressive tumor immunity, the proportion of CD8+ T-cells in tumor tissues is significantly increased from 5.6 ± 2.1% (control) to 37.5 ± 6.9% (p < 0.001) following IONP-mediated PTT. Interestingly, our data suggest that Treg elimination and CD8+ T-cell activation can only be achieved through sequential PTT, not a single dose of PTT, suggesting that the suppressive contribution from the newly responding immune cells following initial antitumor treatment, and the sequential PTT may provide an effective way to not only deplete primary resident suppressive cells, but also newly responding ones. Our data further suggests that the combination therapy against primary tumors elicits a systemic immune rejection of cancer cells at distal sites, while this dramatic immune response is lost when CD8+ T-cells are depleted using anti-CD8 antibody, suggesting the immune rejection triggered by IONP-mediated PTT is through CD8+ T-cell-mediated immune responses. Our data also suggests that the majority of the combination treatment-cured mice can reject re-challenged 4T1 cancer cells, indicating memory T-cell immune surveillance. In conclusion, this study may provide an effective and translational nanomedicine-based strategy to effectively regulate suppressive tumor immunity and turn “cold” breast tumors into “hot”, thereby to significantly enhance checkpoint blockade-based breast cancer immunotherapy. Citation Format: Hongwei Chen, Xin Luan, Hayley Paholak, Joseph Burnett, Nicholas Stevers, Kanokwan Sansanaphongpricha, Miao He, Alfred Chang, Qiao Li, Duxin Sun. Enhancing breast cancer immunotherapy via IONP-mediated photothermal therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3220.

Therapeutic targeting of polyamines in malignant pleural mesothelioma xenograft models.

e20060 Background: Although the use of asbestos has been restricted, the incidence of malignant pleural mesothelioma (MPM) is still rising. The US FDA approved a combination of pemetrexed with cisplatin for treatment of unresectable MPM. And development of novel adjuvant therapeutic options for resected early-stage disease is also urgently needed. From our preliminary data, ornithine decarboxylase (ODC) is highly expressed in MPM xenografts and clinical tumor samples. Upregulation of ODC increases polyamines production and enhances tumor growth. a-difluoromethylornithine (DFMO) is a specific ODC inhibitor. This study aims to disclose the adjuvant (minimal residual disease setting) and therapeutic (metastatic setting) effects of DFMO in MPM xenografts. Methods: In adjuvant therapy setting, nude mice were fed with DFMO in drinking water 7 days before subcutaneous inoculation of 200,000 tumor cells. In therapeutic setting, 107 corresponding cells were injected subcutaneously into nude mice which were randomized for treatment after established tumor growth. Mice with tumor size > 600mm3 were considered reaching humane endpoint. Spermidine levels, protein expression, cytokines concentration, and apoptosis were investigated by Dot plot, Western blot, ELISA, and TUNEL assay respectively. Results: In adjuvant therapy setting, DFMO suppressed tumor growth and increased median survival in both 211H and H226 xenografts. In H226 xenografts, 43% of treated mice have not yet reached humane endpoint, mimicking long-term survival. Upon DFMO treatment, decrease in spermidine level, increase in nitrotyrosine content, and activation of apoptosis were observed in both xenografts. In addition, increase in nitrosocysteine level, intratumoral IL-6, keratinocyte chemoattractant and TNFα, DNA lesions and inhibition of Akt/mTOR pathway were induced by DFMO in H226 xenografts, which may explain higher potency of DFMO in these xenografts. In therapeutic setting, DFMO also suppressed tumor growth in both xenografts with similar mechanisms. Conclusions: DFMO may have a potential role as adjuvant therapy in MPM especially epithelioid mesothelioma.

Intravenous delivery of the toll-like receptor 7 agonist SC1 confers tumor control by inducing a CD8+ T cell response

ABSTRACTTLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application.Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8+ T cells and CD11b+ conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased.We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8+ T cells, which we found to be strongly enriched in the tumors of SC1-treated mice.In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound.

Regulation of Tumor-Associated Myeloid Cell Activity by CBP/EP300 Bromodomain Modulation of H3K27 Acetylation

Myeloid-derived suppressor cells (MDSCs) are found in most cancer malignancies and support tumorigenesis by suppressing immunity and promoting tumor growth. Here we identify the bromodomain (BRD) of CBP/EP300 as a critical regulator of H3K27 acetylation (H3K27ac) in MDSCs across promoters and enhancers of pro-tumorigenic target genes. In preclinical tumor models, invivo administration of a CBP/EP300-BRD inhibitor (CBP/EP300-BRDi) alters intratumoral MDSCs and attenuates established tumor growth in immunocompetent tumor-bearing mice, as well as in MDSC-dependent xenograft models. Inhibition of CBP/EP300-BRD redirects tumor-associated MDSCs from a suppressive to an inflammatory phenotype through downregulation of STAT pathway-related genes and inhibition of Arg1 and iNOS. Similarly, CBP/EP300-BRDi decreases differentiation and suppressive function of human MDSCs invitro. Our findings uncover a role of CBP/EP300-BRD in intratumoral MDSCs that may be targeted therapeutically to boost anti-tumor immunity.

Suppression of Myeloid Cell Arginase Activity leads to Therapeutic Response in a NSCLC Mouse Model by Activating Anti-Tumor Immunity

BackgroundTumor orchestrated metabolic changes in the microenvironment limit generation of anti-tumor immune responses. Availability of arginine, a semi-essential amino acid, is critical for lymphocyte proliferation and function. Levels of arginine are regulated by the enzymes arginase 1,2 and nitric oxide synthase (NOS). However, the role of arginase activity in lung tumor maintenance has not been investigated in clinically relevant orthotopic tumor models.MethodsRNA sequencing (RNA-seq) of sorted cell populations from mouse lung adenocarcinomas derived from immunocompetent genetically engineered mouse models (GEMM)s was performed. To complement mouse studies, a patient tissue microarray consisting of 150 lung adenocarcinomas, 103 squamous tumors, and 54 matched normal tissue were stained for arginase, CD3, and CD66b by multiplex immunohistochemistry. Efficacy of a novel arginase inhibitor compound 9 in reversing arginase mediated T cell suppression was determined in splenocyte ex vivo assays. Additionally, the anti-tumor activity of this compound was determined in vitro and in an autochthonous immunocompetent KrasG12D GEMM of lung adenocarcinoma model.ResultsAnalysis of RNA-seq of sorted myeloid cells suggested that arginase expression is elevated in myeloid cells in the tumor as compared to the normal lung tissue. Accordingly, in the patient samples arginase 1 expression was mainly localized in the granulocytic myeloid cells and significantly elevated in both lung adenocarcinoma and squamous tumors as compared to the controls. Our ex vivo analysis demonstrated that myeloid derived suppressor cell (MDSC)s cause T cell suppression by arginine depletion, and suppression of arginase activity by a novel ARG1/2 inhibitor, compound 9, led to restoration of T cell function by increasing arginine. Treatment of KrasG12D GEMM of lung cancer model with compound 9 led to a significant tumor regression associated with increased T cell numbers and function, while it had no activity across several murine and human non-small cell (NSCLC) lung cancer lines in vitro.ConclusionsWe show that arginase expression is elevated in mouse and patient lung tumors. In a KRASG12D GEMM arginase inhibition diminished growth of established tumors. Our data suggest arginase as an immunomodulatory target that should further be investigated in lung tumors with high arginase activity.

Antagonism of IAPs Enhances CAR T-cell Efficacy.

Chimeric antigen receptor (CAR) T-cell therapy has proven successful in the treatment of hematological malignancies, notably acute lymphoblastic leukemia and B-cell lymphoma. However, the efficacy of CAR T cells against solid tumors is poor, likely due to tumor-associated immunosuppression. Here, we demonstrated that antagonizing the "inhibitor of apoptosis proteins" with the clinical smac-mimetic, birinapant, significantly enhanced the antitumor activity of CAR T cells in a tumor necrosis factor (TNF)-dependent manner. Enhanced tumor cell death occurred independently of the perforin-mediated granule exocytosis pathway, underscoring the cytotoxic potential of CAR T-cell-derived TNF. Combining CAR T-cell therapy with birinapant significantly reduced established tumor growth in vivo, where either therapy alone was relatively ineffective. Using patient biopsy-derived tumoroids, we demonstrated the synergistic potential of combining CAR T-cell therapy with smac-mimetics. Taken together, we identified CAR T-cell-derived TNF as a potent antitumor effector, which can be further harnessed by smac-mimetics.

Inhibitors of ribosome biogenesis repress the growth of MYCN-amplified neuroblastoma

Abnormal increases in nucleolar size and number caused by dysregulation of ribosome biogenesis has emerged as a hallmark in the majority of spontaneous cancers. The observed ribosome hyperactivity can be directly induced by the MYC transcription factors controlling the expression of RNA and protein components of the ribosome. Neuroblastoma, a highly malignant childhood tumor of the sympathetic nervous system, is frequently characterized by MYCN gene amplification and high expression of MYCN and c-MYC signature genes. Here, we show a strong correlation between high-risk disease, MYCN expression, poor survival, and ribosome biogenesis in neuroblastoma patients. Treatment of neuroblastoma cells with quarfloxin or CX-5461, two small molecule inhibitors of RNA polymerase I, suppressed MycN expression, induced DNA damage, and activated p53 followed by cell cycle arrest or apoptosis. CX-5461 repressed the growth of established MYCN-amplified neuroblastoma xenograft tumors in nude mice. These findings suggest that inhibition of ribosome biogenesis represent new therapeutic opportunities for children with high-risk neuroblastomas expressing high levels of Myc.

MMP-9 inhibition promotes anti-tumor immunity through disruption of biochemical and physical barriers to T-cell trafficking to tumors.

Matrix metalloproteinase-9 (MMP-9), whose expression is frequently dysregulated in cancer, promotes tumor growth, invasion, and metastasis by multiple mechanisms, including extracellular matrix remodeling and growth-factor and cytokine activation. We developed a monoclonal antibody against murine MMP-9, which we found decreased growth of established primary tumors in an orthotopic model of HER2-driven breast cancer (HC11-NeuT) in immunocompetent mice. RNA sequencing (RNAseq) profiling of NeuT tumors and additional mouse model tumors revealed that anti-MMP-9 treatment resulted in upregulation of immune signature pathways associated with cytotoxic T-cell response. As there is a need to boost the low response rates observed with anti-PDL1 antibody treatment in the clinical setting, we assessed the potential of anti-MMP-9 to improve T-cell response to immune checkpoint inhibitor anti-PDL1 in NeuT tumors. Anti-MMP-9 and anti-PDL1 cotreatment reduced T-cell receptor (TCR) clonality and increased TCR diversity, as detected by TCR sequencing of NeuT tumors. Flow cytometry analyses of tumors showed that the combination treatment increased the frequency of CD3+ T cells, including memory/effector CD4 and CD8 T cells, but not regulatory T cells, among tumor-infiltrating leukocytes. Moreover, in vitro enzymatic assays corroborated that MMP-9 cleaves key T-cell chemoattractant CXC receptor 3 ligands (CXC ligand [CXCL] 9, CXCL10, and CXCL11) and renders them inactive in T-cell migration assays. Consistent with our in vitro experiments, analysis of NeuT tumor protein lysates showed that anti-MMP-9 treatment increases expression of CXCL10 and other T cell–stimulating factors, such as interleukin (IL)-12p70 and IL-18. We show that inhibition of MMP-9, a key component of the tumor-promoting and immune-suppressive myeloid inflammatory milieu, increases T-helper cell 1 type cytokines, trafficking of effector/memory T cells into tumors, and intratumoral T-cell diversity.

Amphiphilic nanoparticle delivery enhances the anticancer efficacy of a TLR7 ligand via local immune activation

Although immunotherapy shows great promise for the long-term control of cancer, many tumors still fail to respond to treatment. To improve the outcome, the delivery of immunostimulants to the lymph nodes draining the tumor, where the antitumor immune response is initiated, is key. Efforts to use nanoparticles as carriers for cancer immunotherapy have generally required targeting agents and chemical modification of the drug, and have unfortunately resulted in low delivery and therapeutic efficiency. Here, we report on the efficacy of gold nanoparticles with approximately 5 nm hydrodynamic diameter coated with a mixture of 1-octanethiol and 11-mercaptoundecanesulfonic acid for the delivery of an immunostimulatory TLR7 ligand to tumor-draining lymph nodes. The drug was loaded without modification through nonspecific adsorption into the ligand shell of the nanoparticles, taking advantage of their amphiphilic nature. After loading, nanoparticles retained their stability in solution without significant premature release of the drug, and the drug cargo was immunologically active. Upon subcutaneous injection into tumor-bearing mice, the drug-loaded particles were rapidly transported to the tumor-draining lymph nodes. There, they induced a local immune activation and fostered a cytotoxic T-cell response that was specific for the tumor. Importantly, the particle-delivered TLR7 ligand blocked the growth of large established tumors and significantly prolonged survival compared to the free form of the drug. Thus, we demonstrate for the first time that nanoparticle delivery of a TLR7 immunostimulant to the tumor-draining lymph nodes enhances antitumor immunity and improves the outcome of cancer immunotherapy.

Targeting CCR8 Induces Protective Antitumor Immunity and Enhances Vaccine-Induced Responses in Colon Cancer.

CCR8 is a chemokine receptor expressed principally on regulatory T cells (Treg) and is known to be critical for CCR8+ Treg-mediated immunosuppression. Recent studies have demonstrated that CCR8 is uniquely upregulated in human tumor-resident Tregs of patients with breast, colon, and lung cancer when compared with normal tissue-resident Tregs. Therefore, CCR8+ tumor-resident Tregs are rational targets for cancer immunotherapy. Here, we demonstrate that mAb therapy targeting CCR8 significantly suppresses tumor growth and improves long-term survival in colorectal tumor mouse models. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4+ and CD8+ T cells, and a significant decrease in the frequency of tumor-resident CD4+CCR8+ Tregs. Tumor-specific CD8+ T cells displayed lower expression of exhaustion markers as well as increased functionality upon restimulation. Treatment with anti-CCR8 mAb prevented de novo induction and suppressive function of Tregs without affecting CD8+ T cells. Initial studies explored a combinatorial regimen using anti-CCR8 mAb therapy and a Listeria monocytogenes-based immunotherapy. Anti-CCR8 mAb therapy synergized with L. monocytogenes-based immunotherapy to significantly delay growth of established tumors and to prolong survival. Collectively, these findings identify CCR8 as a promising new target for tumor immunotherapy and provide a strong rationale for further development of this approach, either as a monotherapy or in combination with other immunotherapies.Significance: Inhibition of CCR8 represents a promising new cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity and prolong patient survival. Cancer Res; 78(18); 5340-8. ©2018 AACR.

Serial MRI Imaging Reveals Minimal Impact of Ketogenic Diet on Established Liver Tumor Growth.

Rodent models of liver tumorigenesis have reproducibly shown that dietary sugar intake is a powerful driver of liver tumor initiation and growth. In contrast, dietary sugar restriction with ketogenic diets or calorie restriction generally prevents liver tumor formation. Ketogenic diet is viewed positively as a therapeutic adjuvant; however, most ketogenic diet studies described to date have been performed in prevention mode rather than treatment mode. Therefore, it remains unclear whether a ketogenic diet can be administered in late stages of disease to stall or reverse liver tumor growth. To model the clinically relevant treatment mode, we administered a ketogenic diet to mice after liver tumor initiation and monitored tumor growth by magnetic resonance imaging (MRI). Male C57BL/6 mice were injected with diethylnitrosamine (DEN) at 2 weeks of age and fed a chow diet until 39 weeks of age, when they underwent MRI imaging to detect liver tumors. Mice were then randomised into two groups and fed either a chow diet or switched to a ketogenic diet from 40–48 weeks of age. Serial MRIs were performed at 44 and 48 weeks of age. All mice had tumors at study completion and there were no differences in total tumor burden between diet groups. Although a ketogenic diet has marked protective effects against DEN-induced liver tumourigenesis in this mouse model, these data demonstrate that ketogenic diet cannot stop the progression of established liver tumors.

The targeting of endoglin on vascular endothelial cells affects the infiltration of M2 macrophages into the breast cancer microenvironment by modulating the interleukin-6 (IL-6) level

Background: The effect and underlying mechanisms of endoglin on angiogenesis and immunity during tumour progression were investigated. Methods: Differences in the growth of established EO771 breast tumours between endoglin-knockout (Eng-iKO e ) and control mice were evaluated. The tumour tissues were harvested on the day 7th and 14th after engraftment, at which times the growth differences between the groups were significant. The expression of markers of angiogenesis (CD31), endoglin (CD105), tumour associated macrophages (TAMs) (F4/80) and M2 macrophages (CD206) and of interleukin-6 (IL-6), IL-10, and IL-6/Janus kinase 2 (JAK2)/signal transducer and activators of transcription 3 (STAT3) proteins in tumour tissues of Eng-iKO e and control mice were evaluated. Results: We found that tumour angiogenesis and growth were both inhibited in the Eng-iKO e group relative to the control group on day 7, whereas, no significant between-group difference was observed on day 14. Moreover, in breast cancer tissues of the Eng-iKO e group, the numbers of M2 macrophages were significantly decreased on day 7 and increased on day 14, and the expression of IL-6 and IL-10 were lower on day 7 and higher on day 14. Positively strong correlations were found between the IL-6 level and the number of M2 macrophages both on days 7 (P=0.017, r=0.80) and 14 (P e group compared with the control group, which was in accordance with the changes of M2 macrophage numbers. Conclusions: The effect of endoglin-targeted anti-angiogenic therapy was weakened by affecting the infiltration of M2 macrophages in breast cancer through modulation of the IL-6 level. This indicated that IL-6 could not only predict the efficacy after anti-angiogenic therapies, but also acts as a potential target to improve efficacy of anti-angiogenic therapies.

Therapeutic vaccination with 4–1BB co-stimulation eradicates mouse acute myeloid leukemia

ABSTRACTImmunomodulatory therapies can effectively control haematological malignancies. Previously we reported the effectiveness of combination immunotherapies that centre on 4-1BB-targeted co-stimulation of CD8 + T cells, particularly when simultaneously harnessing the immune adjuvant properties of Natural Killer T (NKT) cells. The objective of this study was to assess the effectiveness of agonistic anti-4-1BB antibody-based combination therapy against two aggressive forms of acute myeloid leukemia (AML).Anti-4-1BB treatment alone resulted in transient suppression of established AML-ETO9a tumor growth in 50% of mice, however the majority of these mice subsequently succumbed to disease. Combining alpha-galactosylceramide (α-GalCer)-loaded tumor cell vaccination with anti-4-1BB antibody treatment increased the proportion of responding mice to 100%, and protection led to long-term, tumor-free survival, demonstrating complete eradication of AML. This finding was extended to established mixed lymphocytic leukemia (MLL)-AF9 tumors, whereby vaccine plus anti-4-1BB combination similarly resulted in 100% protection. The addition of anti-PD-1 to anti-4-1BB treatment, although improving survival outcomes compared to anti-4-1BB alone, was not as effective as NKT cell vaccination.The effectiveness of 4-1BB combination therapies was dependent on IFN-γ signaling within host cells, but not tumors. Vaccine plus anti-4-1BB therapy elicited potent generation of functional effector and memory CD8 + T cells in all tumor-associated organs. Therapy induced KLRG1+ effector CD8 T cells were the most effective at controlling disease. We show that combining NKT cell-targeting vaccination with anti-4-1BB provides excellent therapeutic responses against AML and MLL in mice, and these results will guide ongoing efforts in finding immunotherapeutic solutions against acute myeloid leukemias.

Abstract 3773: RORγ agonist enhances antitumor effects of agonist anti-OX40 antibody

Abstract Combination immunotherapy strategies are being developed to improve the clinical outcome in cancer patients. T-cell costimulation through OX40 has been shown to promote expansion and proliferation of effector T-cells leading to enhanced effector functions, memory generation and immune inflammatory antitumor responses. However, treatment with anti-OX40 as a single agent has not led to major positive clinical outcomes. Therefore, anti-OX40 is an ideal candidate for combination immunotherapy. In preclinical models, we have recently shown that combining anti-PD-1 concurrently with anti-OX40 negates the effects of agonist anti-OX40, making identification of combination partners crucial for antitumor therapy. RORγt, a master transcription factor, is known to drive Type 17 T-cell differentiation. Synthetic, small-molecule RORγ agonists have been shown to enhance Type 17 T-cell effector functions and survival, decrease immune suppressive mechanisms and modulate expression of a number of costimulatory and coinhibitory molecules. We hypothesized that combining RORγ agonist could enhance the antitumor effects of anti-OX40. Using a murine model where vaccine is used to prime the immune system, we assessed the effects of anti-OX40 antibody combined with a RORγ agonist (LYC-54143) on growth of established tumors and survival. We found that the combined treatment resulted in a significantly delayed tumor growth and prolonged mice survival. Analysis of the tumor microenvironment revealed that combination therapy significantly increased the numbers of total CD4+ T-cells including RORγt+ and highly activated INFγ+ cells, decreasing Treg numbers. Moreover, we found that RORγ agonist resulted in an increase in the numbers of total, antigen-specific, granzyme B+, and IFNγ+ CD8+ T-cells in the TME. These results show that inducing Th17 cells within the TME produces better anti-OX40 effect, suggesting that agonist anti-OX40 and RORγ agonist treatment is a novel potent combination for cancer immunotherapy. Citation Format: Pankaj Gaur, Vivek Verma, Rahul Nandre, Pooja Vir, Hua Wang, Baolin Kang, Laura Carter, Xiao Hu, Xikui Liu, Seema Gupta, Samir Khleif. RORγ agonist enhances antitumor effects of agonist anti-OX40 antibody [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 3773.

Abstract 2623: CHP-IL12 combined with surgery suppresses tumor metastasis and prolongs survival

Abstract Interleukin 12 (IL-12) is the most promising cytokine for treating tumors by itself or arming immunogenic cells but its toxicity and moderate response limit its clinical use. To avoid or minimize the toxicity issue and boost the anti-tumor immune response, our lab has discovered a comprehensive tumor-targeting carcinoma homing peptide (CHP). Fusion of this peptide encoding DNA sequence to IL12 encoding sequence in frame yields the proprietary product—CHP-IL12 (US patent 9,657,077 B2), or tumor-targeting IL12, which increased the accumulation of IL12 in tumor environment and decreased liver toxicity compared to wild-type IL12 (wt-IL12) gene therapy. Here, we report the therapeutic efficacy of this CHP-IL12 on metastasis and to comprehend the underlying mechanism description between CHP-IL12 and wildtype (wt)-IL12 for boosting anti-tumor response, two orthotopic and spontaneous tumor metastasis models (one adult and one pediatric tumor) were utilized. CHP-IL12 gene therapy showed greater efficacy in controlling growth of established tumors compared to wt-IL12. CHP-IL12 gene therapy, in combination with surgery, reduced number of metastatic nodules in lungs, livers and bones. CHP-IL12, compared to wt-IL12, conferred a potential immunostimulatory effect involving 1) reduction of T-reg cells, myeloid-derived suppressor cells (MDSC) and tumor associated macrophages (TAM); 2) recruitment of activated CD8+ T cells to the tumor. Moreover, CHP-IL12 increased IL12, IFNγ level in tumor environment and decreased IL12, IFNγ level in serum, which is the major cause of toxicity. Furthermore, in combination with surgery, CHP-IL12 gene therapy led to long-term survival and enhanced immunological memory development of tumor bearing mice compared to wt-IL12 gene therapy. These results collectively indicate that CHP-IL12 fusion gene therapy coupled with surgery has a powerful potency in inhibition of tumor metastasis and prolonging survival and a low potency in inducing toxicity. Therefore, CHP-IL12 fusion gene therapy in combination with surgery may translate into clinic as a promising anti-tumor strategy for both adult and pediatric tumors. Citation Format: Qingnan Zhao, Jiemiao Hu, Jeffry J. Cutrera, Abhisek Mitra, Xueqing Xia, Shulin Li. CHP-IL12 combined with surgery suppresses tumor metastasis and prolongs survival [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 2623.

Abstract 1122: MST4 phosphorylation of ATG4B regulates autophagic activity, tumorigenicity, and radio resistance in glioblastoma

Abstract Autophagy-mediated intracellular catabolism sustains the rapid growth of established tumors and tumors in response to multiple stresses including genotoxic/cytotoxic therapies. Here we identify and validate ATG4B, a key regulator that stimulates autophagic process by promoting autophagosome through reversible modification of ATG8 as a novel substrate of mammalian sterile20-like kinase (STK) 26/MST4 that is less known for its function in cellular process (none in autophagy) and unknown authentic substrates in cancer. We show that MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities suppresses autophagic process and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM tumor xenografts markedly slows tumor growth and provides significant survival benefit to animal subjects. This study not only describes a novel regulatory mechanism by which the MST4-ATG4B axis accelerates autophagic process, regulates GBM tumorigenicity, and responses to radiotherapy (RT), but also explores imminent clinical utility of combination of ATG4B inhibition with RT to suppress orthotopic GBM tumor xenografts. This study should prove generalizable to other types of cancer and have positive impacts in advancing our knowledge of cancer biology and designing new cancer treatments. Citation Format: Tianzhi Huang, Chung Kwon Kim, Angel A. Alvarez, Rajendra P Pangeni, Xuechao Wan, xiao song, Taiping shi, Yongyong Yongyong, Namratha Sastry, Craig Horbinski, Songjian Lu, Roger Stupp, John Kessler, Ryo Nishikawa, Ichiro Nakano, Erik Sulman, Xinghua Lu, Charles David James, Xiao-Ming Yin, Bo Hu, Shi-Yuan Cheng. MST4 phosphorylation of ATG4B regulates autophagic activity, tumorigenicity, and radio resistance in glioblastoma [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 1122.