Delayed Tumor Progression Research Articles

Monoacylglycerol lipase regulates cannabinoid receptor 2-dependent macrophage activation and cancer progression

Metabolic reprogramming greatly contributes to the regulation of macrophage activation. However, the mechanism of lipid accumulation and the corresponding function in tumor-associated macrophages (TAMs) remain unclear. With primary investigation in colon cancer and confirmation in other cancer models, here we determine that deficiency of monoacylglycerol lipase (MGLL) results in lipid overload in TAMs. Functionally, macrophage MGLL inhibits CB2 cannabinoid receptor-dependent tumor progression in inoculated and genetic cancer models. Mechanistically, MGLL deficiency promotes CB2/TLR4-dependent macrophage activation, which further suppresses the function of tumor-associated CD8+ T cells. Treatment with CB2 antagonists delays tumor progression in inoculated and genetic cancer models. Finally, we verify that expression of macrophage MGLL is decreased in cancer tissues and positively correlated with the survival of cancer patients. Taken together, our findings identify MGLL as a switch for CB2/TLR4-dependent macrophage activation and provide potential targets for cancer therapy.

Abstract 5920: Antitumor efficacy of a dual stromal and tumor targeted oncolytic measles virus in breast and colon cancer models

Abstract Efficient systemic delivery of oncolytic viruses remains an unresolved issue and an obstacle to the success of oncolytic virotherapy. One of the main barriers that lead to decreased oncolytic viral delivery to tumor cells is the tumor stromal component. A potential strategy to circumvent this problem is to use a retargeted oncolytic virus that is able to target, and replicate in tumor stromal cells. The aim of this study is to evaluate the consequences of tumor stromal/vascular targeting by oncolytic measles virus via urokinase receptor (uPAR) as they relate to improved viral delivery and antitumor efficacy. We rescued and characterized a novel dual targeting MV-un-muPA, which binds human cancer cells via CD46 and murine tissues via murine uPAR. This novel virus variant was generated as a tool to investigate the ability of a virus to target tumor stromal cells, especially tumor microvasculature (via murine uPAR) and deliver the infection to tumor cells (via CD46) in vivo. In vitro, MV-un-muPA infected and induced cytotoxicity to human or murine cancer cells in a species specific manner. Efficient MV-un-muPA replication was demonstrated in multiple human and murine cancer cells as well as murine fibroblasts in vitro. MV-un-muPA mediated viral transfer was demonstrated between murine endothelial or murine fibroblast to (RFP expressing) human cancer cells. To further validate the consequences of tumor stromal/vascular targeting by oncolytic measles virus via uPAR as they relate to improved viral delivery, the effects of MV-un-muPA vs a non-targeted MV virus (MV-GFP) were assessed in human breast (MDA-MD231) and colon (HT-29) cancer xenograft models. In these models, tumor cells express human CD46 and the host stroma expresses murine uPAR. Tumor bearing mice were treated with MV-GFP (targeting tumor cells via CD46), dual targeting virus MV-un-muPA (target tumor stroma via murine uPAR, and tumor cells via CD46) by intravenous administration (3 doses). Systemic administration of MV-un-muPA resulted in a significant delay in tumor progression as well as improved survival compared to mice treated with vehicle control or MV-GFP. Tumor based studies showed increased levels of viral RNA, tumor apoptosis, resulted in a significantly reduced stroma and the reduction of cancer associated fibroblasts and endothelial cells, in tumors treated with the dual targeted virus, compared to MV-GFP. In conclusion, our results demonstrate for the first time that dual targeting of tumor stroma and tumor cells by MV-un-m-uPA enhance in vivo viral delivery and oncolytic virus antitumor efficacy in two in vivo models of cancer. This serves as clear proof that vascular/stromal targeting is an effective strategy to enhance viral delivery and antitumor effects and support future studies to further uncover the potential of stromal targeted oncolytic viruses. Citation Format: Yuqi Jing, Valery Chavez, Natasha Karishma Khatwani, Jaime Merchan. Antitumor efficacy of a dual stromal and tumor targeted oncolytic measles virus in breast and colon cancer models [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 5920.

Abstract 1761: Combination of a T cell activating immunotherapy with immune modulators alters the tumor microenvironment and promotes more effective tumor control in preclinical models

Abstract Combinations of immune therapies for cancer treatment will likely improve clinical responses, and a treatment that stimulates a robust T cell response may be a key component of therapy in patients with poorly infiltrated tumors. DPX-Survivac is a T cell activating therapy targeting survivin, formulated in DepoVax™ (DPX), an oil based delivery platform. In clinical studies, the MHC class I peptide antigens in DPX-Survivac induced strong and sustained T cell responses when used in combination with metronomic cyclophosphamide (mCPA) in ovarian cancer patients. Epacadostat is an indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor which has shown to reduce immune suppression in tumors and has demonstrated encouraging results in clinical trials. Using preclinical mouse tumor models, we evaluated the combination of these three immune therapies. C57Bl/6 mice were implanted subcutaneously with murine pancreatic adenocarcinoma (Panc02) cells. Groups of mice were vaccinated with DPX-Survivac vaccine (containing murine H2D peptides) by subcutaneous injection and treated with mCPA (20 mg/kg/day, PO) and epacadostat (6 mg/day, PO). The combination of the three treatments provided a significant delay in tumor progression, and improvement in survival over untreated animals. Similar findings were also observed in the HPV16 E7 expressing C3 tumor model, using an HPV16 minimal peptide epitope (HPV16 E749-57) formulated in DPX. In this model, mice were terminated at defined endpoints to evaluate systemic immune responses in the spleen by IFN-γ ELISPOT and profile tumor infiltration by flow cytometric analysis. Although antigen-specific immune responses in the spleen were not increased by the triple combination in comparison to the DPX-based vaccine, there was a significant impact on several immune subtypes found in the tumor. Notably, antigen-specific CD8+ T cells (as detected by dextramer analysis) were increased and regulatory CD4+CD25+FoxP3+ T cells (Tregs) were decreased. In comparison, the Treg population in the spleen was highest in the triple therapy group (p=0.0046 compared to DPX/mCPA alone). This may indicate a selective exclusion of Tregs from the tumor microenvironment is induced by epacadostat, which can facilitate the anti-tumor immune response mediated by CD8+ T cells induced by DPX. Other immune modulating therapies, such as anti-PD-L1, may further enhance the tumor control induced by this treatment. The combination of DPX-based, T cell activating therapy with epacadostat, a drug that reduced tumor immune suppression is a rational, synergistic combination that is currently being evaluated in advanced ovarian cancer patients in the DeCidE1 clinical trial (NCT0278520). Citation Format: Alecia MacKay, Genevieve Weir, Holly Koblish, Ava Vila- Leahey, Valarmathy Kaliaperumal, Cynthia Tram, Peggy Scherle, Marianne Stanford. Combination of a T cell activating immunotherapy with immune modulators alters the tumor microenvironment and promotes more effective tumor control in preclinical models [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 1761.

PO-418 Targeting heat shock protein 70 (HSP70) in melanoma with functionalized theranostic superparamagnetic iron oxide nanoparticles

IntroductionNanotechnologies based on superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as a promising tool for the theranostics of advanced and metastatic melanoma. Decoration of the nanoparticle surface with specific moieties that have specific anti-tumour immunotherapeutic activity could provide a theranostic potential for the nanocomplexes. Targeting Hsp70 that is presented on the cell membrane of tumour cells but on corresponding normal cells could provide the specificity of melanoma treatment. Combination of functionalized particles with blocking of immune checkpoint inhibitors could further enhance the therapeutic benefit.Material and methodsDeveloped nanoparticles conjugated with granzyme B (GrB-SPIONs) were in vitro assessed (employing confocal and electron microscopies, flow cytometry) for specific targeting of the membrane-bound Hsp70 in B16 melanoma cells. Intratumoral administration of GrB-SPIONs as a monotherapy or in combination with anti-CTLA-4 monoclonal antibodies and adoptive T-cell transfer was performed in the orthotopic model of B16 melanoma in C57/Bl6 mice. IHC sections were performed to assess the intratumoral distribution of nanoparticles as well as the caspase-3 expression in the melanoma following the treatment.Results and discussionsInternalised GrB-SPIONs in Hsp70-positive B16 melanoma cells induced apoptotic death in dose- and time-dependent manner (as shown by flow cytometry). Monotherapy with intratumorally injected GrB-SPIONs in B16 animal model resulted in delayed tumour progression (MRI volumometry). Biodistribution analysis employing highly sensitive magnetic resonanse imaging (7T) confirmed the retention of particles in the tumour (that was further proved by histological studies). Subsequent therapy with anti-CTLA-4 antibodies and adoptive T-cell transfer synergistically improved the efficacy of GrB-SPIONs nanocomplexes. Inhibitory immune checkpoint blockade significantly enhanced the apoptosis in tumour cells as was demonstrated by the caspase-3 IHC analysis.ConclusionThese findings reveal that GrB-SPIONs can specifically target Hsp70-positive melanoma cells. Combinatorial regimen of targeted nanocomplexes and immune checkpoint inhibitors has a high therapeutic potency that could be translated into clinial trials.

PO-411 Combinatorial therapy with immune checkpoint blockade and theranostic tumor-targeted nanoparticles in eradication of tumours

IntroductionTargeted superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as a promising detection tool for the molecular magnetic resonance imaging (MRI). Decoration of the surface with specific moieties that have anti-tumour immunotherapeutic activity could provide a theranostic potential for the nanocomplexes. Combination of functionalized particles with blocking of immune checkpoint inhibitors could further enhance the therapeutic benefit.Material and methodsSynthesised nanoparticles conjugated with granzyme B (GrB-SPIONs) were in vitro assessed (employing confocal and electron microscopies, flow cytometry) for specific targeting of the membrane-bound Hsp70 in cancer cells. Application of GrB-SPIONs as a monotherapy or in combination with single dose (10Gy) radiotherapy (SARRP, X-strahl) was performed in the orthotopic models of human U87 glioblastoma in NMRI nu/nu mice, GL261 glioma in C57/Bl6 mice, C6 glioma in rats. Combinatorial therapy of anti-CTLA-4 and anti-PD-1 anitbodies with GrB-SPIONs and radiotherapy was performed.Results and discussionsInternalised GrB-SPIONs in Hsp70-positive tumour cells induced apoptotic death in dose- and time-dependent manner (as shown by flow cytometry). Monotherapy with GrB-SPIONs in animal models resulted in delayed tumour progression (MRI volumometry) and increased survival. Biodistribution analysis employing highly sensitive non-linear magnetic response measurements (NLR-M2) confirmed the retention of particles in the tumour (that was further proved by histological studies). Addition of radiotherapy further promoted therapeutic benefit of systemically administered nanoparticles. Subsequent therapy with anti-CTLA-4 and anti-PD-1 antibodies synergistically improved the efficacy of GrB-SPIONs nanocomplexes with radiotherapy. Inhibitory immune checkpoint blockade significantly enhanced inflitration of tumour tissue with activated CD8 +and natural killer (NK1.1) cells and decreased suppressive PD-1 +cells.ConclusionThese findings reveal that GrB-SPIONs can specifically target Hsp70-positive cancer cells that could be used simultaneously for nonivasive MRI diagnosis of tumours and therapy. Combinatorial regimen of targeted nanocomplexes and immune checkpoint inhibitors has a high theraputic potency that could be translated into clinial trials.

Abstract 5619: Additional mechanisms of action of SGN-CD48A in multiple myeloma and improved antitumor activity in combination with daratumumab

Abstract SGN-CD48A is a novel multiple myeloma (MM) antibody-drug conjugate (ADC) composed of a CD48-directed antibody conjugated to 8 molecules of a cytotoxic antimitotic drug, monomethyl auristatin E (MMAE), via a next generation β-glucuronidase-cleavable linker. Previously, we showed that SGN-CD48A has potent single agent antitumor activity against MM cells, and produces durable complete remissions in mouse xenograft models. Here, we describe the bystander effect activity and immunogenic cell death (ICD) potential of SGN-CD48A in MM models. In addition, combination studies with approved MM therapeutic antibodies were explored. ADC bystander effect is the additional cytotoxic activity on antigen-negative cells in the presence of antigen-positive tumor cells. To measure bystander effect, we generated CD48 knock-out (KO) myeloma cell lines, which also express luciferase (Luc+). SGN-CD48A demonstrated bystander effect cell killing of the CD48KO-Luc+ cells when co-cultured with parental CD48+ MM cell lines in vitro. Next, a novel imaging-based method for measuring in vivo bystander effect within the mouse bone marrow compartment was developed using surgical intratibial implantation of admixed parental CD48+ and engineered CD48KO-Luc+ MM cells. SGN-CD48A in vivo bystander effect activity was evaluated over time using bioluminescence imaging and ex vivo flow cytometry of bone marrow aspirates. We then measured SGN-CD48A activation of ICD and endoplasmic reticulum (ER) stress signaling markers. SGN-CD48A treatment increased exposure of intracellular ER membrane markers calreticulin and heat-shock protein 70 (HSP70) on the MM cell surface, hallmarks of ICD. Myeloma cells already have high basal levels of ER stress markers due to constitutive production of monoclonal immunoglobulin. However, we observed increased phospho-JNK and a minor elevation of ATF-4 expression following SGN-CD48A treatment. Finally, we tested SGN-CD48A antitumor activity in combination with MM therapeutic antibodies, daratumumab and elotuzumab, in a MOLP8-Luc+ disseminated xenograft mouse model. SGN-CD48A, daratumumab, and elotuzumab showed delay of tumor progression as single agents in this system. However, the combination of SGN-CD48A with daratumumab produced durable complete remissions in all animals. In contrast, combination of SGN-CD48A with elotuzumab did not produce additional antitumor activity. In summary, SGN-CD48A has potential antitumor mechanisms of action beyond direct delivery of cytotoxic MMAE to CD48+ myeloma cells, and preclinical data has identified daratumumab as a plausible combination partner. Citation Format: Devra J. Olson, Bernard A. Liu, Margo Zaval, Anthony Cao, Jesse Gurgel, Julia Cochran, Nicole Stevens, Martha Anderson, Timothy S. Lewis. Additional mechanisms of action of SGN-CD48A in multiple myeloma and improved antitumor activity in combination with daratumumab [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 5619.

Abstract 4936: Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers

Abstract A hallmark of many non-immunogenic cancers is the lack of tumor infiltrating lymphocytes (TIL) and/or failure to mount a robust anti-tumor T cell response via multiple mechanisms. The presence of T regulatory cells and myeloid derived suppressor cells (MDSCs) serve to dampen the immune response, and furthermore, tumor antigen-specific T cell tolerance limits the efficacy of therapeutic cancer vaccines. CD40 signaling is critical to the decision of whether cytotoxic T lymphocytes become primed or tolerized. Administration of monoclonal CD40 agonistic antibody (Ab) has been shown to promote CD8 activation in vivo, and likely alters the myeloid component of the tumor microenvironment. Our study asks the question of whether combining a T cell inducing vaccine and PD1 inhibition with CD40 agonistic Ab can induce T cell priming and TIL activation in non-immunogenic solid malignancies. We utilized mouse models of pancreatic ductal adenocarcinoma (PDAC) and breast cancer to assess the effects of drug combinations on intratumoral immune responses. Tumor-bearing mice were treated with a GM-CSF secreting vaccine (GVAX) + anti-PD1 Ab alone or in combination with CD40 Ab, or isotype control Ab, and monitored for survival. A separate cohort of mice were analyzed by immunohistochemistry and multi-color flow cytometry to assess T cell infiltration/activation and myeloid maturation. In a hemisplenectomy model of PDAC in which tumor cells were surgically implanted into wild-type recipients, mice treated with isotype control Abs succumbed to disease with extensive liver and peritoneal metastases at 35-70 days. GVAX + anti-PD1 Ab treatment displayed some efficacy, although 70% of mice eventually developed fatal liver metastases. In contrast, CD40 Ab was highly active, with 90% long-term survival afforded by a single administration of Ab, and mice treated with GVAX + anti-PD1 Ab + CD40 Ab had 100% survival. Similar trends in treatment efficacy were observed following subcutaneous tumor implantation of PDAC tumor cells in the lower limb. In an orthotopic model in which HER2/neu-expressing breast tumor cells were implanted into the mammary fat pad of syngeneic neu-N mice, we demonstrated delayed tumor progression and increased median survival in mice treated with GVAX + anti-PD1 Ab + CD40 Ab relative to either therapy alone. Further characterization of immune populations was carried out by high dimensional flow cytometric analysis utilizing PhenoGraph clustering and visualized by t-SNE. Changes were observed in monocytic and dendritic cell infiltration and maturation in the tumors of combination-treated mice. A significant decrease in granulocytic MDSCs was associated with response, as well as an increase in mature antigen presenting cells. In conclusion, GVAX, anti-PD1 and CD40 agonist Ab have potential synergy in modulating anti-tumor immunity in non-immunogenic cancers. Citation Format: Hayley S. Ma, Evanthia Roussos Torres, Bibhav Poudel, Tara Robinson, Brian Christmas, Kayla Cruz, Skylar Woolman, Christine Rafie, Blake Scott, Valerie Wall, Todd Armstrong, Elizabeth Jaffee. Combination CD40 agonist and PD-1 antagonist antibody therapy enhances vaccine induced T cell responses in non-immunogenic cancers [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 4936.

PO-425 Combined administration of anti-IL6 and anti-PD-L1 antibodies prevents ketogenic failure, reduces tumour progression, and increases overall survival in an autochthonous murine pancreatic cancer model

IntroductionTumour associated IL-6 downregulates hepatic ketogenesis and thus predisposes to metabolic stress during caloric deficiency. Stress induced glucocorticoids are immune suppressive and can cause failure of cancer immunotherapy. Here, we investigate if this convergence of host metabolism and anti-tumour immunity offers a target for combination therapy with anti-IL-6 and anti-PD-L1 antibodies.Material and methodsCombined anti-IL-6 and anti-PD-L1 antibodies (n=13) or equimolar control antibodies (n=12) were administered to mice bearing autochthonous pancreatic cancer (KPC: KrasLSL.G12D/+; p53R172H/+; Pdx-Cre/+). Mice in both study arms received one day 1 dose of gemcitabine. Groups were matched with regard to tumour location and size, body weight, sex, and age. Tumour volumes were monitored by ultrasound. Body weight and food intake were recorded. Histopathological analyses of the liver and tumour were performed and the biochemical profile of mice was characterised. For statistical analysis, differences between groups were determined using an unpaired two-tailed Student’s t-test and were considered significant if p-value≤0.05. Overall survival (OS) was analysed on an intention-to-treat basis using the Kaplan-Meier method and by applying Log-rank (Mantel-Cox) statistics. The correlation between IL-6-JAK-STAT-3 pathway transcription and overall survival of patients from The Cancer Genome Atlas (TCGA) database was examined.Results and discussionsOverall survival increased significantly in anti-IL-6 and anti-PD-L1 treated mice compared to control mice (OS: 25 days vs. 11 days, p=0.04). Day 7 measurements revealed delayed tumour progression in the combination immunotherapy cohort (volume increase in%: 118+/-5.4 vs. 138+/-5.6, p=0.02). Detailed metabolic analyses are ongoing. The combined analysis of expression from 55 505 transcripts from 9345 patients using GSEA identified an association between IL-6 pathway transcription and shorter OS (p<0.0001). IL-6 expression was statistically significantly associated with shorter OS in non-small cell lung cancer (p=0.036, n=496) and associated with shorter OS in pancreatic cancer (p=0.064, n=178).ConclusionCombination treatment with anti-IL-6 and anti-PD-L1 antibodies significantly increased OS and slowed tumour growth in an autochthonous mouse model of pancreatic cancer. The results of our work provide further justification to explore this treatment combination in patients with cancer associated elevation of IL-6.

Abstract 4712: AT1965, a novel B cell-activating immunotherapy, exerts potent anticancer activity

Abstract Emerging clinical evidence implicate B cells in long term survival in cancer. However, a therapeutic activation of a B cell response against cancer has not been explored. Here we describe the results with AT1965, which exerts a potent anticancer effect via the activation of B cells. AT1965 completely regressed aggressive 4T1 breast tumors established in balb/c mice. Immunophenotyping revealed that IGKC, a B cell marker, was the dominant immune biomarker that is over-expressed following AT1965 treatment. The lack of functional B cells in vivo, via the deletion of the JH gene results in the loss of heavy chain production, blocked this anti-cancer activity of AT1965. Activation of a B cell response is associated with long term memory, which should prevent relapse. To test this, tumor-bearing animals were treated with AT1965 to induce regression; Not a single animal relapsed, i.e. developed the tumor when re-challenged with the tumor thirty days after the primary tumors had regressed. T and B cells are natural partners in an immune response. We therefore tested the combination of a PD1 immune checkpoint inhibitor and AT1965 in an immunocompetent lung cancer model. Only 12.5% of the animals treated with AT1965 or the PD1-inhibitor exhibited a complete regression of lung cancer, while 37.5% exhibited delayed tumor progression. In contrast, 50% of the animals underwent complete tumor regression and 25% exhibited tumor growth inhibition when the two drugs were combined, consistent with the basic immunology premise that both B and T cells work together to mount a complete immune response. Our results indicate that B cell activation could emerge as the next paradigm in cancer immunotherapy. Citation Format: Monideepa Roy, Vineeth Krishna, Aniruddha Sengupta, Sudip Roy, Ashish Kulkarni, Shiladitya Sengupta. AT1965, a novel B cell-activating immunotherapy, exerts potent anticancer activity [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 4712.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

PO-201 Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase

IntroductionThe ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signalling have established SHP2 as an essential player in oncogenic signalling pathways. Recently, novel potent allosteric SHP2-inhibitors have been presented as a viable therapeutic option for RTK-driven cancers, but were shown to be ineffective in KRAS mutant tumour cell lines in vitro.Material and methodsVarious mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC) were employed to investigate the contribution of Ptpn11/SHP2 to carcinogenesis and tumour maintenance, and to determine its utility as a therapeutic target for treatment of established tumours in vivo. CRISPR/Cas9 mediated knockout of PTPN11 in human PDAC cell lines, drug-synergy screening and human PDAC organoid and tissue xenograft model therapeutic trials substantiated the rationale of dual SHP2/MEK inhibition in a human context.Results and discussionsWe report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumours. Genetic deletion of Ptpn11 profoundly inhibited tumour development in mutant KRAS-driven murine models PDAC and NSCLC. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumours delayed tumour progression but was not sufficient to achieve tumour regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumour growth control in murine and human patient-derived organoids and xenograft models of PDAC and NSCLC.ConclusionOur data suggest clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS mutant cancers.

Abstract 5549: Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments

Abstract LTX-315 is an oncolytic peptide that induces immunogenic cancer cell death and is currently evaluated in phase 1 clinical trials for its ability to trigger recruitment of tumor-specific T cells to tumors. Previous experimental studies identified LTX-315's ability to control cancer in mice grafted with various tumor cell lines. Here we extend our analyses to conditional genetic mouse models of both melanoma (driven by Braf and Pten alterations) and soft tissue sarcoma (driven by Kras and P53 alterations). In these genetic models, cancer cells are derived from somatic cells that are transformed in their normal tissue microenvironment and progress to high-grade tumors that are poorly infiltrated by T cells and typically resist prescribed chemo- and immunotherapeutic treatments. We report that LTX-315 not only delays tumor progression substantially in both models, but also profoundly alters the tumor microenvironment, most notably characterized with CD8+ T cell, NK cell and dendritic cell infiltration. Transition from a ‘cold' to a ‘hot' tumor microenvironment was also observed in patients with melanoma, sarcoma and breast cancer. Furthermore, CD8+ T cell depletion in mice abrogated long-term antitumor efficacy of LTX-315, indicating that these cells are required for drug-induced tumor control. Importantly, the ability to convert non-T-cell-infiltrated tumors into ones that display antitumor T cell immunity opens the possibility to prime and make unresponsive tumors sensitive for systemic immune treatments. Citation Format: Mikael J. Pittet, Hsin-Wei Liao, Baldur Sveinbjørnsson, Øystein Rekdal. Antitumor efficacy of the oncolytic peptide LTX-315 in genetic mouse models that resist conventional chemo- and immunotherapeutic treatments [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 5549.

Vectorized Delivery of Alpha-GalactosylCeramide and Tumor Antigen on Filamentous Bacteriophage fd Induces Protective Immunity by Enhancing Tumor-Specific T Cell Response

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels, and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (α-GalCer), a lipid antigen-stimulating invariant natural killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/α-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of α-GalCer and a MHC class I restricted tumor-associated antigenic determinant to antigen-presenting cells via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free α-GalCer and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe, inexpensive, and versatile tool.

Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase.

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers.

Commensal dysbiosis modulates the tumor microenvironment in breast cancer and diminishes efficacy of PD-L1 inhibition

Abstract For patients with HR+ breast cancer, non-responsiveness to PD-1/PD-L1 blockade cannot be attributed to poor immunogenicity, as immune-stimulatory gene signatures are associated with greater long-term survival and enhanced responsiveness to therapy. Targeting myeloid-driven immune suppression has also been shown to enhance efficacy of PD-L1 blockade in non-responsive tumors. However, we find that in HR+ breast cancer, synergy with PD-L1 blockade is negatively influenced by a pre-established loss of commensal homeostasis, or commensal dysbiosis. Using a clinically relevant non-responsive HR+ breast tumor model, establishing commensal dysbiosis prior to breast tumor initiation drives significant and irreversible T cell dysfunction within primary tumors. Additionally, commensal dysbiosis alters the cellular composition within the tumor microenvironment, culminating in the recruitment and polarization of M2-like macrophages with distinct tumor-suppressive attributes. Systemically, tumor-bearing animals with established commensal dysbiosis have significantly higher levels of prostaglandin E2 with the serum, which is independent of tumor volume. However, blockade of COX-2 only synergizes with PD-L1 blockade in animals without commensal dysbiosis. Unexpectedly, we found that commensal dysbiosis enhances COX-1/PGE2 signaling within the mammary epithelium during early tumor progression. Inhibition of COX-1-mediated PGE2 synthesis during early stages of tumor progression resulted in delayed tumor progression only in dysbiotic tumor-bearing mice, suggesting that dysbiosis-induced COX-1 mediated tissue inflammation is sufficient to drive irreversible immune suppression during advanced breast cancer.

IL-36γ promotes a local immune response via the formation of tertiary lymphoid structures in colorectal carcinoma.

Abstract Tertiary lymphoid structures (TLS) form at sites of chronic inflammation, and locally prime the immune system independently of secondary lymphoid organs. The intratumoral presence of TLS has been associated with a positive prognostic outcome for patients with solid tumors, including colorectal carcinoma. Thus, the therapeutic induction of TLS would be hypothesized to confer clinical benefit to patients. Indeed, the therapeutic introduction of the IL-1 family cytokine IL-36 gamma into the tumor microenvironment (TME) of murine MC38 colon carcinoma leads to delayed tumor progression and the development of TLS at 5 days post-treatment. The TLS present with characteristics of secondary lymphoid organs, including PNAd+ high endothelial venules (HEV) surrounded by CD3+ T cells and CD11c+ dendritic cells, but are considered as “non-classical” due to the absence of a defined B cell zone. The formation of TLS is dependent upon the adaptive immune response, as IL-36 gamma-treated tumors grown in Rag2−/− animals fail to present with PNAd+ vasculature within the tumor. Thus, crosstalk between the immune system and cells of the vasculature is key to the efficacy of the IL-36 gamma-induced anti-tumor immune response. In human colorectal carcinoma, IL-36 gamma was also observed to promote the local anti-tumor immune response. Expression of IL-36 gamma by vascular endothelial cells of HEV was associated with an increased density of CD20+ B cells in TLS. Specific IL-36 gamma production by CD68+ macrophages was associated with an increased infiltration of CD4+ central memory T cells into the TME. Therefore, IL-36 gamma is a pro-inflammatory mediator involved in the anti-tumor immune response in both murine and human colorectal cancer.

Generating suppression-resistant natural killer cells as an enhanced immunotherapeutic for neuroblastoma

Abstract Refractory neuroblastoma (NB) has a poor prognosis and there is a need to explore novel therapies. Natural killer (NK) cells serve as a promising immunotherapy for NB, however they are impaired by the immunosuppressive TGFb-rich NB tumor microenvironment. Critically, TGFβ impairs NK cell proliferation and cytolytic activity. To overcome these detrimental effects, we genetically modified NK cells to express a variant TGFβ receptor – composed of a truncated extracellular TGFbRII domain fused to a synthetic Notch-like receptor coupled to RELA – which may be able to associate with exogenous TGFβ and convert downstream signals to enhance NK cell function. NK cells were isolated from umbilical cord blood (CB; universal donor source) and expanded ex vivo. The modified TGFβ receptor was cloned into an SFG vector backbone. Expanded CB-derived NK cells were transduced with the modified TGFβ receptor (42.5±5.5%). Transduction did not negatively affect the endogenous expression of the activating receptors NKG2D, NKp44, or NKp30 (p&amp;gt;0.05) or NK cell proliferative capacity (p&amp;gt;0.05). Non-transduced (NT) NK cells were cytotoxic against K562 target cells in a dose-dependent manner (45.2±4.69% killing at 40:1), however NK cell cytolytic activity was impaired following exposure to TGFβ (p&amp;lt;0.05). In contrast, transduced-NK cells exhibited dose-dependent cytotoxicity (40.2±4.42% killing at 40:1), which was unaffected following exposure to TGFβ (p&amp;gt;0.05). Administration of transduced-NK cells in a xenograft model of human NB delayed tumor progression by &amp;gt;14 days and significantly prolonged survival compared to control NK cells (p=0.04). In summary, this novel immunotherapeutic may translate as a potent treatment modality for patients with NB.

Nuclear receptor NR2F6 inhibition potentiates responses to PD-L1/PD-1 cancer immune checkpoint blockade

Analyzing mouse tumor models in vivo, human T cells ex vivo, and human lung cancer samples, we provide direct evidence that NR2F6 acts as an immune checkpoint. Genetic ablation of Nr2f6, particularly in combination with established cancer immune checkpoint blockade, efficiently delays tumor progression and improves survival in experimental mouse models. The target genes deregulated in intratumoral T lymphocytes upon genetic ablation of Nr2f6 alone or together with PD-L1 blockade reveal multiple advantageous transcriptional alterations. Acute Nr2f6 silencing in both mouse and human T cells induces hyper-responsiveness that establishes a non-redundant T-cell-inhibitory function of NR2F6. NR2F6 protein expression in T-cell-infiltrating human NSCLC is upregulated in 54% of the cases (n = 303) and significantly correlates with PD-1 and CTLA-4 expression. Our data define NR2F6 as an intracellular immune checkpoint that suppresses adaptive anti-cancer immune responses and set the stage for clinical validation of targeting NR2F6 for next-generation immuno-oncological regimens.

Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti–PD-1 treatment

In a large proportion of cancer patients, CD8 T cells are excluded from the vicinity of cancer cells. The inability of CD8 T cells to reach tumor cells is considered an important mechanism of resistance to cancer immunotherapy. We show that, in human lung squamous-cell carcinomas, exclusion of CD8 T cells from tumor islets is correlated with a poor clinical outcome and with a low lymphocyte motility, as assessed by dynamic imaging on fresh tumor slices. In the tumor stroma, macrophages mediate lymphocyte trapping by forming long-lasting interactions with CD8 T cells. Using a mouse tumor model with well-defined stromal and tumor cell areas, macrophages were depleted with PLX3397, an inhibitor of colony-stimulating factor-1 receptor (CSF-1R). Our results reveal that a CSF-1R blockade enhances CD8 T cell migration and infiltration into tumor islets. Although this treatment alone has minor effects on tumor growth, its combination with anti-PD-1 therapy further increases the accumulation of CD8 T cells in close contact with malignant cells and delays tumor progression. These data suggest that the reduction of macrophage-mediated T cell exclusion increases tumor surveillance by CD8 T cells and renders tumors more responsive to anti-PD-1 treatment.

Multimodal selenium nanoshell-capped Au@mSiO2 nanoplatform for NIR-responsive chemo-photothermal therapy against metastatic breast cancer

Multimodal therapeutic agents based on novel nanomaterials for multidrug resistance have attracted increasing attention in cancer therapy. In this study, we describe the construction of a programmed mesoporous silica-capped gold nanorod covered with nano-selenium overcoat (Se@Au@mSiO2) nanoparticles as a multifunctional nanoplatform to incorporate materials with specific chemotherapeutic, chemoprevention, and photoablation/hyperthermia functions that collectively contribute to enhance anticancer efficacy in multidrug-resistant breast cancer. The triple-combination-based nanosized Se@Au@mSiO2/DOX effectively accumulates in the tumor and the release of the therapeutic cargo could be remotely manipulated by mild near-infrared (NIR) irradiation. Se@Au@mSiO2/DOX notably enhances the cell killing effect through induction of cell apoptosis. In addition, Se@Au@mSiO2/DOX inhibits tumor cell growth through cell cycle arrest and induction of apoptosis via suppression of the Src/FAK/AKT signaling pathways. Synergistic Se-photothermal-chemotherapy combination exhibits significant tumor growth suppression and delayed tumor progression in vivo. Immunohistochemistry analysis shows elevated numbers of caspase-3 and PARP-immunolabeled cells and decreased Ki-67 + and CD31 + cancer cells in the tumor mass. No noticeable signs of organ damage or toxicity are observed after treatment with Se@Au@mSiO2/DOX (NIR+), which is further supported by hematology and biochemical analyses. Thus, Se@Au@mSiO2/DOX has potential for the clinical treatment of metastatic breast cancers with little or no adverse effects.