Intra-Tumoral Nerve-Tracing in a Novel Syngeneic Model of High-Grade Serous Ovarian Carcinoma

Dense tumor innervation is associated with enhanced cancer progression and poor prognosis. We observed innervation in breast, prostate, pancreatic, lung, liver, ovarian, and colon cancers. Defining innervation in high-grade serous ovarian carcinoma (HGSOC) was a focus since sensory innervation was observed whereas the normal tissue contains predominantly sympathetic input. The origin, specific nerve type, and the mechanisms promoting innervation and driving nerve-cancer cell communications in ovarian cancer remain largely unknown. The technique of neuro-tracing enhances the study of tumor innervation by offering a means for identification and mapping of nerve sources that may directly and indirectly affect the tumor microenvironment. Here, we establish a murine model of HGSOC and utilize image-guided microinjections of retrograde neuro-tracer to label tumor-infiltrating peripheral neurons, mapping their source and circuitry. We show that regional sensory neurons innervate HGSOC tumors. Interestingly, the axons within the tumor trace back to local dorsal root ganglia as well as jugular–nodose ganglia. Further manipulations of these tumor projecting neurons may define the neuronal contributions in tumor growth, invasion, metastasis, and responses to therapeutics.

600 BR101801 stimulates anti-tumor immunity and enhances the efficacy of radiation in a syngeneic model

BackgroundBR101801 is an inhibitor of PI3K γ/δ and DNA-PK. It has received clinical approval from the U.S. FDA as an anticancer drug candidate, and phase 1a/1b is ongoing in the U.S. and South Korea. According to the prior studies PI3K γ/δ inhibition exhibits anticancer immune effects by changing the tumor microenvironment [1]. In addition, ionizing radiation (IR) activates the immune response by causing the destroyed cells to act as antigens [2]. Therefore, the combination of BR101801 and IR can induce cancer cell death and amplify anticancer immune effects. This study aims to demonstrate efficacy of the BR101801 as a potent cancer immunotherapy.MethodsThe enzymatic potency of PI3K isotype and DNA-PK was analyzed by Eurofins. The effects of BR101801 on cell viability were evaluated in 4T1 (breast cancer) and CT-26 (colon cancer) cells for 72 h using WST-8 assay. For in vivo studies, the tumor (4T1 or CT-26)-bearing syngeneic mice were treated with BR101801. To evaluate the synergistic effect, CT-26 tumor-bearing syngeneic mice were treated with vehicle, BR101801, IR (2 Gy or 7.5 Gy), and BR101801 + IR. Immune cells from the spleen or tumor were quantified by flow cytometry.ResultsIn vitro selectivity and target potency of BR101801 on different PI3K isotypes and DNA-PK were studied in a cell-free system. The biochemical IC50 values of BR101801 for PI3K -γ, -δ, and DNA-PK were 15 nM, 2 nM, and 6 nM, respectively. In vitro 50% of maximal inhibition of cell proliferation (GI50) in 4T1 and CT26 cell lines were both above 10 μM. In 4T1 and CT-26 syngeneic models, BR101801 showed the highest tumor inhibitor efficacy (Figure 1). In particular, regulatory T cells (Tregs) & Myeloid derived suppressor cells (MDSC) were decreased and CD8+ T cells were increased in the spleens isolated from the tumor-bearing mice. Compared with other PI3K inhibitors, BR101801 had the highest efficacy, confirming that it changes the immune microenvironment. Moreover, BR101801 was synergistic in combination with 2 Gy or 7.5 Gy of IR in the syngeneic model. Notably, Tregs & Macrophage2 were decreased and CD8+ T cells were increased in the tumor tissue, confirming that the anticancer efficacy.Abstract 600 Figure 1Synergistic effect with ionizing radiation In VivoThe combination of BR101801 and ionising radiation showed synergistic effects in the CT-26 Syngeneic model. BR101801 increases anti-cancer immune cells, CD8 + T cells, and decreases immune suppressor cells Tregs and macrophages through a combination of radiation, resulting in immuno-cancer effects.ConclusionsBR101801 demonstrated an anticancer immune effect by changing the tumor microenvironment and showed synergistic effects with radiation combination therapy. We will confirm the anticancer immunity effect in ongoing clinical trials.ReferencesOkkenhaug K, Graupera M, Vanhaesebroeck B. Targeting PI3K in Cancer: Impact on Tumor Cells, Their Protective Stroma, Angiogenesis, and Immunotherapy. Cancer Discov. 2016; 10: 1090–1105.McKelvey K, Hudson A, Back M, Eade T, Diakos C. Radiation, inflammation and the immune response in cancer. Mammalian Genome. 2018;9:843–865Ethics ApprovalThe protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional animal Car and Use Committee (IACUC) of BoRyung Pharm. prior to conduct.[Approval number:BR18130]

848 Novel small molecule HPK1 inhibitor PCC-1 induces strong anti-tumor activity

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1), is a negative regulator of T and B cell receptor signaling.1 2 3 A strong anti-tumor immunogenic response and tumor rejection was observed in mice with HPK1 gene knocked out.3 Treatment of HPK1 kinase dead mice with immune check-point blockers (ICBs) demonstrated enhanced tumor growth inhibition.3 Hence, HPK1 is an attractive therapeutic strategy for immuno-oncology based treatment in cancers. In comparison to our previous HPK1 small molecule inhibitor, PCC,4 we present here a differentiated novel HPK1 inhibitor, PCC-1 with good anti-T cell kinases selectivity and stronger anti-tumor efficacy in CT26 tumor model. In addition, using the syngeneic model of MC38 expressing human PD-L1, we present for the first time, the combination efficacy of a HPK1 inhibitor with the clinical ICB, Atezolizumab.MethodsIntuitive medicinal chemistry complemented by structure-based drug design was used to identify & develop potent inhibitors of HPK1 with optimal kinase selectivity, PK and in vivo efficacy profile. The SAR efforts were guided by biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo target engagement and pharmacodynamic data was generated using CT26 and MC38-hPD-L1 tumor models.ResultsPCC-1 has sub-nanomolar HPK1 inhibition potency and strong target engagement resulting in pSLP76 inhibition, enhanced anti-tumor cytokine production of IL-2 and/or IFNgamma in Jurkat cells, human PBMCs and human whole blood. PCC-1 also demonstrated nanomolar potency in inducing a complete reversal of PGE2 or adenosine mediated immunosuppression. Oral dosing of PCC-1 as a single agent, induced strong tumor growth inhibition (TGI) in the syngeneic model of CT26 and MC38-hPD-L1 tumor models. Combination of PCC-1 with anti-CTLA4 in CT26 tumor model induced significantly greater TGI than anti-CTLA4 alone. Moreover, as a first, the combination of PCC-1 with clinical ICB, Atezolizumab in MC38-hPD-L1 induced enhanced rejection of tumors. These results strongly suggest PCC-1 as a promising candidate for HPK1 inhibition and as a combination partner with ICBs in clinic.ConclusionsPCC-1 is a novel, orally active HPK1 inhibitor that demonstrates excellent stand-alone efficacy and enhances current immunotherapy efficacy. Further evaluation of PCC-1 is ongoing to advance towards clinic.AcknowledgementsWe thank Dnyaneshwar Dahale, Sanjay Patale, Sandip Patil, Vidya Kattige, Jiju Mani, Namrata Singh, Ekta Kashyap, Sandeep Thorat, Pankaj Jain and Pramod Sagar for their contributions to the projectTrial RegistrationN/AReferencesKiefer F, et al. The EMBO Journal 1996.Hu, et al. Genes and Development 1996.Sawasdikosol, Burakoff. eLife 2020;9:e55122.Sachin S Chaudhari, et al. Poster#1709, AACR Annual Meeting April-May 2021.Ethics ApprovalThe studies involving animals have obtained ethics approval from Institutional Animal Ethics Committee (IAEC), The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India, GRC/IAEC/472/2020-1. Participants of the studies have given informed consent before taking part.

Phenotypic Switching of B16F10 Melanoma Cells as a Stress Adaptation Response to Fe3O4/Salicylic Acid Nanoparticle Therapy

Melanoma is a melanocyte-derived skin cancer that has a high heterogeneity due to its phenotypic plasticity, a trait that may explain its ability to survive in the case of physical or molecular aggression and to develop resistance to therapy. Therefore, the therapy modulation of phenotypic switching in combination with other treatment modalities could become a common approach in any future therapeutic strategy. In this paper, we used the syngeneic model of B16F10 melanoma implanted in C57BL/6 mice to evaluate the phenotypic changes in melanoma induced by therapy with iron oxide nanoparticles functionalized with salicylic acid (SaIONs). The results of this study showed that the oral administration of the SaIONs aqueous dispersion was followed by phenotypic switching to highly pigmented cells in B16F10 melanoma through a cytotoxicity-induced cell selection mechanism. The hyperpigmentation of melanoma cells by the intra- or extracellular accumulation of melanic pigment deposits was another consequence of the SaIONs therapy. Additional studies are needed to assess the reversibility of SaIONs-induced phenotypic switching and the impact of tumor hyperpigmentation on B16F10 melanoma's progression and metastasis abilities.

Comparing syngeneic and autochthonous models of breast cancer to identify tumor immune components that correlate with response to immunotherapy in breast cancer

Background The heterogeneity of the breast tumor microenvironment (TME) may contribute to the lack of durable responses to immune checkpoint blockade (ICB); however, mouse models to test this are currently lacking. Proper selection and use of preclinical models are necessary for rigorous, preclinical studies to rapidly move laboratory findings into the clinic. Methods Three versions of a common syngeneic model derived from the MMTV-PyMT autochthonous model were generated by inoculating 1E6, 1E5, or 1E4 cells derived from the MMTV-PyMT mouse into wildtype recipient mice. To elucidate how tumor latency and TME heterogeneity contribute to ICB resistance, comprehensive characterization of the TME using quantitative flow-cytometry and RNA expression analysis (NanoString) was performed. Subsequently, response to ICB was tested. These procedures were repeated using the EMT6 breast cancer model. Results The 3 syngeneic versions of the MMTV-PyMT model had vastly different TMEs that correlated to ICB response. The number of cells used to generate syngeneic tumors significantly influenced tumor latency, infiltrating leukocyte populations, and response to ICB. These results were confirmed using the EMT6 breast cancer model. Compared to the MMTV-PyMT autochthonous model, all 3 MMTV-PyMT syngeneic models had significantly more tumor-infiltrating lymphocytes (TILs; CD3+, CD4+, and CD8+) and higher proportions of PD-L1-positive myeloid cells, whereas the MMTV-PyMT autochthonous model had the highest frequency of myeloid cells out of total leukocytes. Increased TILs correlated with response to anti-PD-L1 and anti-CTLA-4 therapy, but PD-L1expression on tumor cells or PD-1 expression of T cells did not. Conclusions These studies reveal that tumor cell number correlates with tumor latency, TME, and response to ICB. ICB-sensitive and resistant syngeneic breast cancer models were identified, in which the 1E4 syngeneic model was most resistant to ICB. Given the lack of benefit from ICB in breast cancer, identifying robust murine models presented here provides the opportunity to further interrogate the TME for breast cancer treatment and provide novel insights into therapeutic combinations to overcome ICB resistance.

Effects of Gallotannin-Enriched Extract of Galla Rhois on the Activation of Apoptosis, Cell Cycle Arrest, and Inhibition of Migration Ability in LLC1 Cells and LLC1 Tumors

Gallotannin (GT) and GT-enriched extracts derived from various sources are reported to have anti-tumor activity in esophageal, colon and prostate tumors, although their anti-tumor effects have not been determined in lung carcinomas. To investigate the anti-tumor activity of GT-enriched extract of galla rhois (GEGR) against lung carcinomas, alterations in the cytotoxicity, apoptosis activation, cell cycle progression, migration ability, tumor growth, histopathological structure, and the regulation of signaling pathways were analyzed in Lewis lung carcinoma (LLC1) cells and LLC1 tumor bearing C57BL/6NKorl mice, after exposure to GEGR. A high concentration of GT (69%) and DPPH scavenging activity (IC50=7.922 µg/ml) was obtained in GEGR. GEGR treatment exerted strong cytotoxicity, cell cycle arrest at the G2/M phase and subsequent activation of apoptosis, as well as inhibitory effects on the MAPK pathway and PI3K/AKT mediated cell migration in LLC1 cells. In the in vivo syngeneic model, exposure to GEGR resulted in suppressed growth of the LLC1 tumors, as well as inhibition of NF-κB signaling and their inflammatory cytokines. Taken together, our results provide novel evidence that exposure to GEGR induces activation of apoptosis, cell cycle arrest, and inhibition of cell migration via suppression of the MAPK, NF-κB and PI3K/AKT signaling pathways in LLC1 cells and the LLC1 syngeneic model.

Targeting autocrine amphiregulin robustly and reproducibly inhibits ovarian cancer in a syngeneic model: roles for wildtype p53

Ovarian cancer (OvCA) remains one of the most devastating malignancies, but treatment options are still limited. We report that amphiregulin (AREG) can serve as an effective and safe pharmacological target in a syngeneic murine model. AREG is highly abundant in abdominal fluids of patients with advanced OvCa. In immunocompetent animals, depletion or overexpression of AREG respectively prolonged or shortened animal survival. A new antibody we generated in AREG-knockout mice recognized murine AREG and reproducibly prolonged animal survival in the syngeneic model. The underlying mechanism likely involves binding of wildtype p53 to AREG’s promoter and autocrine activation of the epidermal growth factor receptor (EGFR), a step blocked by the antibody. Accordingly, depletion of p53 downregulated AREG secretion and conferred tolerance, whereas blocking an adaptive process involving CXCL1, which transactivates EGFR, might increase therapeutic efficacy. Consistent with these observations, analysis of OvCa patients revealed that high AREG correlates with poor prognosis of patients expressing wildtype TP53. In conclusion, clinical tests of the novel antibody are warranted; high AREG, normal TP53, and reduced CXCL1 activity might identify patients with OvCa who may derive therapeutic benefit.