Median Animal Survival Research Articles

Abstract 3717: Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing

Abstract The potential impact of the immune tumor microenvironment (iTME) was assessed with respect to the anticancer efficacy of sudocetaxel zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin (SORT1)-mediated endocytic pathway. In the triple-negative breast cancer (TNBC)-derived MDA-MB-231 immunocompromised xenograft tumor model, TH1902 induced complete tumor regression for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed higher staining of CD45 leukocytes and of STING, a master regulator in the cancer-immunity cycle, in comparison to docetaxel. Additionally, in a murine B16-F10 melanoma syngeneic tumor model, weekly administration of TH1902 as a single agent also demonstrated superior tumor growth inhibition than did docetaxel. Although B16-F10 is considered a non-immunogenic “cold” tumor model, we observed a net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages. Moreover, in TH1902-treated tumors, increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increased tumor growth inhibition and median animal survival. Accordingly, TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the cell surface expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the iTME and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Veronique Lullier, Richard Beliveau, Christian Marsolais, Borhane Annabi. Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3717.

Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing.

The anticancer efficacy of Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin-mediated process, was assessed in a triple-negative breast cancer-derived MDA-MB-231 immunocompromised xenograft tumor model where complete tumor regression was observed for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed high staining of STING, a master regulator in the cancer-immunity cycle. A weekly administration of TH1902 as a single agent in a murine B16-F10 melanoma syngeneic tumor model demonstrated superior tumor growth inhibition than did docetaxel. A net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages was observed. Increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increases in tumor growth inhibition and median animal survival. TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the immune tumor microenvironment and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes.

DNAR-07. MOLECULAR BIOMARKERS ASSOCIATED WITH TARGETED DISRUPTION OF ALTERNATIVE LENGTHENING OF TELOMERES IN ATRX-DEFICIENT GLIOMA MODELS

Abstract Alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism of telomere maintenance that occurs in approximately 10-15 percent of all cancers. ALT enables the replicative immortality of cancer cells and is characterized by elevated levels of DNA replication stress and telomere-localized DNA double-strand breaks (DSBs). In gliomas, ALT frequently co-occurs with inactivating mutations in ATRX, which is thought to promote ALT induction by disrupting ATRX-mediated histone H3.3 deposition at telomeres, thus leading to telomere synthesis by homology-directed repair mechanisms. To identify potential therapeutic vulnerabilities specific to ALT-positive tumors, we identified candidate ALT-positive cancer cell lines in the Cancer Dependency Map (DepMap) dataset and validated the presence or absence of ALT phenotypic markers, including ALT-associated PML bodies (APBs) and nuclear foci of single-stranded telomeric DNA (ssTeloC). Comparing ALT-positive cells to all other cancer cell lines in the DepMap dataset, we identified the replication fork remodeling enzymes SMARCAL1 and FANCM as gene dependencies with the highest degree of ALT-specificity. To validate SMARCAL1 as an ALT-specific dependency, we used inducible RNAi to deplete SMARCAL1 in ALT-positive glioma cells. SMARCAL1 depletion caused a marked increase in the abundance and intensity of ssTeloC nuclear foci, as well as increased γH2AX abundance in ALT+ glioma cells but not telomerase-positive cells. Inducible SMARCAL1 depletion did not coincide with detectable increases in apoptotic markers (e.g. caspase-3 cleavage). However, SMARCAL1 depletion caused a significant increase in the abundance of nuclear abnormalities indicative of mitotic catastrophe (e.g. micronuclei, nucleoplasmic bridges, and fragmented/multilobular nuclei). In an orthotopic model of ALT-positive glioma, doxycycline-inducible RNAi targeting SMARCAL1 extended the median survival of tumor-bearing animals to 155 days compared to 111 days in the non-targeting control condition. These data validate the therapeutic targetability of SMARCAL1 in ALT-positive gliomas and support the development of small molecule SMARCAL1 inhibitors for treatment of ALT-positive tumors.

PRL2 inhibition elevates PTEN protein and ameliorates progression of acute myeloid leukemia.

Overexpression of phosphatases of regenerating liver 2 (PRL2), detected in numerous diverse cancers, is often associated with increased severity and poor patient prognosis. PRL2-catalyzed tyrosine dephosphorylation of the tumor suppressor PTEN results in increased PTEN degradation and has been identified as a mechanism underlying PRL2 oncogenic activity. Overexpression of PRL2, coincident with reduced PTEN protein, is frequently observed in patients with acute myeloid leukemia (AML). In the current study, a PTEN-knockdown AML animal model was generated to assess the effect of conditional PRL2 inhibition on the level of PTEN protein and the development and progression of AML. Inhibition of PRL2 resulted in a significant increase in median animal survival, from 40 weeks to greater than 60 weeks. The prolonged survival reflected delayed expansion of aberrantly differentiated hematopoietic stem cells into leukemia blasts, resulting in extended time required for clinically relevant leukemia blast accumulation in the BM niche. Leukemia blast suppression following PRL2 inhibition was correlated with an increase in PTEN and downregulation of AKT/mTOR-regulated pathways. These observations directly established, in a disease model, the viability of PRL2 inhibition as a therapeutic strategy for improving clinical outcomes in AML and potentially other PTEN-deficient cancers by slowing cancer progression.

Abstract LB101: Cell-generated IL12 combined with PD-1 inhibition produces local and abscopal immune activation to eradicate metastatic melanoma and pancreatic cancer

Abstract Background: Cytokines have been FDA approved for cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years (1, 2). To overcome stability and toxicity limitations seen with high dose cytokine immunotherapy, we developed a delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These clinically translatable cytokine factories are able to safely deliver high local doses of pro-inflammatory cytokines, such as interleukin-12 (IL12), and allow for controlled and predictable dosing in vivo. Results: Tumor-adjacent administration of IL12-based cytokine factories in vivo created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. In addition, administration of cytokine factories in combination with anti-PD1 checkpoint inhibitors caused reduction of tumor burden by over 60% when delivered as a monotherapeutic to mice with metastatic melanoma (3). Further, when administered in combination with local anti-PD1 checkpoint inhibitors, these cytokine factories led to reduction of intraperitoneal tumor burden by over 80% after only 10 days of treatment. Finally, we evaluated the ability of this therapy to treat pancreatic tumors which are notoriously known to be resistant to immunotherapy. We found that the median survival for control animals was 45 days. However, 8/8 of animals treated with IL12 cytokine factories survived throughout the duration of the study (110 days), demonstrating a significant extension of overall survival time. Conclusions: Our findings demonstrate efficacy of cytokine factories as single agent and combination therapeutics in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans.

Abstract 1695: Guanylate binding protein 1 modulate the proteasomal machinery and causes the cytochrome C degradation

Abstract Guanylate Binding Protein 1 (GBP1) is known as an interferon γ induced GTPase. It plays a crucial role in innate immunity against pathogenic infections but its role in cancer and therapeutic response is not well understood. Therefore, to study the role of GBP1, we investigated genetically manipulated ovarian cancer cells. We have employed in vitro and in vivo studies along with a proteomics approach to uncover the protein-protein interactions and their crosstalk with GBP1. The data suggest that GBP1 knockdown (KD) inhibits the clonogenic potential of cancer cells. In vivo studies including subcutaneous xenograft and intraperitoneal models revealed that GBP1 overexpressing (OV) tumor cells growing faster and reduce the median survival of animals compared to vehicle control (VC). Whereas GBP1 KD tumors were progressing slowly with longer median survival compared to scrambled control (SC). To study the molecular interactions of GBP1, we performed a proteomics-based Cellular thermal shift assay (CETSA) on GBP1 OV and KD ovarian cancer cells. We found that GBP1 interacts with members of proteasome including PSMB1, PSMB6, PSMA3, and Cytochrome C (CYCS). Protein analysis showed that GBP1 over-expressing cells had less ubiquitinylated proteins compared to VC, whereas GBP1 KD cells accumulate the ubiquitinylated proteins compared to SC. We also found that the GBP1 KD cells are sensitive to paclitaxel treatment and accumulated ubiquitinylated proteins compared to SC. Moreover, GBP1 KD inhibits the overall proteasomal activity. Paclitaxel-treated GBP1 OV cells accumulated the ubiquitination of CYCS, followed by clearance of cytosolic CYCS. Furthermore, global proteome profiling also suggests that GBP1 KD induces apoptotic pathways whereas GBP1 OV induces pathways associated with the stress response. Overall, the results from our studies elucidate the role of GBP1 in chemotherapeutic stress response via the activation of proteasomal machinery. Citation Format: Dhanir Tailor, Fernando Jose Garcia-Marques, Abel Bermudez, Sharon J. Pitteri, Sanjay V. Malhotra. Guanylate binding protein 1 modulate the proteasomal machinery and causes the cytochrome C degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1695.

Activity of 2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propandyоl (chlonisol) versus lomustine in intracranial transplanted tumor model in mice

Background. Alkylating drugs have been used in tumor chemo therapy for many decades, and the search for effective compounds continues.The aim of the study was to study the activity of the developed new compound – 2-[3-(2-chloroethyl)-3-nitrosoureido]-1,3-propanediol (chlonisol) in comparison with lomustine (CCNU) from the nitrosoalkylurea group, which is similar in chemical structure, in the model of intracranially transplanted Ehrlich's tumor and sarcoma 180 in mice.Methods. According to the developed technique, 64 female mice of the BALB/c line were punctured in the skull under anesthesia and inoculated with tumor cells of Ehrlich's carcinoma or sarcoma 180 in 0.9 % sodium chloride solution. After 24 hours, the test substances were administered at the maximum tolerated doses – chlonisol (20 mg/kg, i.p.) and lomustine (50 mg/kg, orally), once. The effect was compared with the control (solvent injection).Results. Chlonisol significantly increased the median overall survival (MOS) of animals after intracranial transplantation of both Ehrlich's tumor (by 39 %) and sarcoma 180 (by 84 %) compared with control (p<0.0001). Chlonisol reduced the risk of death in mice by 73 % compared to control in Ehrlich tumor transplantation and by 83 % in sarcoma 180 (p<0.0001). In contrast, lomustine did not show a significant therapeutic effect in intracranial transplantation of both tumors.Conclusion. The high activity of chlonisol in comparison with lomustine gives reason to consider it as a potential cytostatic agent in the treatment of nervous system tumors.

DDDR-35. TARGETING GBM INVASION BY INHIBITING KCNA1 WITH 4-AMINOPYRIDINE: AN FDA APPROVED DRUG THAT EASILY PASS THROUGH THE BBB

Abstract Diffuse invasion is a hall mark of glioblastoma (GB) and one of the primary causes of poor clinical outcomes in GBM patients. Tumor cells migrate deep into the normal brain tissues are frequently protected by the BBB, making them particularly difficult to treat. New therapeutic targets are needed. Our previous studies using spatially dissected and functionally validated matching pairs of invasive and tumor core GBM cells identified KCNA1 as a shared gene that is selectively over-expressed in the invasive GBM cells in 6 patient derived orthotopic xenograft (PDOX) mouse models of pediatric GBM (Huang YL et al, Adv Science 2021). A subsequent analysis of adult GBM RNAseq data from IVY Atlas revealed a significantly elevated expression of KCNA1 (4.9 fold) in the invasive edges of patient GBM tumors. It is also one of the 11 core molecules identified in GEO and TCGA databases through an integrated bioinformatic analysis (Yang J, Front Onc 2021). To determine the anti-invasive activities of targeting KCNA1, we treated three highly invasive adult GBM PDOX models with 4-aminopyridine (4-AP), an old lipid soluble drug that easily penetrate the BBB, at 5 mg/kg, i.p., 5 days/week for 8 weeks acing alone or in combination with fractionated radiation (at 2 Gy/day x days). As single agent, 4-AP significantly extended median animal survival times in 1/3 GBM models (69 to 77 days, P = 0.033). Combination with XRT did not significantly improve the animal survival times in the three models. Systematic analysis of GBM invasion in mouse brains of the 3 PDOX models before, during and after 4-AP treatment revealed remarkable inhibition of tumor invasion. Our data highlighted the role of KCNA1 in promoting GBM invasion and support the fine tuning of 4-AP dose, schedule, and length of treatment to serve as a novel component of anti-GBM invasion therapies.

1 Oral - A radiohapten capture system for CAR T cells that tracks them in vivo and improves efficacy

Background: CAR T cell therapy has become an important therapeutic tool for the treatment of B-cell neoplasms. However, the development of CAR T cell therapy outside of hematologic malignancies has stalled. As such, there is an unmet need to understand why CAR T cell therapy fails, which would be aided through the creation of tools to track them in vivo. Additionally, endowing CAR T cells with potent orthogonal anti-tumor activity has emerged as a strategy to improve upon CAR T cell efficacy. To accomplish both goals, we engineered a class of theranostic “THOR” CAR T cells that express a membrane-bound scFv, huC825, that binds DOTA-radiohaptens with pM affinity; these hapten chelands can be conjugated with radionuclides with therapeutic potential or diagnostic imaging capabilities. Methods: We engineered 19BBZ anti-CD19 CAR T cells to express huC825 (THOR CAR T cells) and confirmed transduction of these cells with flow cytometry. To assess the cytolytic ability and effector function of THOR CAR T cells, we used the Matador co-culture assay and measured the cytokine secretion of these co-cultures. We tested the potential to track THOR CAR T cells in vivo using PET/CT following intravenous DOTA-based radiohapten tracer administration in immunodeficient (NSG) mice bearing subcutaneous CD19+ Raji tumors. We also tested the ability of THOR CAR T cells to synergize with therapeutic radionuclide therapy. We evaluated the anti-tumor efficacy using whole animal bioluminescence imaging, tumor size measurements, and median animal survival. Results: We successfully transduced THOR CAR T cells, which demonstrated similar in vitro anti-tumor effects and cytokine secretion as parent 19BBZ CAR T cells. In vivo tracking experiments with 86Y-DOTA-Bn radiotracer showed that THOR CAR T cells can be observed at day 7 post T cell administration, peak at day 14, and persist until at least day 28. 86Y-DOTA-Bn uptake was specific to THOR CAR T cells, as minimal uptake was observed in mice transplanted with 19BBZ CAR T cells. Importantly, we noted rapid renal clearance of unbound tracer resulting in high contrast images. In efficacy assays in vivo, we demonstrated the synergy between 225Ac-Proteus-DOTA and THOR CAR T cells, as shown by a significant decrease in tumor burden and increase in median survival (THOR CAR treated = 26 days, treated with THOR CAR plus 225Ac-Proteus-DOTA = 32 days, p = .04, n = 6 per group). In contrast, mice receiving 19BBZ CAR T cells with or without 225Ac-Proteus-DOTA showed no improvements in antitumor efficacy. Conclusions: We show the pharmacokinetics of THOR CAR T cells can be probed in vivo following systemic administration of radiotracer using serial PET/CT imaging. Furthermore, we demonstrate a CAR T cell can be potentiated by systemically administered cytotoxic radionuclide that are then delivered selectively to tumors by the THOR cells. Conflict of interest: Ownership: MSK has filed for patent protection on behalf of M.M.D, S.M.C., D.R.V., B.H.S., N.K.C., D.A.S., S.M.L. and S.K. for inventions related to this work. Advisory Board: N.K.C. is an advisor to, or owns equity in Abpro, Alexs Lemonade Stand Foundation, Biotec Pharmacon, Eureka Therapeutics, Keystone Symposia, Partner Therapeutics, St. Jude-VIVA Forum, and Y-mAbs Therapeutics that may work in areas related to this study. Other Substantive Relationships: S.M.C. has licensed IP to Y-mAbs.

Abstract 1066: BMS-754807, a small-molecule inhibitor of insulin-like growth factor-1 receptor/insulin receptor is cytotoxic to esophageal adenocarcinoma cells and enhances nab-paclitaxel response in experimental esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is one of the fastest growing cancers in the Western world and the overall 5-year survival rate of EAC is below 20 percent. Epidemiological studies have linked obesity with EAC. Insulin-like growth factor (IGF) signaling is an important mediator in obesity-associated EAC. Paclitaxel (PT) has been used in combination with carboplatin as a standard combination therapy for advanced EAC. PT required emulsification with solvents which have resulted in serious adverse effects in patients. Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is an albumin-stabilized, cremophor-free and water-soluble nanoparticle formulation of PT. Nab-paclitaxel has recently shown greater efficacy over PT in EAC. In this study, we evaluated the potential of targeting IGF signaling and improvement in nab-paclitaxel response by the addition of BMS-754807 in experimental EAC. Methods: We first evaluated the phosphorylation status of IGF-1R/IR protein by Western blot in a panel of EAC cell lines. BMS-754807 and nab-paclitaxel, alone or in combination were tested for effects on cell growth, cell apoptosis, cell migration and cell cycle analyses of EAC cell lines detected respectively by WST-1 assay, Western blotting, would healing scratch assay and propidium iodide flow cytometry. We then explored the antitumor efficacy of BMS-754807 and nab-paclitaxel monotherapy and in combination in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. Results: BMS-754807 dose-dependently inhibited in-vitro cell proliferation of EAC cell lines having phosphorylation of IGF-1R/IR protein. The co-administration of BMS-754807 and nab-paclitaxel effectively enhanced cell growth inhibition, cleavage of caspase-3 and PARP, in-vitro wound healing inhibition and cell cycle arrest at sub G0/G1 phase. BMS-754807 in combination with nab-paclitaxel treatment resulted in significantly higher antitumor efficacy by increasing intratumoral apoptosis and survival benefit compared with BMS-754807 or nab-paclitaxel treatment alone. In subcutaneous xenografts using OE19 cells, average net tumor growth after two weeks in different therapy groups was 558.67 mm3 in control, 208.47 mm3 after BMS-754807 (p=0.043), 104.60 mm3 after nab-paclitaxel (p=0.013), and 14.30 mm3 after BMS-754807 plus nab-paclitaxel (p=0.0005). There was a significant increase in median animal survival after BMS-754807 plus nab-paclitaxel treatment (85 days) compared to control (47 days, p=0.0034), BMS-754807 monotherapy (57 days, p=0.0021) or nab-paclitaxel (68 days, p=0.0339) monotherapy. Conclusion: These results support the potential of BMS-754807 in combination with nab-paclitaxel as an effective option for EAC therapy. Citation Format: Md Sazzad Hassan, Nicholas Cwidak, Chloe Johnson, Saisantosh Ponna, Niranjan Awasthi, Urs von Holzen. BMS-754807, a small-molecule inhibitor of insulin-like growth factor-1 receptor/insulin receptor is cytotoxic to esophageal adenocarcinoma cells and enhances nab-paclitaxel response in experimental esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1066.

Percutaneous delivery of self-propelling hemostatic powder for managing non-compressible abdominal hemorrhage: a proof-of-concept study in swine

IntroductionNon-compressible intra-abdominal hemorrhage (NCIAH) is a major cause of preventable death on the battlefield and in civilian trauma. Currently, it can only be definitively managed with surgery, as there are limited strategies for controlling ongoing NCIAH in the prehospital environment. We hypothesized that a self-propelling thrombin-containing powder (SPTP) could increase survival in a swine model of NCIAH when delivered percutaneously into the closed abdomen using an engineered spray system. Materials and MethodsNineteen swine underwent surgical laparotomy followed by a Grade V liver injury that created massive hemorrhage, before closing the abdomen with sutures. Animals either received treatment with standard of care fluid resuscitation (n=9) or the SPTP spray system (n=10), which consisted of a spray device and a 14 Fr catheter. Using the spray system, SPTP was delivered into a hemoperitoneum identified using a focused assessment with sonography in trauma (FAST) exam. Lactated Ringer's solution was administered to all animals to maintain a mean arterial pressure (MAP) of >50 mmHg. The primary outcome was percentage of animals surviving at three hours following injury. ResultsIn the swine model of NCIAH, a greater percentage of animals receiving SPTP survived to three hours, although differences were not significant. The SPTP spray system increased the median survival of animals from 1.6 hr in the fluid resuscitation group to 4.3 hr. The SPTP spray system delivered a total mass of 18.5 ± 1.0 g of SPTP. The mean change in intra-abdominal pressure following SPTP delivery was 5.2 ± 1.8 mmHg (mean ± SEM). The intervention time was 6.7 ± 1.7 min. No adverse effects related to the SPTP formulation or the spray system were observed. SPTP was especially beneficial in animals that had either severely elevated lactate concentrations or low mean arterial pressure of <35 mmHg shortly after injury. ConclusionsThis demonstrates proof-of-concept for use of a new minimally invasive procedure for managing NCIAH, which could extend survival time to enable patients to reach definitive surgical care.

EXTH-56. ZIKA VIRUS TREATMENT SIGNIFICANTLY PROLONGS SURVIVAL IN A GLIOBLASTOMA PATIENT DERIVED XENOGRAFT MODEL

Abstract The poor median survival for patients with glioblastoma (GBM) of 15 months has not budged for the past 15 years, when the current standard treatment was first approved. There is no standard of care chemotherapy for recurrent GBM. We previously showed that Zika virus (ZIKV) tropism for GBM cells is mediated through the receptor tyrosine kinase, AXL. This infection is cytotoxic. In this study we show that ZIKV is an effective oncolytic virus in a patient derived xenograft model. Fox N1 Nude homozygous female mice 6-8-weeks-old were grouped into 4 experimental arms: two patient derived cell lines, each with a ZIKV treated and a control group. There were 12 mice in each arm. Animals received subcutaneous flank injections of GBM 8049 or its AXL CRISPR knockout 8049 AXLKO (2x106 cells). When tumors reached 200 mm3, mice received intra-tumoral injection of 2.5x106 ZIKV particles or saline. ZIKV induced complete tumor remission in 22 of 24 animals (8049: 11/12; 8049 AXLKO: 11/12). There was no tumor remission in the saline treated animals. Median survival of 8049 and 8049 AXLKO ZIKV treated mice was 124 days and 125 days, respectively. This is compared to median survival of control animals 8049: 42 days; 8049 AXLKO: 46 days (P= 0.001). Among ZIKV treated mice, there were two recurrences: one in the 8049 tumor (24 days after significant tumor remission) and one 8049 AXLKO tumor (7 days after significant tumor remission). We conclude that ZIKV should be considered a candidate oncolytic virus for GBM.

DIPG-14. TARGETING POLO-LIKE KINASE 1 IN COMBINATION WITH KEY ONCOGENIC DRIVERS IN DIPG: FROM SINGLE AGENT TO COMBINATION STRATEGIES

Diffuse Intrinsic Pontine Glioma (DIPG) are devastating paediatric brainstem tumours. Loss of function mutations in DIPG decrease genetic stability and impair DNA damage response pathways promoting tumourigenesis. Polo-like Kinase 1 (PLK1) is a pivotal controller of cell growth, regulating key intermediaries of DNA replication, homologous repair, the cell cycle and cell division. We have found DIPG cultures consistently overexpress PLK1 with inhibition resulting in decreased tumour cell growth, heightened cell cycle arrest and apoptosis. Single agent treatment using PLK1 inhibitors unprecedentedly doubled the median survival of animals harbouring DIPG tumours. Through gene expression analysis, we’ve showed PLK1 inhibition affected multiple pathways which control the cell cycle, cell death regulation, microtubule organization and regulation of cell migration. We found these pathways of differentially expressed genes were significantly enriched for known targets of both E2F1 and E2F4. Analysis of gene expression and proteomic studies also revealed PLK1 inhibition decreased the activation and expression of key tumour promoting mediators within multiple phases of the cell cycle, decreased expression of tumour promoters including MYC and the PI3K/mTOR pathway and reactivated tumour suppressors p53 and PTEN. Assessing these changes in the treated transcriptome and proteome, we aim to develop multiple potentially translatable combination treatment strategies for DIPG. We have performed mechanistic studies and identified synergism with PLK1 inhibitors and the epigenetic regulator panobinostat, bet/bromodomain inhibitor JQ1, dual PI3K/mTOR inhibitor bimiralisib and PI3K inhibitor BKM120. Finally, we found PLK1 inhibitors act as potent radiosensitizers, enhancing the therapeutic effects of radiotherapy in vitro and in vivo.

Influences of the Culturing Media in the Virulence and Cell Wall of Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa.

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are etiological agents of sporotrichosis, a human subcutaneous mycosis. Although the protocols to evaluate Sporothrix virulence in animal models are well described, the cell preparation before inoculation is not standardized, and several culturing media are used to grow yeast-like cells. Here, we found that carbon or nitrogen limitation during fungal cell preparation negatively impacted the ability of S. schenckii and S. brasiliensis to kill Galleria mellonella larvae, but not S. globosa. The fungal growth conditions associated with the short median survival of animals were accompanied by increased hemocyte countings, phenoloxidase activity, and cytotoxicity. The fungal growth under carbon or nitrogen limitation also affected the cell wall composition of both S. schenckii and S. brasiliensis and showed increased exposure of β-1,3-glucan at the cell surface, while those growing conditions had a minimal impact on the S. globosa wall, which had higher levels of this polysaccharide exposed on the wall regardless of the culture condition. This polysaccharide exposure was linked to the increased ability of insect hemocytes to uptake fungal cells, suggesting that this is one of the mechanisms behind the lower virulence of S. globosa or cells from the other species grown in carbon or nitrogen limitation.

High Efficacy of Liposomal Annamycin (L-ANN) in Combination with Cytarabine in Syngeneic p53-Null AML Mouse Model

High Efficacy of Liposomal Annamycin (L-ANN) in Combination with Cytarabine in Syngeneic p53-Null AML Mouse Model

Abstract 4219: Efficacy of CR13626, a novel oral brain penetrant multi-kinase inhibitor, in a mouse model of glioblastoma

Abstract BACKGROUND: Currently there is no cure for glioblastoma (GBM), the most malignant primary brain cancer. Glioblastoma etiopathogenesis involves mutations and alterations of key signaling pathways including tyrosine kinases (TKs). Notably, aberrant activation of receptor tyrosine kinases (RTKs) such as EGFR (amplified in 40% of glioblastoma cases) and KDR represents one of the driving forces for cellular proliferation and angiogenesis. The ability to cross the blood brain barrier (BBB) and appropriate pharmacodynamics (target selectivity) and pharmacokinetics (brain penetration) are critical issues for the generation of potential drug candidates against glioblastoma. CR13626 is a novel brain penetrant small molecule able to inhibit different TKs relevant to the development of this cancer. The present study describes the in vitro and in vivo properties of CR13626 and its antitumor activity in a mouse model of glioblastoma. METHODS: We identified the TKs targeted by CR13626 against a panel of 173 kinase enzymes. The effects of CR13626 on cellular proliferation were assessed in different 2D human GBM cell lines (U87MG, U373, U87MG vIII) by ViaCount, and in U87MG 3D spheroids by CellTiter-Glo 3D viability assay. The antitumor activity in vivo was determined in an orthotopic mouse model of GBM based on the injection of U87MG-Luciferase cells in nude mice. Animals were orally treated with CR13626 (50 mg/kg/daily) for 10 days, starting on day 9 post-implantation. Measurement of bioluminescence (BLI) at day 19 (end of dosing) and during follow-up (days 26-33) allowed the monitoring of tumor progression. CR13626 pharmacokinetics and brain exposure were assessed by LC/MS/MS in plasma and brain homogenate tissues of CD1 and tumor-bearing nude mice. RESULTS: CR13626 potently inhibited tyrosine kinases relevant to GBM development, with IC50 values in the nanomolar range: FYN (69 nM), YES (3.6 nM), KDR (82 nM) and EGFR (6 nM). CR13626 reduced the growth of different human glioblastoma cell lines with GI50 values in the range 1-3 µM. In vivo, CR13626 treatment led to a time-dependent reduction of tumor growth, reaching 60% on the last BLI evaluation 33 days post-implantation (i.e. 15 days after the end of dosing). A 25% significant increase in the median survival of animals compared to the vehicle group was also observed. The antitumor effects of CR13626 were in line with the exposure of tumor-bearing mice to the compound. Pharmacokinetic studies in CD1 mice showed good oral bioavailability (72%) and brain penetration (brain/plasma ratio of 1.4) for CR13626. CONCLUSION: The ability of CR13626 to cross the BBB without being a substrate of efflux transporters that mediate tumor resistance, to simultaneously inhibit the activity of different TKs involved in GBM development, and to reduce tumor growth eventually leading to an increased survival of animals, warrant its further development as a drug candidate in glioblastoma. Citation Format: Chiara Galimberti, Tiziana Piepoli, Giuseppe M. Montagna, Silvia Zerbi, Ornella Letari, Roberto Artusi, Milena Colovic, Stefano Persiani, Gianfranco Caselli, Lucio C. Rovati. Efficacy of CR13626, a novel oral brain penetrant multi-kinase inhibitor, in a mouse model of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4219.

Effects of 5-Hydroxypyrimidine Derivatives on Tumor Growth and Lifespan of C57BL/6 Mice with Epidermoid Lung Carcinoma.

The effects of 5-hydroxypyrimidine derivatives SNK-411 (2-isobutyl-4,6-dimethyl-5-hydroxypyrimidine) and SNK-578 (2-isobutyl-4,6-dimethyl-5-hydroxypyrimidine chlorohydrate) on the tumor growth and survival of male C57BL/6 mice with transplanted Lewis lung epidermoid carcinoma (LLC) were studied in animals receiving intraperitoneal treatment on days 2-15 of tumor development. Compound SNK-578 in a dose of 10 mg/kg significantly inhibited tumor growth (by 3.6 times; 72.2%) in 7 days after the treatment was discontinued, while compound SNK-411 in a dose of 25 mg/kg only negligibly reduced tumor volume (by 41.7%). A combination of course of SNK-411 (25 mg/kg) and single intraperitoneal dose of doxorubicin (4 mg/kg) significantly inhibited the tumor growth (by 2.2 times; 55.2%), while the combination of SNK-578 (10 mg/kg) with doxorubicin (4 mg/kg) was in fact ineffective. The median survival of animals with untreated LLC was 28 days. Median survival of mice injected with SNK-578 (10 mg/kg) was 43 days, hence, the lifespan of mice with LLC was by 38.6% longer after the treatment. Two of ten mice in this group developed no tumors.

ABC Transporter Inhibition Plus Dexamethasone Enhances the Efficacy of Convection Enhanced Delivery in H3.3K27M Mutant Diffuse Intrinsic Pontine Glioma.

An impermeable blood-brain barrier and drug efflux via ATP-binding cassette (ABC) transporters such as p-glycoprotein may contribute to underwhelming efficacy of peripherally delivered agents to treat diffuse intrinsic pontine glioma (DIPG). To explore the pharmacological augmentation of convection-enhanced delivery (CED) infusate for DIPG. The efficacy of CED dasatinib, a tyrosine kinase inhibitor, in a transgenic H3.3K27M mutant murine model was assessed. mRNA expression of ABCB1 (p-glycoprotein) was analyzed in 14 tumor types in 274 children. In Vitro viability studies of dasatinib, the p-glycoprotein inhibitor, tariquidar, and dexamethasone were performed in 2 H3.3K27M mutant cell lines. Magnetic resonance imaging (MRI) was used to evaluate CED infusate (gadolinium/dasatinib) distribution in animals pretreated with tariquidar and dexamethasone. Histological assessment of apoptosis was performed. Continuous delivery CED dasatinib improved median overall survival (OS) of animals harboring DIPG in comparison to vehicle (39.5 and 28.5 d, respectively; P=.0139). Mean ABCB1 expression was highest in K27M gliomas. In Vitro, the addition of tariquidar and dexamethasone further enhanced the efficacy of dasatinib (P<.001). In Vivo, MRI demonstrated no difference in infusion dispersion between animals pretreated with dexamethasone plus tariquidar prior to CED dasatinib compared to the CED dasatinib. However, tumor apoptosis was the highest in the pretreatment group (P<.001). Correspondingly, median OS was longer in the pretreatment group (49 d) than the dasatinib alone group (39 d) and no treatment controls (31.5 d, P=.0305). ABC transporter inhibition plus dexamethasone enhances the efficacy of CED dasatinib, resulting in enhanced tumor cellular apoptosis and improved survival in H3.3K27M mutant DIPG.

Abstract 2339: High fat diet modulates host immunity and impairs the efficacy of combined radiation and immunotherapy

Abstract Introduction: Obesity is associated with a higher incidence of cancer, increased cancer mortality, impaired responses to radiation therapy (RT), and worse toxicity after RT. Paradoxically, recent studies suggest that obese patients may actually have improved outcomes with immunotherapy (IO), such as programmed death (PD)-1 inhibition, highlighting a link between the host’s metabolic state and immune homeostasis. We therefore hypothesized that a high fat diet could be used to alter the host’s immune response to improve the efficacy of combined RT and IO. Materials/Methods: 12-week old female Balb/c mice were injected with 5x104 syngeneic, luciferase tagged, triple-negative, murine breast cancer cells (4T1). Upon development of a palpable tumor, the mice were randomized to either an ad lib (AL) or high fat (HF), defined as 45% calories from fat, diet. Additionally, each cohort was treated with either diet alone (D), diet plus RT (4 Gy in 3 daily fractions), diet plus PD-1 inhibitor, or a combination of all three (Combo). On day 26, the mice were injected with luciferin and tumor growth was measured using the IVIS bioluminescence in vivo imaging system. At time of sacrifice, primary tumors and spleen were harvested for downstream analysis of immune markers via RNA microarray and flow cytometry. Results: The median overall survival (OS) for animals fed either an HF or AL diet was 36 and 39 days, respectively (NS). The difference in OS however became much more apparent between the individual treatment arms: HF+αPD1 vs AL+αPD1 (37d vs 41.5d, p=0.041); HF+RT vs AL+RT (36d vs 45d, p=0.039); HF+Combo vs AL+Combo (39d vs 48d, p=0.037). HF fed mice also developed metastases sooner than AL fed mice across all arms (D: 22d vs 30d; RT: 24d vs 32d; αPD1: 24d vs 34d; Combo: 27d vs 37d, p=0.047). Microarray analysis of primary tumor tissue revealed an at least 1.5 fold increase expression of cytokines associated with inflammation including IFN-y, TNF-α, IL-17F, IL-22 and IL-1β in the HF diet groups while AL fed mice demonstrated increased expression of the anti-inflammatory cytokines IL-10 and TGF-β. Consistent with the increase in inflammatory markers after a HF diet, flow cytometric analysis of splenocytes revealed a significant decrease in the number of CD4+CD25+Foxp3+ regulatory T cells in the HF group compared with AL fed mice (3.3% versus 7.68%, respectively; p = 0.006). Notably, these Tregs also produced less IL-10 compared with those from AL mice (24.4% vs 54.5%, p=0.037). Conclusions: We demonstrate that a HF diet impairs the efficacy of RT, IO, and combined therapy. This is associated with the development of a pro-inflammatory state demonstrated by increased expression of pro-inflammatory cytokines and a decrease in the number and activity of regulatory T cells. Based on these findings, we conclude that diet can directly impact treatment efficacy and should be considered as a variable in future clinical studies. Note: This abstract was not presented at the meeting. Citation Format: Gregor Manukian, Brittany A. Simone, Tiziana DeAngelis, Kevin Ko, Charles Kivolowitz, Sylvester Jusu, Nicole L. Simone. High fat diet modulates host immunity and impairs the efficacy of combined radiation and immunotherapy [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 2339.

Abstract LB-077: Dipeptidyl Peptidase Inhibitor BXCL701 synergizes with an OX40-agonist antibody resulting in synergistic anti-tumor response and survival in an animal model of colorectal cancer by bridging the innate and adaptive arms of the immune system

Abstract The oral DPP8/9 and FAP inhibitor BXCL701 in combination with an anti-PD-1 antibody (aPD-1) has demonstrated inhibition of tumor growth as well as up-regulation of immuno-stimulatory cytokines and tumor-infiltrating immune cells in animal models [AACR 2017, ASCO 2018]. In the present study, the primary cytokines/chemokines influenced by BXCL701 administered as a single agent have been explored and provide the rational for an efficacy study of BXCL701 in combination with an OX40-agonist antibody (aOX40). Cytokines/chemokines were evaluated in serum samples from animals in an acute PK/PD study revealing that BXCL701 stimulated several pro-inflammatory factors. The most stimulated cytokine was IL-18 which is known to bridge the innate and adaptive immune systems through induction of IFN-γ and the CD134 (OX40) pathway. (J of Immunol, 2006, 177: 234). Therefore, an in vivo study was conducted by combining BXCL701, aOX40 and aPD-1 in a preclinical model (MC38) of colorectal cancer. Evaluation of tumor size on day 23 compared to vehicle control showed aOX40 and BXCL701 treated animals to have tumor growth inhibition (TGI) of 0.8% (p = 0.97), and 8% (p = 0.61) respectively. Importantly, BXCL701 in combination with aOX40 resulted in synergistic inhibition of 51% (p = 0.001) when compared to vehicle. A 43% TGI (p = 0.006) was observed in animals treated with the combination of aPD-1 and aOX40. Addition of BXCL701 to the double combination of aOX40 and aPD-1 resulted into 59% TGI (p = 0.0005). At study end, BXCL701 demonstrated a significant improvement in the survival of animals when combined with aOX40. The median survival of vehicle treated animals was determined to be 30 days. BXCL701 and aOX40-treated animal groups had median survival of 35 days and 29 days, respectively (BXCL701 vs Vehicle p = 0.0958, aOX40 vs Vehicle p = 0.8560). The median survival of animals treated with combination of BXCL701 and aOX40 was significantly increased to 46 days in comparison to BXCL701 alone (p &amp;lt; 0.0001), while combination of aPD1 and aOX40 treated animals had a median survival of 36 days (aPD-1 and aOX40 vs Vehicle p = 0.0991). The triple combination of BXCL701, aOX40 and aPD1 resulted in the median survival of 46 days (p &amp;lt; 0.0001 vs Vehicle). In conclusion, the inclusion of BXCL701 in the drug combinations was necessary for a survival benefit because while the aOX40 and aPD-1 combination showed a significant TGI on day 23, this did not translate into an overall survival benefit. Therefore, BXCL701 contributed to a clear synergistic anti-tumor and survival response within the combinations. Ostensibly, BXCL701-regulated cytokines are responsible for the observed synergism with anOX40 treatment. The present study suggests the clinical evaluation of BXCL701 in combination with an OX40-agonist in solid tumors. Citation Format: Veena R. Agarwal, John MacDougall, Dimple Bhatia, Snigdha Gupta, Zeenia Zagga, Nirmal keshava. Dipeptidyl Peptidase Inhibitor BXCL701 synergizes with an OX40-agonist antibody resulting in synergistic anti-tumor response and survival in an animal model of colorectal cancer by bridging the innate and adaptive arms of the immune system [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 LB-077.