Delayed Tumor Progression Research Articles

Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors

BackgroundThe oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells.MethodsEnvisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed.ResultsWe showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway.ConclusionBased on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Targeting the Ang2/Tie2 Axis with Tanshinone IIA Elicits Vascular Normalization in Ischemic Injury and Colon Cancer.

Pathological angiogenesis, as exhibited by aberrant vascular structure and function, has been well deemed to be a hallmark of cancer and various ischemic diseases. Therefore, strategies to normalize vasculature are of potential therapeutic interest in these diseases. Recently, identifying bioactive compounds from medicinal plant extracts to reverse abnormal vasculature has been gaining increasing attention. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza, has been shown to play significant roles in improving blood circulation and delaying tumor progression. However, the underlying mechanisms responsible for the therapeutic effects of Tan IIA are not fully understood. Herein, we established animal models of HT-29 human colon cancer xenograft and hind limb ischemia to investigate the role of Tan IIA in regulating abnormal vasculature. Interestingly, our results demonstrated that Tan IIA could significantly promote the blood flow, alleviate the hypoxia, improve the muscle quality, and ameliorate the pathological damage after ischemic insult. Meanwhile, we also revealed that Tan IIA promoted the integrity of vascular structure, reduced vascular leakage, and attenuated the hypoxia in HT-29 tumors. Moreover, the circulating angiopoietin 2 (Ang2), which is extremely high in these two pathological states, was substantially depleted in the presence of Tan IIA. Also, the activation of Tie2 was potentiated by Tan IIA, resulting in decreased vascular permeability and elevated vascular integrity. Mechanistically, we uncovered that Tan IIA maintained vascular stability by targeting the Ang2-Tie2-AKT-MLCK cascade. Collectively, our data suggest that Tan IIA normalizes vessels in tumors and ischemic injury via regulating the Ang2/Tie2 signaling pathway.

Experience with Sorafenib in Patients with Advanced Hepatocellular Carcinoma Treated at the National Cancer Institute of Panama (Sorafen Study)

Sorafenib is an oral multi-kinase inhibitor that increases survival and delays tumor progression in patients with advanced hepatocarcinoma (HCC). This study aimed to determine the progression-free survival (PFS) and overall survival (OS) and to analyze OS-related factors in this population between 2014 and 2019. We performed a retrospective review of patients with advanced HCC who were treated with sorafenib at the National Cancer Institute of Panama (Instituto Oncologico Nacional de Panama – IONP) from January 2014 to December 2019. The data were collected from electronic health records of the IONP. In total, 77 patients with a mean age of 65 years were analyzed. Sixty-three percent of the patients were men, and most of them had an Eastern Cooperative Oncology Group (ECOG) performance status of 1 and had not undergone any prior treatment. Forty-four percent patients had Child-Pugh Class A. The most frequent progression site was the liver (27%), followed by the lungs. Mean PFS was 1 month, and mean OS was 3 months. The clinical benefit was 39% and Overall response rate was 3,9%; for those with stable disease, it was 35,1%, and 3,9% showed a partial response. More encouraging results were obtained from patients with higher functional statuses (ECOG status 0-1) and who were in milder stages of liver disease (Child-Pugh Class A). The most common adverse events were fatigue, hand-foot syndrome, nausea and vomiting, and arterial hypertension. The survival results assessed in our institution for patients with advanced hepatocellular carcinoma treated with sorafenib are below of those published in the literature; however, after selecting the cases with ECOG 0-1 and Child A stage, our results were more in line with those of the international literature, being the most important prognostic factors in our patients. Fatigue and hand-foot syndrome were the most common adverse events. The key to the success of this therapy lies in an adequate selection of patients.

Altered Regulation of HIF-1α in Naive- and Drug-Resistant EGFR-Mutant NSCLC: Implications for a Vascular Endothelial Growth Factor-Dependent Phenotype.

The treatment of patients with EGFR-mutant NSCLC with vascular endothelial growth factor (VEGF) inhibitors in combination with EGFR inhibitors provides a greater benefit than EGFR inhibition alone, suggesting that EGFR mutation status may define a patient subgroup with greater benefit from VEGF blockade. The mechanisms driving this potentially enhanced VEGF dependence are unknown. We analyzed the effect of EGFR inhibition on VEGF and HIF-1α in NSCLC models invitro and invivo. We determined the efficacy of VEGF inhibition in xenografts and analyzed the impact of acquired EGFR inhibitor resistance on VEGF and HIF-1α. NSCLC cells with EGFR-activating mutations exhibited altered regulation of VEGF compared with EGFR wild-type cells. In EGFR-mutant cells, EGFR, not hypoxia, was the dominant regulator of HIF-1α and VEGF. NSCLC tumor models bearing classical or exon 20 EGFR mutations were more sensitive to VEGF inhibition than EGFR wild-type tumors, and a combination of VEGF and EGFR inhibition delayed tumor progression. In models of acquired EGFR inhibitor resistance, whereas VEGF remained overexpressed, the hypoxia-independent expression of HIF-1α was delinked from EGFR signaling, and EGFR inhibition no longer diminished HIF-1α or VEGF expression. In EGFR-mutant NSCLC, EGFR signaling is the dominant regulator of HIF-1α and VEGF in a hypoxia-independent manner, hijacking an important cellular response regulating tumor aggressiveness. Cells with acquired EGFR inhibitor resistance retained elevated expression of HIF-1α and VEGF, and the pathways were no longer EGFR-regulated. This supports VEGF targeting in EGFR-mutant tumors in the EGFR inhibitor-naive and refractory settings.

Cfi-400945 Induces Cytokinesis Failure By Activating Hippo Signaling Pathway in Diffuse Large B-Cell Lymphoma

Targeting mitosis has been found to be a therapeutic strategy for cancer since microtubule poisons have proved efficacy in a broad range of malignancies. Cytokinesis failure can increase chromosomal instability beyond a critical threshold that triggers tumor suppression. Thus, agents developed to block cytokinesis can stop proliferation of cancer cells. We previously demonstrated that the polo-like kinase 4 (PLK4) inhibitor, CFI-400945, triggers growth inhibition and apoptotic death in diffuse large B-cell lymphoma (DLBCL). Since PLK4 is involved in centrosome duplication for cell division, we aimed to explore the effects of CFI-400945 on cytokinesis in DLBCL cells. Here, we demonstrated that CFI-400945 induces cytokinesis failure in DLBCL. CFI-400945-treated cells led to binucleated/multinucleated cells (Fig. 1a). Previous study showed that the Hippo signaling pathway played a critical role in cytokinesis. Consistent with this finding, we found that CFI-400945 led to phosphorylation of LATS1 and YAP (Fig. 1b). Confocal microscopy confirmed the reduced nuclear YAP upon treatment of CFI-400945 (Fig. 1c). These results show that reduced nuclear YAP levels and activities contribute to growth inhibition by CFI-400945 in DLBCL cells. In addition, we further explored the synergy of CFI-400945 and doxorubicin (DOX) both in vitro and in vivo. A combined treatment of CFI-400945 and DOX significantly inhibited DLBCL cell survival and induced more apoptosis compared with CFI-400945 or DOX alone (Fig. 1d, Fig. 1e).In the mice xenograft model, the combination of CFI-400945 and DOX significantly delayed tumor progression compared to those treated with vehicle or DOX alone (Fig. 1f). Tumors from combination-treated mice displayed an increase in γ-H2AX staining and a reduction in Ki67 staining (Fig. 1g). Strikingly, tumor cells were generally larger, heterogeneous in size and frequently binucleated (Fig. 1h). Combined, our results indicate for the first time that CFI-400945 treatment induces cytokinesis failure and activation of Hippo signaling pathway, leading to vulnerability to mitotic catastrophe. Moreover, this study proves an attractive combination treatment that simultaneous targeting PLK4 with CFI-400945 could exacerbate mitotic defects to improve response of anthracycline-based chemotherapy in DLBCL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Divergent Roles of PI3K Isoforms in PTEN-Deficient Glioblastomas

SUMMARYLoss of PTEN, the negative regulator of PI3K activity, is frequent in glioblastomas (GBMs). However, the role of the two major PI3K isoforms, p110α and p110β, in PTEN-deficient gliomagenesis remains unknown. We show that PTEN-deficient GBM largely depends on p110α for proliferation and p110β for migration. Genetic ablation of either isoform delays tumor progression in mice, but only ablating both isoforms completely blocks GBM driven by the concurrent ablation of Pten and p53. BKM120 (buparlisib) treatment only modestly prolongs survival in mice bearing intracranial Pten/p53 null tumors due to partial pathway inhibition. BKM120 extends the survival of mice bearing intracranial tumors in which p110β, but not p110α, has been genetically ablated in the Pten/p53 null glioma, indicating that BKM120 fails to inhibit p110β effectively. Our study suggests that the failure of PI3K inhibitors in GBM may be due to insufficient inhibition of p110β and indicates a need to develop brain-penetrant p110α/β inhibitors.

Chromatin Regulator CHD1 Remodels the Immunosuppressive Tumor Microenvironment in PTEN-Deficient Prostate Cancer.

Genetic inactivation of PTEN is common in prostate cancer and correlates with poorer prognosis. We previously identified CHD1 as an essential gene in PTEN-deficient cancer cells. Here, we sought definitive in vivo genetic evidence for, and mechanistic understanding of, the essential role of CHD1 in PTEN-deficient prostate cancer. In Pten and Pten/Smad4 genetically engineered mouse models, prostate-specific deletion of Chd1 resulted in markedly delayed tumor progression and prolonged survival. Chd1 deletion was associated with profound tumor microenvironment (TME) remodeling characterized by reduced myeloid-derived suppressor cells (MDSC) and increased CD8+ T cells. Further analysis identified IL6 as a key transcriptional target of CHD1, which plays a major role in recruitment of immunosuppressive MDSCs. Given the prominent role of MDSCs in suppressing responsiveness to immune checkpoint inhibitors (ICI), our genetic and tumor biological findings support combined testing of anti-IL6 and ICI therapies, specifically in PTEN-deficient prostate cancer. SIGNIFICANCE: We demonstrate a critical role of CHD1 in MDSC recruitment and discover CHD1/IL6 as a major regulator of the immunosuppressive TME of PTEN-deficient prostate cancer. Pharmacologic inhibition of IL6 in combination with immune checkpoint blockade elicits robust antitumor responses in prostate cancer.This article is highlighted in the In This Issue feature, p. 1241.

Enhanced YB1/EphA2 axis signaling promotes acquired resistance to sunitinib and metastatic potential in renal cell carcinoma

VHL mutations are the most common tumorigenic lesions in clear cell renal cell carcinoma (ccRCC) and result in continued activation of the HIF/VEGF pathway and uncontrolled cancer progression. Receptor tyrosine kinase (RTK) inhibitors such as sunitinib have been demonstrated to target tumorigenic signaling pathways, delay tumor progression, and improve patient prognosis in metastatic renal cell carcinoma (mRCC). Although several mechanisms of sunitinib resistance have been reported, the solutions to overcome this resistance remain unclear. In our study, we found that increased expression of Y-box binding protein 1 (YB1, a multidrug resistance associated protein) and EphA2 (a member of the erythropoietin-producing hepatocellular (Eph) receptor family, belonging to the RTK family) mediated sunitinib resistance and mRCC exhibited a large phenotypic dependence on YB1 and EphA2. In addition, our findings confirm that YB1 promotes the invasion, metastasis and sunitinib resistance of ccRCC by regulating the EphA2 signaling pathway. Furthermore, pharmacological inhibition of EphA2 through the small molecule inhibitor ALW-II-41-27 reduced the proliferation of sunitinib-resistant tumor cells, suppressed tumor growth in vivo, and restored the sensitivity of sunitinib-resistant tumor cells to sunitinib in vitro and in vivo. Mechanistically, YB1 increases the protein levels of EphA2 by maintaining the protein stability of EphA2 through inhibition of the proteasomal degradation pathway. Collectively, our findings provide the theoretical rationale that ccRCC metastasis and RTK-directed therapeutic resistance could be prospectively and purposefully targeted.

High FLT3 Levels May Predict Sorafenib Benefit in Hepatocellular Carcinoma.

To identify a predictive biomarker of sorafenib for hepatocellular carcinoma personalized therapy. The patients treated with or without sorafenib after hepatocellular carcinoma recurrence from multicenters were matched with propensity score matching analysis. The expression levels of Fms-like tyrosine kinase 3 (FLT3) in hepatocellular carcinoma specimens of the matched patients (n = 276) were analyzed by IHC. The optimal cut-off point of FLT3 levels for overall survival (OS) was defined via Cutoff Finder. Subgroup analysis of OS was employed to investigate the association between FLT3 levels and sorafenib benefit. The predictive value was assessed via Cox regression models with an interaction term. Hepatocellular carcinoma and paratumoral normal tissues were used to investigate the expression and copy-number variation of FLT3. Patient-derived xenograft (PDX) models were used to confirm the association between FLT3 levels and sorafenib response. Patients with FLT3-high hepatocellular carcinoma exhibited a superior OS upon sorafenib treatment. High FLT3 levels were predictive of sorafenib benefit in terms of OS (P interaction = 0.00006). Copy-number losses and decreased expression of FLT3 in hepatocellular carcinoma were detected in about 64% of patients. Moreover, the PDXs derived from tumors with high FLT3 levels also displayed a better response to sorafenib. Sorafenib may be able to delay tumor progression in patients with FLT3-high hepatocellular carcinoma. This potential biomarker needs to be further validated in independent cohorts prior to helping stratify patients for precision therapy in advanced hepatocellular carcinoma.

Abstract 6276: Brain penetrant MDM2 inhibitor RG7388 extends survival benefit of radiation treatment in select glioblastoma patient-derived xenograft models

Abstract Despite aggressive treatment that involves surgery, radiation and temozolomide therapy, a significant morbidity from glioblastoma (GBM) recurrence highlights the pressing unmet medical need to develop effective novel therapies for GBM. Murine Double Minute 2 (MDM2) is an important regulator of the p53 tumor suppressor, which promotes cell cycle arrest and apoptosis in response to DNA damage. Here we have assessed the efficacy of RG7388, a purported brain penetrant MDM2-inhibitor, alone or combined with radiation therapy (RT) in patient-derived xenograft (PDX) models of GBM. In vitro, RG7388 suppressed viability of GBM PDX short-term stem cell cultures of p53 wildtype lines with MDM2-amplification (GBM46 and 108, IC50 25 and 11 nM, respectively) or without MDM2-amplification (GBM10 and 14, IC50 43.5 and 9.1 nM, respectively). Serum proteins appear to reduce efficacy of RG7388, which is supported by the notion that adding 2.5% bovine serum albumin to a serum free stem cell cultures increased the IC50 by 3 to 5 fold. At the molecular level, both RG7388 and RT could induce p53 signaling in GBM10, but a more robust induction was observed with the combination. In athymic nude mice, RG7388 readily distributes into normal brain as measured by LCMS-MS at 1 hour after five daily oral doses (70 mg/kg) the average RG7388 concentrations in brain and plasma were 2167 ng/g and 4423 ng/ml, respectively. In an initial efficacy study using flank tumors established from GBM10, one week of dosing at 70 mg/kg/day RG7388 alone was ineffective, while combining RG7388 with focal radiation (4 daily doses of 5 Gy each) had a moderate 15 day delay in tumor progression. However, in an MDM2-amplified GBM108 line with a relatively intact blood-brain barrier, one week of daily RG7388 dosing, either alone or in combination with RT was remarkably effective (median survival 71 days with RG7388 alone vs. 29 days with vehicle, p<0.0001; and 262 days for RG7388/RT vs. 74 days with RT, p=0.017). Taken together, RG7388 alone or in combination with RT is highly effective in an MDM2-amplified pre-clinical model of GBM. Determining impact of serum protein binding on drug pharmacokinetics and further evaluation of pharmacodynamic effects and orthotopic therapy in additional p53 wildtype PDX lines will help to increase our understanding if RG7388/RT therapy can be used in a larger population of GBM tumors. Citation Format: Shiv K. Gupta, Ann C. Mladek, Surabhi Telele, Afroz S. Mohammad, Lihong He, Zeng Hu, Katrina K. Bakken, Danielle M. Burgenske, Brett L. Carlson, William F. Elmquist, Jann N. Sarkaria. Brain penetrant MDM2 inhibitor RG7388 extends survival benefit of radiation treatment in select glioblastoma patient-derived xenograft models [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 6276.

Abstract 4235: A combination of CAR-NK and CAR-T cells results in rapid and persistent anti-tumor efficacy while reducing CAR-T cell mediated cytokine release and T-cell proliferation

Abstract CAR-NK cells show rapid and potent anti-tumor cytotoxic activity even at very low effector-to-target ratios, however, they do not typically expand upon encounter with antigen and lack long term persistence commonly associated with CAR-T cells. Conversely, CAR-T cells generally exhibit persistent killing activity, even though they are activated somewhat more slowly. In addition, the explosive expansion of CAR-T cells is frequently associated with a variety of toxicities (e.g. CRS, CRES, etc.). To address these shortcomings, Nkarta has developed a novel platform to combine both CAR-NK and CAR-T cells as a novel cancer therapeutic. To evaluate the overall outcomes of combining CAR-NK and CAR-T cells as an anti-tumor therapy, we have developed two novel in vitro assays: (a) IncuCyte-based matrixed cytotoxicity assay, which allows direct assessment of tumor killing kinetically with different E:T and NK:T ratios; (b) IncuCyte-based tumor re-challenge assay, which provides a convenient and quick assessment of anti-tumor persistence. For proof-of-concept studies, a CD19 directed CAR (CAR19) was retrovirally engineered into NK and T cells. Our newly developed assays revealed that the combination of CAR-NK and CAR-T cells clearly outperformed CAR19-NK or CAR19-T cells alone in anti-tumor efficacy and persistence. Additionally, the combination also showed an improved cytokine profile. To further investigate the cellular mechanism(s) underlying the beneficial effects of combining CAR-NK and CAR-T cells, we performed FACS-based phenotyping and a Violet Cell Trace based proliferation assay. When challenged with tumor cells, the presence of CAR19-NK cells reduced the proliferation of CAR19-T cells. Surprisingly, the presence of CAR19-T cells could trigger the proliferation of CAR19-NK cells. Finally, we evaluated the NK plus T platform in vivo with a Nalm6 xenograft model. Treatment with CAR19-NK cells combined with a low dose of CAR19-T cells (5e5 cells/animal) delayed tumor progression. Combined treatment with a medium dose of CAR19-T cells (2.5e6 cells/animal) largely prevented tumor relapse, and consequently enhanced the overall survival of the animals. In summary, our in vitro and in vivo studies strongly demonstrate that the NK plus T platform can harness the advantages of both CAR-NK and CAR-T cells, allowing for both rapid and persistent killing while potentially minimizing the toxicities associated with CAR-T cells. Citation Format: Guangnan Li, Xiumin Wu, Ivan H. Chan, James B. Trager. A combination of CAR-NK and CAR-T cells results in rapid and persistent anti-tumor efficacy while reducing CAR-T cell mediated cytokine release and T-cell proliferation [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 4235.

Abstract LB-392: Optimizing dual immuno-oncology combinations with a virtual tumor

Abstract The purpose of the study was to investigate the application of a ‘virtual tumor' (‘VT') technology to optimizing the dosing and scheduling of dual immuno-oncology combinations in order to maximize the immunopotentiation and anti-tumor efficacy.The tumor microenvironment and local immune response play a key role in cancer biology, and immune resistance is a feature of many cancers. Immune checkpoints suppress the ability of the immune system to destroy cancer cells, and are a clear therapeutic target. However, response rates for such therapies are generally low. While anti-tumor efficacy can be improved by combining agents that target immune checkpoints with selected conventional anti-cancer therapies[1], the potential of immune-checkpoint blockade could be further unlocked through combination instead with other immunotherapies[2]. However, dosing and scheduling of immunotherapy combinations need to be optimized to take full advantage of a limited window for immunopotentiation[1]. Modelling can be used to deliver insights to help optimize such combination immunotherapies. We have developed a preclinical and a clinical VT technology that can predict how a tumor will respond to drug exposure[3]. This integrated PK/PD simulation platform can be used to optimize drug dosing and scheduling, and to design new combination therapies. The VT technology integrates pharmacokinetic and pharmacodynamic effects and models the way individual cells behave within a tumor population. These agent-based methods are particularly suitable for modeling not only tumor cells, but also other cell populations - such as those involved in the immune response - and also interactions between cells. Here we describe our recent development and application of the VT technology for modeling preclinical efficacy of novel immuno-oncology combinations.Building upon previous work, in which the VT platform was extended by the addition of a module that captured the synergistic interaction of PD(L)-1 blockade with conventional anticancer therapies[4], we have further expanded the model to allow simulation of immuno-checkpoint block with other immunotherapies. This module captures the mechanisms by which immunotherapies activate the innate antitumor immune response, either through immune-checkpoint block or by targeting immunosuppression by MDSC within the tumor microenvironment. Through a preclinical case study derived from the literature[5] and additional data available in the public domain, we demonstrate that the extended VT can be applied to simulate the efficacy of PD-1 blockade combined with an anti-C5a agent, and show that the combination treatment delays tumor progression. Furthermore, we demonstrate that the model can be applied to optimize the timing of the two therapies in order to maximize overall efficacy. Our enhanced VT capability represents a key step towards a strategic tool for optimizing dosing and scheduling of immuno-oncology combinations. [1] Ott, PA et al., Journal for ImmunoTherapy of Cancer 5, 16 (2017). [2]. Galon, J. & Bruni, D. Nature Reviews Drug Discovery (2019) [3]. Orrell, D. & Fernandez, E. Innovations in Pharmaceutical Technology 60-62 (2011). [4]. Brightman, FA et al., Abstract 4866. Cancer Res 75, 4866-4866 (2015). [5] Ajona, D. et al. Cancer Discov 7, 694-703 (2017). Citation Format: Fernando Ortega, Frances A. Brightman, Christophe Chassagnole. Optimizing dual immuno-oncology combinations with a virtual tumor [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 LB-392.

Abstract 5027: The mu-opioid receptor antagonist methylnaltrexone can decrease tumorigenesis in head and neck squamous cell carcinoma

Abstract Head and neck cancer (HNC) is the sixth leading cause of cancer in the world, and the third most common neoplasia in developing countries [1]. Patients with HNC may suffer significant pain, for which opioids are commonly given. However, recent reports suggest that opioids may promote cancer progression by action on the mu-opioid receptor (MOR) [2, 3]. The goal of this research was to test the hypothesis that the MOR antagonist methylnaltrexone (MTNX) can decrease tumorigenesis in HN squamous cell carcinoma (SCC). First, we confirmed via RNAseq and western blot analyses that FaDu cells (a HNSCC line) express MOR. Then, we performed cyclic adenosine monophosphate (cAMP) activation enzyme-linked immunosorbent assay to evaluate the activity of MOR after activation with [D-Ala2N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), a selective MOR agonist. Short term (1hr) treatment with DAMGO decreased cAMP levels, suggesting full activity of the receptor. We conducted similar cAMP activity assays using MNTX. Our dose-dependent (10nM-1µM) experiments revealed that 1hr exposure of cells to MTNX inhibited cAMP activity, but not after 48 hrs of treatment, consistent with an inverse or partial agonist effect at low doses. To assess the impact of our findings on FaDu cell behaviors, we conducted in vitro proliferation, colony formation, and migration assays. MTNX at different concentrations (0.1nM, 1nM, 10nM, 100nM and 1µM) inhibited FaDu cell proliferative and metastatic cell behaviors (p<0.05). To translate our results to in vivo, we inoculated the tongue of athymic nude mice with FaDu cells (200,000 cells/30 mL). Starting the day of cell inoculation, we randomly treated the animals with MNTX (subcutaneously, 1µg/kg, 10 µg/kg, and 100µg/kg) versus placebo (saline only) for 17 consecutive days. In vivo experiments demonstrated that administration of MTNX at three different concentrations delayed tumor progression and reduced tumor growth and formation compared to placebo (p<0.0001). Overall, our results suggest that MTNX inhibits tumorigenesis of HNSCC, possibly by acting on MOR. 1. Siegel, R.L. et al, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30. 2. Lennon, F.E., et al, The mu-opioid receptor in cancer progression: is there a direct effect? Anesthesiology, 2012. 116(4): p. 940-5. 3. Patino, M.A., et al., The impact of intraoperative opioid use on survival after oral cancer surgery. Oral Oncol, 2017. 74: p. 1-7. Citation Format: Aysegul Gorur, Miguel Patino, Ted Shi, Hideaki Takahashi, Jeffrey N. Myers, Juan P. Cata. The mu-opioid receptor antagonist methylnaltrexone can decrease tumorigenesis in head and neck squamous cell carcinoma [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 5027.

A systematic review and meta-analysis protocol of clinical characteristics and prognostic significance of mammalian target of rapamycin for gastric cancer patients.

Background:The high morbidity and mortality of Gastric cancer (GC) is seriously endangered human health. Owing to the low rate of early diagnosis and human body can resistant to the anti-tumor drugs, so an early diagnostic biology marker is essential. However, recently studies indicated that Mammalian target of rapamycin (mTOR) is usually frequently deregulated in many cancers, especially in GC. And the efficacy of mTOR inhibitor was promising in a phase II clinical trial which could inhibited the proliferation of GC cells and delayed tumor progression. Therefore, mTOR were identified as a potential prognosis biomarker for GC, and its inhibitor will be promising in anti-GC therapy. The main aim of this systematic review and meta-analysis is to investigate the relationships between the expression level and prognostic value of mTOR in patients with GC.Methods:Four electronic databases were systematically searched as follow: the PubMed, EMbase, Web of Science databases, and the Cochrane Library. All the data will be extracted by independent researchers from the eligible studies with the inclusion and exclusion criteria. And the data will be analyzed through STATA 12.0 software.Results and conclusion:This meta-analysis indicated that overexpressed mTOR was significantly in predicting a poorer prognosis for GC patients. The expression level of mTOR should be considered as a potential independent prognostic predictor for GC patients.Protocol registration number:CRD42020159690.

Intestinal microbiota regulates anti‐tumor effect of disulfiram combined with Cu2+ in a mice model

BackgroundA growing number of studies show that intestinal microbiota affect the therapeutic effects of antineoplastic agents. Disulfiram (tetraethylthiuram disulfide, DSF) is an old alcohol‐aversion drug that has been shown to be effective against various types of cancers in preclinical studies, while few studies are carried out to explore its mechanism.MethodsA mice model of melanoma xenograft was generated and treated with antibiotics (Abx), disulfiram/copper (DSF/Cu2+), Abx + DSF/Cu2+, and the tumor volume and survival curve were observed. Hematoxylin‐eosin (HE) staining and western blotting (WB) were used to observe the protein changes related to cell morphology, inflammation, and apoptosis in tumor tissues. Quantitative real time polymerase chain reaction (qPCR) was used to detect the expression of pro‐inflammatory cytokines in tumors. High‐throughput sequencing was used to detect the effects of Abx and DSF/Cu2+ on intestinal microbiota.ResultsThe DSF/Cu2+ and Abx + DSF/Cu2+ markedly delayed tumor progression and prolonged mice survival, of which the combination of Abx and DSF/Cu2+ possessed the best anti‐tumor effect. Abx + DSF/Cu2+ significantly reduced the pro‐inflammatory cytokines Interleukin‐1β (IL‐1β), IL‐6 and tumor necrosis factor α (TNF‐α) in tumors, and significantly reduced the expression of phosphorylated‐protein kinase B (p‐AKT)/protein kinase B (AKT), toll‐like receptors 4 (TLR‐4), and phosphorylated‐ nuclear factor kappa‐B (p‐NFκB)/NFκB in tumors. Moreover our high‐throughput sequencing first indicated that the sound anti‐cancer effect of Abx + DSF/Cu2+ had a strong connection with the increased abundance of intestinal beneficial bacteria Akkermansia, as well as the reduced abundance of opportunistic pathogenic bacteria Campylobacterales, Helicobacteraceae, and Coriobacteriaceae.ConclusionsThe disturbed intestinal microbiota (increased abundance of opportunistic pathogens Campylobacterales, Helicobacteraceae, and Coriobacteriaceae) and the over‐activated TLR4/NF‐κB signaling pathway in tumor tissues deteriorated the cancer development, and the using of antibiotics is benefit to enhance the therapeutic effect of DSF on tumors via inhibiting the growth of opportunistic pathogenic bacteria.

EZH2-Deficient T-cell Acute Lymphoblastic Leukemia Is Sensitized to CHK1 Inhibition through Enhanced Replication Stress.

Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2-deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small-molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2-deficient, but not EZH2 wild-type, T-ALL cells in vivo, as well as in a primary cell model of PRC2-mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene-expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Finally, we demonstrate this phenotype is mediated through derepression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2-mutated T-ALL. SIGNIFICANCE: Loss-of-function mutations of PRC2 genes are associated with chemotherapy resistance in T-ALL, yet no specific therapy for this aggressive subtype is currently clinically available. Our work demonstrates that loss of EZH2 activity leads to MYCN-driven replication stress, resulting in increased sensitivity to CHK1 inhibition, a finding with immediate clinical relevance.This article is highlighted in the In This Issue feature, p. 890.

Abstract A01: Specific targeting of adipose tissue-associated metastasis using bile salt nanoparticles

Abstract Purpose: Mortality in ovarian cancer occurs in the setting of recurrent disease. Recurrent disease is known to arise from residual disease, which persists after the conclusion of standard of care, consequently rebuilding the tumor. Adipose tissue provides cancer cells with a unique microenvironment that can enhance survival by upregulating antiapoptosis genes or by decreasing drug bioavailability. Indeed, adipose-rich organs such as the omentum are not only preferential sites for metastasis but also main sites of residual disease in ovarian cancer. We hypothesized that targeting adipose-rich tissues could limit residual disease and prevent recurrence. Thus, the objective of this study was to develop effective drug delivery platforms that can specifically target residual disease in adipose-rich tissues. Methods: The delivery system is a nanoparticle platform composed of polymerized ursodeoxycholic acid (PUDCA) synthesized by esterification of UDCA monomer. Paclitaxel (PTX) or deep-infrared FL dye (DIR) was encapsulated to a final concentration of 24 ug or 5 mg per mg PUDCA, respectively. In vitro dose response studies were performed using mCherry+ human OCSC1-F2 ovarian cancer cells for 72 h. DMSO-resuspended PTX (DMSO-PTX) was used as control. In vivo studies were performed in athymic nude mice bearing i.p. tumors from mCherry+ OCSC1-F2 cells. For biodistribution studies, DIR-PUDCA was administered as one-time i.p. injection of 1.3 mg of DIR-PUDCA followed by ex vivo measurement of FL intensity after 14 days. DMSO-resuspended DIR (DMSO-DIR) was used as control. For efficacy studies, the groups were: (1) vehicle control; (2) taxol (PTX in Cremaphor; 12mg/kg) + DMSO-PTX (31.2 ug PTX); and (3) taxol + PUDCA-PTX. Treatment was administered i.p. twice a week for a total of 4 doses. Tumor burden and residual disease were determined by quantification of mCherry ROI FL area. Results: PUDCA-PTX demonstrated significantly improved efficacy both in vitro and in vivo. PUDCA-PTX demonstrated a six-fold reduction in in vitro GI50 compared to DMSO-PTX (23.04 nM PUDCA-PTX vs. 137.2 nM DMSO-PTX). Biodistribution studies showed delivery of DIR specifically to adipose-rich tissues (DIR mean FL intensity (MFI) for greater and lesser omentum = 76570±17808 vs. mesentery MFI = 22326±2956; p = 0.0297). Efficacy studies showed that PUDCA-PTX was significantly more effective in inhibiting tumor progression (p = 0.0191) and, more importantly, in preventing recurrent disease compared to DMSO-PTX. Finally, PUDCA-PTX was able to significantly reduce omental-residual disease compared to DMSO-PTX (p=0.049). Conclusion: We demonstrate for the first time that PUDCA is able to target residual disease localized in adipocyte-rich tissues and that this significantly delays tumor progression and prevents recurrence. These results underscore the value of i.p.-delivered PUDCA as a drug delivery platform for enhanced targeting of cancer cells within adipose-rich tissues and its value in the management of ovarian cancer patients. Citation Format: Roslyn Tedja, Ayesha B. Alvero, Jung Seok Lee, Mary Pitruzzello, Tarek Fahmy, Gil G. Mor. Specific targeting of adipose tissue-associated metastasis using bile salt nanoparticles [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A01.

Gene Expression in Human Polymorphonuclear Neutrophils (PMNs) Stimulated by Bacillus Calmette-Guérin (BCG).

Neutrophils are the most abundant leukocytes in the blood. Moreover, neutrophils form the first line of host immune defense against bacterial and fungal invasion, and also play an important part in inflammatory and immune system responses. Intravesical bacillus Calmette-Guérin (BCG) has been shown to reduce and delay tumor progression to muscle-invasive disease after transurethral resection of bladder tumors (TRUBTs). Following intravesical BCG, neutrophils gather around tissues infected by BCG in the early stage of inflammatory and immune responses. In our previous study, we reported that BCG induced the formation of neutrophil extracellular traps (NETs), which play an important role in tumor treatment. Therefore, in the present study, we analyzed the gene expression profile of neutrophils stimulated by BCG through high-throughput arrays, which helped us determine the potential roles of neutrophils in BCG immunotherapy. The results showed that the expression of neutrophil genes led to changes in the early stage of BCG stimulation. The changed genes were involved in many functions of neutrophils such as mobility, proliferation, and secretion of cytokines, chemokines, and adhesion molecules. These changes in neutrophil biological functions may play an essential role in BCG induction of inflammatory and immune responses, and in anti-tumor processes.

Cerenkov luminescence imaging is an effective preclinical tool for assessing colorectal cancer PD-L1 levels in vivo

BackgroundPreclinical and clinical studies have demonstrated that immunotherapy has effectively delayed tumor progression, and the clinical outcomes of anti-PD-1/PD-L1 therapy were related to PD-L1 expression level in the tumors. A 131I-labeled anti-PD-L1 monoclonal antibody tracer, 131I-PD-L1-Mab, was developed to study the target ability of noninvasive Cerenkov luminescence imaging in colorectal cancer xenograft mice.MethodAnti-PD-L1 monoclonal antibody labeled with 131I (131I-PD-L1-Mab), and in vitro binding assays were used to evaluate the affinity of 131I-PD-L1-Mab to PD-L1 and their binding level to different colorectal cancer cells, and compared with flow cytometry, Western blot analysis, and immunofluorescence staining. The clinical application value of 131I-PD-L1-Mab was evaluated through biodistribution and Cerenkov luminescence imaging, and different tumor-bearing models expressing PD-L1 were evaluated.Results131I-PD-L1-Mab showed high affinity to PD-L1, and the equilibrium dissociation constant was 1.069 × 10-9 M. The competitive inhibition assay further confirmed the specific binding ability of 131I-PD-L1-Mab. In four different tumor-bearing models with different PD-L1 expression, the biodistribution and Cerenkov luminescence imaging showed that the RKO tumors demonstrated the highest uptake of the tracer 131I-PD-L1-Mab, with a maximum uptake of 1.613 ± 0.738% IA/g at 48 h.ConclusionsThere is a great potential for 131I-PD-L1-Mab noninvasive Cerenkov luminescence imaging to assess the status of tumor PD-L1 expression and select patients for anti-PD-L1 targeted therapy.

Antitumor and Anti-Metastatic Effects of Citral-Loaded Nanostructured Lipid Carrier in 4T1-Induced Breast Cancer Mouse Model.

Cancer nano-therapy has been progressing rapidly with the introduction of many novel drug delivery systems. The previous study has reported on the in vitro cytotoxicity of citral-loaded nanostructured lipid carrier (NLC-Citral) on MDA-MB-231 cells and some preliminary in vivo antitumor effects on 4T1 breast cancer cells challenged mice. However, the in vivo apoptosis induction and anti-metastatic effects of NLC-Citral have yet to be reported. In this study, the in vitro cytotoxic, anti-migration, and anti-invasion effects of NLC-Citral were tested on 4T1 breast cancer cells. In addition, the in vivo antitumor effects of oral delivery of NLC-Citral was also evaluated on BALB/c mice induced with 4T1 cells. In vitro cytotoxicity results showed that NLC-Citral and citral gave similar IC50 values on 4T1 cells. However, wound healing, migration, and invasion assays reflected better in vitro anti-metastasis potential for NLC-Citral than citral alone. Results from the in vivo study indicated that both NLC-Citral and citral have anti-tumor and anti-metastasis effects, whereby the NLC-Citral showed better efficacy than citral in all experiments. Also, the delay of tumor progression was through the suppression of the c-myc gene expression and induction of apoptosis in the tumor. In addition, the inhibition of metastasis of 4T1 cells to lung and bone marrow by the NLC-Citral and citral treatments was correlated with the downregulation of metastasis-related genes expression including MMP-9, ICAM, iNOS, and NF-kB and the angiogenesis-related proteins including G-CSF alpha, Eotaxin, bFGF, VEGF, IL-1alpha, and M-CSF in the tumor. Moreover, NLC-Citral showed greater downregulation of MMP-9, iNOS, ICAM, Eotaxin, bFGF, VEGF, and M-CSF than citral treatment in the 4T1-challenged mice, which may contribute to the better anti-metastatic effect of the encapsulated citral. This study suggests that NLC is a potential and effective delivery system for citral to target triple-negative breast cancer.