Xenograph Model Research Articles

Abstract 3586: In vivo targeted detection and imaging of ovarian cancer by SPMR and MRI using anti-folate receptor functionalized iron oxide nanoparticles

Abstract Ovarian cancer is the second most common and most deadly gynecological cancer in women. Early and accurate detection is crucial in improving survival rate and quality of life of patients. Folate receptor alpha (FRα) is a folate transporter that has been reported to be overexpressed in approximately 90% of ovarian cancer, which makes it a suitable molecular target to use in developing tumor imaging methods. Iron oxide nanoparticles (NPs) have been used in a variety of cancer detection/imaging applications. Here we show that anti-FRα targeted superparamagnetic iron oxide nanoparticles (SPION) can be used for in vivo detection by two different imaging methods. Superparamagnetic Relaxometry (SPMR) is a highly sensitive in vivo detection technology that is able to differentiate the magnetic signature of nanoparticles bound to tumor cells from unbound nanoparticles. Same targeted SPIONs accumulated in tumor are able to provide molecular magnetic resonance imaging (MRI) contrast as well. Our results have shown that the anti-FRα NPs can distinguish high and low FRα expression cell lines, such as KB and A549, respectively in an in vitro cell based assay by SPMR, demonstrating good sensitivity, specificity and selectivity. In vivo study using xenograph model with KB and A549 cells implanted subcutaneously on the flank region of female athymic nude mice demonstrated higher level tumor accumulation of anti-FRα NPs in KB tumor compared to A549 tumor, measurable by ex vivo tumor detection using SPMR. Using a PEG NPs (without the target ligand) as control for the mice study have shown that minimum nanoparticle tumor accumulated were observed, demonstrating the specificity of the nanoparticles in vivo. Organ distribution study indicated that anti-FRα NPs mostly accumulated in liver and spleen as these nanoparticles were cleared through these two organs. Other organs such as brain, heart, lung, kidney didn’t show appreciable accumulation. The repeat of same type of xenograph mice model used in the SPMR study is currently under the way using MRI as an alternative in vivo detection method with images taken pre- and post- dosing of the nanoparticles. Based on the specificity and sufficient accumulation of nanoparticles demonstrated in the SPMR study, it is expected that MRI will show similar performance with additional anatomical information.Our anti-FRα NPs provided targeted and specific delivery to cancerous tissue and generated measurable signal by SPMR. Furthermore, demonstration of these anti-FRα NPs generating tumor specific contrast in MRI is currently under the way. These studies lay the groundwork for use of bio-safe magnetic particles as a detection and contrast agent for early ovarian cancer detection with improved accuracy to minimize the needs of invasive biopsies/surgery as well as potential utility for monitoring therapy response or recurrence. Citation Format: Marie Zhang, Kathirvel Kandasamy, Yamitha Perera, Dan Inglese, Eric Smith-Nguyen. In vivo targeted detection and imaging of ovarian cancer by SPMR and MRI using anti-folate receptor functionalized iron oxide nanoparticles. [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 3586.

Sonothrombolysis

Cardiovascular disease and stroke are the two leading causes of death worldwide (doi.org/10.1161/CIRCOUTCOMES.118.005375). Sonothrombolysis, ultrasound which accelerates thrombus breakdown in combination with thrombolytic drugs, has been under extensive investigation in the last two decades. An overview of in vitro, preclinical, and clinical sonothrombolysis studies will be provided. In a xenographic porcine cerebral thromboembolism model employing retracted human clots, 220 kHz ultrasound, in conjunction with Definity® increased the probability of early successful reperfusion with rt-PA. Combined treatment with rt-PA, Definity®, and ultrasound invivo increased the rate of reperfusion up to 45 min faster than clots treated with rt-PA or saline alone. Tsivgoulis et al. (doi.org/10.1161/STROKEAHA.120.030960) performed a meta-analysis of sonothrombolysis safety and efficacy data in patients with acute ischemic stroke from 7 randomized-controlled clinical trials. Sonothrombolysis was associated with a 2-fold increase in the odds of complete recanalization compared with intravenous lytic alone. The likelihood of symptomatic intracranial hemorrhage was not significantly different between the 2 groups (7.3% versus 3.7% odds ratio). However, in spite of the higher recanalization rates no difference in 3-month clinical outcomes was uncovered, though this conclusion might have been underpowered statistically. Strategies to improve clinical sonothrombolysis outcomes will be discussed.

Abstract 6269: A cell double barcoding system for quantitative evaluation of primary tumors & metastasis in animals: Uncovers clonal-specific anti-cancer drug effects

Abstract Imaging in monitoring metastasis in mouse models have low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, e.g. drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. Yet, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograph models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations, and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall cell double barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies. Citation Format: Arkadi Hesin, Santosh Kumar, Valid Gahramanov, Maria Becker, Maria Vilenchik, Ilya Alexandrov, Julia Yaglom, Michael Sherman. A cell double barcoding system for quantitative evaluation of primary tumors & metastasis in animals: Uncovers clonal-specific anti-cancer drug effects [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 6269.

Enriching for tumor-reactive CD8 TIL (AGX148) leads to effective tumor clearance in a patient-derived xenograph model

Abstract Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a subset of patients with melanoma; however, the efficacy of standard bulk TIL therapy can be limited. We hypothesize that the therapeutic efficacy of TIL ACT is dependent on the frequency and functionality of tumor-reactive T cells when infused back into patients. Our approach enhances the activity of the ACT product by enriching for the tumor-reactive CD8 TIL prior to ex vivo expansion by sorting on cells that co-express CD103 and CD39. The CD103/CD39 double positive (DP) CD8 TIL can be found in most human solid tumors but at varying frequencies in any given patient. Surgically resected human tumor samples were provided through a collaboration with the EACRI. Tumor samples were flow sorted and expanded ex vivo and patient-matched autologous tumor lines were generated. Tumors were then cocultured with the expanded autologous ACT product (AGX148) in vitro or used in xenograph mouse experiments. The AGX148 TIL products are effective at recognizing and killing autologous tumor cells, in vitro. Upon adoptive transfer into patient-derived tumor-bearing huIL-2 NOG mice, the transferred human T cells increased in frequency, migrated to the tumor, became activated in the tumor and recognized/destroyed established tumors. Interestingly there was little to no in vivo activity of CD8 TIL that did not express these two markers. We have generated a potent therapeutic ACT TIL product (AGX148) through the selective isolation and expansion of tumor-reactive T cells. This TIL product effectively induces durable curative responses in hosts harboring autologous tumors providing rationale for clinical investigation.

MT2-MMP is differentially expressed in multiple myeloma cells and mediates their growth and progression

ObjectiveMembrane type-matrix metalloproteinases (MT-MMPs) are known as key regulators of cancer progression/metastasis. However, their roles in the growth and progression of multiple myeloma (MM) have not been yet elucidated. Methods and materialsThe expression of 6 MT-MMPs in MM, B cell lines, and normal peripheral blood (PB) cells were measured by RT-PCR, qRT-PCR, flow cytometry, western blotting, and immunocytochemistry. B lymphocytes, CD19−/CD138−, and CD19−/CD138+ cells, known as malignant plasma cells (MPC), were sorted from bone marrow (BM) aspirations of 10 MM patients, and MT2-MMP expression was examined in these cells using qRT-PCR, flow cytometry and immunohistochemistry, and western blotting. Moreover, the expression of MT2-MMP in BM biopsies from 13 normal individuals and 14 MM patients was analyzed by immunohistochemistry. MT2-MMP was also knocked down in U266 cells using siRNA technology and the adhesion, invasion, migration abilities, and cell proliferation were determined and compared with scrambled ones in both in vitro and in vivo studies. ResultsOur results showed that MT2-MMP expression is significantly higher in MM cell lines and MPC cells than B cell lines and other PB- or BM-derived cells. MT2-MMP is expressed in BM biopsies from all 14 patients with MM, and 67.85% ± 32.38 of BM cells were positive for MT2-MMP. In contrast, only 0.38 ± 0.76 of BM biopsies from normal individuals were positive for MT2-MMP. Importantly, MT2-MMP was expressed in all the patients' BM biopsies at the diagnosis, but not in the remission phase. MT2-MMP siRNA significantly decreased adhesion, invasion, migration, and 3D cell proliferation of U266 cells. Moreover, in the xenographic model, MT2-MMP siRNA prevented the growth and development of plasmacytoma. Taken together, these data demonstrate that MT2-MMP is strongly expressed in MM cells and plays important role in the growth and progression of these cells, suggesting that MT2-MMP is an appropriate biomarker in diagnosis and therapeutic interventions of MM.

Epigenetic Priming with Pre-Transplant Oral Panobinostat Followed By Post-Transplant Consolidation

Despite the rich armamentarium of novel agents that are available to treat multiple myeloma (MM), high dose melphalan (HDM) and stem cell transplant (SCT) remain an essential therapy that achieves long-term durable remissions. However, most patients relapse following HDM-SCT due to the persistence of minimal residual disease (MRD). Therefore, there remains an unmet clinical need to overcome chemoresistant MRD and potentially achieve a cure for MM patients. Here, we describe a phase Ib trial designed to epigenetically priming with pre-transplant oral panobinostat to increase MRD sensitivity to HDM-SCT.The contribution of dysregulated gene silencing to epigenomic alterations in cancer development provides a strong rationale for the use of epigenetic modulators, such as the histone deacetylase (HDAC) inhibitor panobinstat as anti-myeloma therapy. Access to DNA is primarily governed by chromatin structure such that its configuration is under control of histone acetylators and deacetylators. Inhibition of histone deacetylases weakens the histone-DNA bonds and decondenses chromatin, i.e., epigenetic priming, which potentially enhances sensitivity to melphalan. Pre-clinical studies have demonstrated a strong synergistic effect between panobionostat (LBH589) and melphalan in in vitro as well as in vivo studies utilizing patient-derived xenograph models, i.e., SCID-hu model of human MM, LAG-1(Sanchez et al. Leukemia research. March 2011). Furthermore, HDAC inhibitors suppress multi-drug resistance protein-1 (MRP1) and it can count as another mechanism of synergy for this class of drugs and alkylating agents in hematologic disorders (Tsubaki M. et al. Leukemia research. Oct 2012). Taken together, these studies support HDAC inhibitors in combination with melphalan to augment anti-myeloma efficacy of HDM-SCT. Here, we propose the combination through a pre-SCT epigenetic priming phase with high dose panobinostat and a post-SCT lower dose panobinostat consolidation phase (Study Schema).Trial design: We propose a Simon 3+3 design to test panobinostat at three dose levels of 20, 30 and 40 mg every other day for four doses prior to high dose melphalan as study schema illustrates. There will not be any intra-patient dose escalation. The study will be extended to another 15 patients at the maximum tolerated dose for the pre-SCT phase. All patients regardless of the pre-SCT panobinostat dose level will received fixed dose of Panobinostat. Primary and Secondary objectives and endpoints are listed in Table 1.Rationale for high dose panobinostat in epigenomic phase: Previous reports of HDACi combined with standard-dose chemotherapy (e.g., Ifosfomide, Carboplatin, and Etoposide for lymphoma or 7+3 for acute myeloid leukemia) had shown prolonged myelosuppression as the main adverse effect, but no significant increase in the non-hematological toxicities. Therefore, we predict the use of peripheral blood progenitor cells in the offered study has potential to circumvent the increased myelotoxicity associated with pre-SCT high dose panobinostat, and it may render the opportunity to test higher doses such as 40 mg every other day before SCT as epigenetic priming strategy.Rational for low dose panobinostat dosing for the consolidation phase: The Panobinostat consolidation design will follow the positive landmark trial by the Australian group (Mithraprabhu S. et al. British J. Hemat. Apr 2021). In their study panobinostat was given at 20 mg three times weekly on alternate weeks for 6 months started 8-12 weeks post-transplant was administered. Forty eight percent of patients improved their depth of response after a median range of 4.3 months of panobinostat. In responders, T lymphocyte histone acetylation increased after both three cycles and six cycles of panobinostat when compare to baseline, with no differences in no-responders. [Display omitted] DisclosuresMalek: Janssen: Other: Advisory board ; Bluespark Inc.: Research Funding; BMS: Honoraria, Research Funding; Amgen: Honoraria; Cumberland Inc.: Research Funding; Sanofi: Other: Advisory Board; Medpacto Inc.: Research Funding; Takeda: Honoraria. Metheny: Incyte: Speakers Bureau; Pharmacosmos: Honoraria. Stricker: Secura Bio: Current Employment. OffLabel Disclosure:Panobinostat has been used as single agent therapy before and after transplant in this trial.

Ruthenium(II) Diphosphine Complexes with Mercapto Ligands That Inhibit Topoisomerase IB and Suppress Tumor Growth In Vivo.

Ruthenium(II) complexes (Ru1-Ru5), with the general formula [Ru(N-S)(dppe)2]PF6, bearing two 1,2-bis(diphenylphosphino)ethane (dppe) ligands and a series of mercapto ligands (N-S), have been developed. The combination of these ligands in the complexes endowed hydrophobic species with high cytotoxic activity against five cancer cell lines. For the A549 (lung) and MDA-MB-231 (breast) cancer cell lines, the IC50 values of the complexes were 288- to 14-fold lower when compared to cisplatin. Furthermore, the complexes were selective for the A549 and MDA-MB-231 cancer cell lines compared to the MRC-5 nontumor cell line. The multitarget character of the complexes was investigated by using calf thymus DNA (CT DNA), human serum albumin, and human topoisomerase IB (hTopIB). The complexes potently inhibited hTopIB. In particular, complex [Ru(dmp)(dppe)2]PF6 (Ru3), bearing the 4,6-diamino-2-mercaptopyrimidine (dmp) ligand, effectively inhibited hTopIB by acting on both the cleavage and religation steps of the catalytic cycle of this enzyme. Molecular docking showed that the Ru1-Ru5 complexes have binding affinity by active sites on the hTopI and hTopI-DNA, mainly via π-alkyl and alkyl hydrophobic interactions, as well as through hydrogen bonds. Complex Ru3 displayed significant antitumor activity against murine melanoma in mouse xenograph models, but this complex did not damage DNA, as revealed by Ames and micronucleus tests.

P38 MAPK and NF-κB Survival Pathways As Targets for Action of Rituximab In Vivo in a Xenographic Model,

Treatment of B-NHL cell lines with rituximab has been shown to inhibit the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene). Chemosensitization was a direct consequence of the inhibition of the Bcl-2 / Bcl-xL anti-apoptotic proteins. It has recently shown for us (Vega, et al BMC-Cancer 2015), that activation and expression of p38 MAPK correlate with a poor prognosis in patients with DLBCL-NHL treated with CHOP alone. However, cellular signaling by rituximab in tissue from patients with B-NHL has not been described, and therefore we have examined in an in vivo model that carries a tumor xenograft for validation. Balb/c nu/nu mice were inoculated s.c. with the B-NHL Raji cell line; one group of mice was left untreated, and another group was treated with rituximab (30 mg/kg) at days 5 and 10 after implantation with 8x106 Raji cells. We monitored the tumor growth, and the animals sacrificed at day 30, the tumors collected for further analysis. Treatment with rituximab resulted in significant growth inhibition of tumor cells compared to the control. The IHC examination of the tissue tumors for the expression of phospho and nonphospho-total-p38 MAPK, NF-κB (phospho p65), and Bcl-xL revealed an overexpression of phospho-p38 MAPK, Bcl-xL, and NF-κB with a strong nuclear localization of phospho-p65. In contrast, tumor tissues derived from rituximab treated mice demonstrated a significant inhibition of phospho-p38 MAPK, Bcl-xL, NF-κB, and a reduction of nuclear localization of phospho-p65. We also analyzed the expression of CD20 in tumors and its correlation with the expression of phospho-p38 MAPK. Furthermore, we inoculated a group of mice with Ramos cells (8X106), and two weeks post-incubation the tumors were collected, and their expression of phospho-p38 MAPK and NF-κB analyzed. A section of the tumor was re-inoculated into another group of mice, and we repeated this procedure three more times. The re-inoculated tumors showed an increased phospho-p38 MAPK and NF-κB expression. These findings established for the first time, in a preclinical model, the rituximab-mediated inhibition of cell survival pathways mediated by p38 MAPK and NF-κB. Moreover, the study also corroborates the role of Bcl-xL expression in resistance and its inhibition by rituximab. It also allows establishing that p38 MAPK activation correlates with tumor malignancy in a tumor re-transplantation model. Overall, this study suggests that the survival pathways of p38 MAPK and NF-κB are targets for rituximab-mediated effects and suggests these proteins can use for intervention in cases of resistance to rituximab and p38 MAPK as well as a prognostic factor.Vega MI, et al Rituximab inhibits p38 MAPK activity in 2F7 B NHL and decreases IL-10 transcription: pivotal role of p38 MAPK in drug resistance. Oncogene. 2004;23(20):3530-40Vega GG, et al. P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma. BMC Cancer. 2015;15:722. DisclosuresNo relevant conflicts of interest to declare.

Vincristine-induced peripheral neuropathy is driven by canonical NLRP3 activation and IL-1β release.

Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.

Following cancer through the body

Cancer Genomics The heterogeneity of mammalian tumors has been well documented, but it remains unknown how differences between individual cells lead to metastasis and spread throughout the body. Quinn et al. created a Cas9-based lineage tracer and used single-cell sequencing to generate phylogenies and follow the movement of metastatic human cancer cells implanted in the lung of a mouse xenograph model. Using this model, they found that within the same cell line, cancer cells exhibited diverse metastatic phenotypes. These subclones exhibited differential gene expression profiles, some of which were previously associated with metastasis. Science , this issue p. [eabc1944][1] [1]: /lookup/doi/10.1126/science.abc1944

Accelerated sonothrombolysis with Definity in a xenographic porcine cerebral thromboembolism model

Adjuvant ultrasound at 2 MHz with or without an ultrasound contrast agent improves the rate of thrombus resolution by recombinant tissue plasminogen activator (rt-PA) in laboratory and clinical studies. A sub-megahertz approach can further expand this therapy to a subset of patients with an insufficient temporal bone window, improving efficacy in unselected patient populations. The aim of this study was to determine if a clinical ultrasound contrast agent (UCA), Definity, and 220 kHz pulsed ultrasound accelerated rt-PA thrombolysis in a preclinical animal model of vascular occlusion. The effect of Definity and ultrasound on thrombus clearance was first investigated in vitro and subsequently tested in a xenographic porcine cerebral thromboembolism model in vivo. Two different microcatheter designs (end-hole, multi-side-hole) were used to infuse rt-PA and Definity at the proximal edge or directly into clots, respectively. Sonothrombolysis with Definity increased clot mass loss relative to saline or rt-PA alone in vitro, only when rt-PA was administered directly into clots via a multi-side-hole microcatheter. Combined treatment with rt-PA, Definity, and ultrasound in vivo increased the rate of reperfusion up to 45 min faster than clots treated with rt-PA or saline. In this porcine cerebral thromboembolism model employing retracted human clots, 220 kHz ultrasound, in conjunction with Definity increased the probability of early successful reperfusion with rt-PA.

BMI1 is directly regulated by androgen receptor to promote castration-resistance in prostate cancer.

B lymphoma Mo-MLV insertion region 1 (BMI1) has been reported to be an oncoprotein. BMI1 represses tumor suppressors to promote cell proliferation, epithelial-mesenchymal transition (EMT), and cancer progression. Although it is known that the expression of BMI1 is increased in many cancer types, the mechanism of BMI1 upregulation is not yet clear. We performed integrative analysis for 3 sets of prostate cancer (PCa) genomic data, and found that BMI1 and androgen receptor (AR) were positively correlated, suggesting that AR might regulate BMI1. Next, we showed that dihydrotestosterone (DHT) upregulated both mRNA and protein levels of BMI1 and that BMI1 was increased in castration-resistant prostate cancer (CRPC) from both human patients and a mouse xenograph model. We further identified an AR binding site in the promoter/enhancer region of BMI1, and confirmed BMI1 as the direct target of AR using gene-editing technology. We also demonstrated that high expression of BMI1 is critical for the development of castration-resistance. Our data also suggest that BMI1-specific inhibitors could be an effective treatment of CRPC.

Abstract 1931: Investigating the PK/PD/efficacy relationship of PI3K inhibitors in vitro, enabled by a microfluidic addition and removal device

Abstract Characterizing the relationship between pharmacokinetics (PK), pharmacodynamics (PD) and efficacy is critical in the discovery and development of new drugs, schedules and combinations. The PK/PD/efficacy relationship has historically been characterized in xenograft models, owing to an absence of viable alternatives. The study of this relationship in vitro, has to date been problematic as the generation of time varying concentrations in multi-well plates has not been possible. We have explored an in vitro methodology utilizing a device (Microformulator, Viibre, Vanderbilt University) capable of precision addition and removal of medium from the wells of a microtiter cell culture plate. Through stepwise addition and removal of medium the device was able to recapitulate PK-like, time varying concentration profiles of one, or more drugs in individual wells. We used this approach to explore the effects of PI3K pathway inhibitors, on p-AKT levels, and viability in a number of cancer lines. The PI3K/mTOR pathway is a central oncogenic pathway deregulated in cancer and p-AKT is a marker of PI3K pathway activity. BYL719 and PI-103, are PI3K inhibitors with varying PK profiles and varying target coverage against PI3K isoforms. The compounds were tested on a pair of cell lines, T47D (breast, sensitive) and Colo205 (gut, insensitive). Both compounds were potent in a bolus dose 72hr assay with BYL719 having an IC50 of 0.63 µM for T47D and 15.8 µM for Colo205. Whilst PI-103 gave IC50 of 0.72 µM for T47D and 4.16 µM for Colo205. Both compounds also reduced, p-AKT in a dose-dependent manner after 2 hours. Using the microfluidic device, we generated PK-like dosing profiles for the two compounds, (BYL719 - t1/2 6 hours) (PI-103 - t1/2 3 hours) which mimicked their in vivo clearance profiles over a 24-hour period. p-AKT levels were initially reduced by both compounds but recovered at 24 hours as the concentration of the compound in the well declined. This replicates findings in xenograph models. The shorter t1/2 of PI-103, resulted in a more rapid recovery of p-AKT than observed for BYL719. Efficacy tests were conducted in which a 24 hour PK-like profile was applied for 3 consecutive days, mimicking daily dosing. For both compounds in T47D cells, the inhibition of cell growth was reduced as compared to a bolus addition at the Cmax concentration. These data demonstrate how PI-103 initially appeared to be an efficacious molecule but its rapid in vivo metabolism meant it could not be progressed in clinical studies. In conclusion, this study demonstrates the microfluidic addition and removal device can be used to recapitulate PK-like profiles in vitro and allow exploration of the PK/PD/efficacy relationship. The availability of an in vitro method will enable these important parameters to be determined at an earlier stage of the drug discovery process. Citation Format: Tomasz M. Kostrzewski, Dharaminder Singh, Paul Golby, Alysha Bray, David Hughes. Investigating the PK/PD/efficacy relationship of PI3K inhibitors in vitro, enabled by a microfluidic addition and removal device [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 1931.

MiR-450a Acts as a Tumor Suppressor in Ovarian Cancer by Regulating Energy Metabolism.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. SIGNIFICANCE: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3294/F1.large.jpg.

Lasofoxifene as a potential treatment for ER+ metastatic breast cancer.

1056 Background: Estrogen receptor positive (ER+) metastatic breast cancers (MBC) that express constitutively active somatic ESR1 mutations at Y537S and D538G allow tumors to progress in the presence of approved endocrine therapies. For patients with ER+ MBC, fulvestrant is the first line of treatment. Palbociclib or other CDK4/6 inhibitors are now being included. Preliminary studies show that lasofoxifene, a selective ERα modulator (SERM), was effective in reducing tumor growth in an endocrine resistant xenograph model expressing ERα mutations Y537S or D538G. Additionally, lasofoxifene more effectively inhibited the development of liver and lung metastases than fulvestrant. Lasofoxifene is currently under evaluation in a phase 2 study. Because certain combinations of a hormonal agents like fulvestrant improved efficacy, we investigated the combination of palbociclib and lasofoxifene as a potential therapeutic for mutant ESR1 MBC. We hypothesized that this combination should improve outcome and compared it to a combination with fulvestrant. Methods: We first determined the optimal dose of lasofoxifene in an intraductal (MIND) xenograph model of MCF-7 cells that express active ERα Y537S and D538G. Subsequently, we performed combination studies with lasofoxifene (10mg/kg 5/week SQ) +/- palbociclib (100mg/kg gavage, 5/week) or fulvestrant (5mg/mouse/week, SQ) +/- palbociclib. Results: Lasofoxifene alone was significantly more effective than fulvestrant at inhibiting the metastasis of both MCF7 Y537S and D538G tumors to the lungs and liver. Lasofoxifene + palbociclib was more effective than fulvestrant + palbociclib at reducing primary tumor growth; both combinations demonstrated an increased response. Lasofoxifene + palbociclib was more effective at inhibiting liver metastasis than either drug alone and was more effective than fulvestrant + palbociclib at reducing metastasis to the liver and lung. Structural studies showed that lasofoxifene effectively disrupts the active conformation of the ERα Y537S ligand-binding domain. Conclusions: These results demonstrate that lasofoxifene, in combination with CDK4/6 inhibitors like palbociclib, has promise for treating endocrine therapy resistant ER+ MBC patients whose tumors express activating ESR1 mutations, more effectively than either drug alone.

Melatonin Differentially Modulates NF-кB Expression in Breast and Liver Cancer Cells.

NF-kB (nuclear factor kappa B) is a transcription factor composed of two subunits, p50 and p65, which plays a key role in the inflammatory process. Melatonin has oncostatic, antiangiogenic and antimetastatic properties, and some recent studies have indicated an inhibitory effect of melatonin on NF-kB in some types of cancer. This work aims to investigate the effects of melatonin treatment on the expression of NFkB in breast and liver cancer models. The breast cancer xenographic model was performed using female Balb/c nude athymic mice injected with MDA-MB-231 cells. The animals were treated with 40 mg/Kg of melatonin for 21 days. Volume of the tumors was measured with a digital caliper. Hepatocarcinoma model was developed by using the HepG2 cells in vitro, treated with 1 mM melatonin for 24 h. The expression of NF-kB protein was verified by immunohistochemistry and immunocytochemistry and quantified by optical densitometry, in vivo study and in vitro study, respectively. NF-kB gene expression was performed by quantitative RT-PCR. The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size (P=0.0022). There was a decrease in NF-kB protein staining (P=0.0027) and gene expression (P=0.0185) in mice treated with melatonin. The opposite results were observed for the hepatocarcinoma model. HepG2 cells treated with melatonin showed an increase in the NF-kB immunostaining when compared to control cells (P=0.0042). Our results indicated that the treatment with melatonin was able to decrease both gene and protein expressions of NF-kB in breast cancer cells and, conversely, increase the transcription factor protein expression in hepatocarcinoma cells. These data highlighted a double role in the expression of NF-kB, depending on the cell type. Further studies are needed to better elucidate the action of melatonin in NF-kB, since this transcription factor acts on different signaling pathways that are fundamental for carcinogenesis.

Abstract B005: Ginger consumption inhibits β-arrestin-2 expression and functions in melanoma and prostate cancer cells

Abstract Ginger active components are widely known for being a potent antioxidant and recently as a potential anticancer agent. [6]-Gingerol, the most abundant and pungent bioactive component of ginger, was shown to inhibit angiogenesis and tumorigenesis in xenograph models of both prostate cancer (PCa) and melanoma. To date, the precise mechanism(s) for the antitumor effect of ginger is not well understood. The adaptor proteins, β-arrestins, have been shown to modulate tumor development and metastasis in both PCa and melanoma. In this study, we sought to determine the role of [6]-gingerol in β-arrestin (βarr) 1 and 2-mediated PCa and melanoma development, progression, and metastasis. To that end, the PCa cells PC-3 and the melanoma B16-F10 cells were treated with different concentrations of [6]-gingerol for 48 hours. Cell lysates were assayed by Western blot analysis for βarr-1 and βarr-2, CXCR1, and CXCR2 expression, as well as MAP kinase and transcription factors activation. The data demonstrated that pretreatment with [6]-gingerol caused a dose-dependent inhibition of βarr-2, but not βarr-1, in both cell lines, which correlated with significant decrease in Akt and NF-κB activation. [6]-Gingerol pretreatment decreased CXCR1 and CXCR2 expression, CXCL-8-induced intracellular calcium mobilization; and delayed wound closure. [6]-Gingerol exposure also decreased PC-3 and B16 metastasis in zebrafish. Altogether the data indicated that the protective effect of [6]-gingerol in tumorigenesis is likely mediated via a βarr-2-dependent mechanism. Citation Format: Tonelia A. Mowart, Timothy Adekoya, Nikia Smith, Tonya S. Lane, Ricardo M. Richardson. Ginger consumption inhibits β-arrestin-2 expression and functions in melanoma and prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B005.

Alterations in the p53-SOCS2 axis contribute to tumor growth in colon cancer

Altered expression of suppressor of cytokine signaling (SOCS) is found in various tumors. However, regulation of SOCS2 by upstream molecules has yet to be clearly elucidated, particularly in tumor cells. SCOCS2 expression was examined in tumor cells transfected with an inducible p53 expression system. The impact of SOCS2 on cell proliferation was measured with in vitro assays. Inhibition of tumorigenicity by SOCS2 knockdown was assessed via a mouse model. Expression profiles were compared and genes differentially expressed were identified using four types of p53-null cells (Saos, HLK3, PC3, and H1299) and the same cells stably expressing p53. Twelve kinds of target genes were simultaneously upregulated or downregulated by p53 in three or more sets of p53-null cells. SOCS2 expression was reciprocally inhibited by inducible p53 expression in p53-null cells, even colon cancer cells. SOCS2 promoter activity was inhibited by wild type but not mutant p53. SOCS2 knockdown inhibited tumor growth in vitro and in an animal xenograph model. SOCS2 overexpression was detected in a murine model of azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer compared to mock-treated controls. SOCS2 expression was heterogeneously upregulated in some human colon cancers. Thus, SOCS2 was upregulated by p53 dysfunction and seemed to be associated with the tumorigenic potential of colon cancer.

Downregulation of glycine decarboxylase enhanced cofilin-mediated migration in hepatocellular carcinoma cells.

Metabolic reprogramming is a hallmark of cancer. Glycine decarboxylase (GLDC), an oxidoreductase, plays an important role in amino acid metabolism. While GLDC promotes tumor initiation and proliferation in non-small cell lung cancer and glioma and it was reported as a putative tumor suppressor gene in gastric cancer, the role of GLDC in hepatocellular carcinoma (HCC) is unknown. In the current study, microarray-based analysis suggested that GLDC expression was low in highly malignant HCC cell lines, and clinicopathological analysis revealed a decrease in GLDC in HCC tumor samples. While the knockdown of GLDC enhanced cancer cell migration and invasion, GLDC overexpression inhibited them. Mechanistic studies revealed that GLDC knockdown increased the levels of reactive oxygen species (ROS) and decreased the ratio of glutathione/oxidized glutathione (GSH/GSSG), which in turn dampened the ubiquitination of cofilin, a key regulator of actin polymerization. Consequently, the protein level of cofilin was elevated, which accounted for the increase in cell migration. The overexpression of GLDC reversed the phenotype. Treatment with N-acetyl-L-cysteine decreased the protein level of cofilin while treatment with H2O2 increased it, further confirming the role of ROS in regulating cofilin degradation. In a tumor xenographic transplant nude mouse model, the knockdown of GLDC promoted intrahepatic metastasis of HCC while GLDC overexpression inhibited it. Our data indicate that GLDC downregulation decreases ROS-mediated ubiquitination of cofilin to enhance HCC progression and intrahepatic metastasis.

Implication of STARD5 and cholesterol homeostasis disturbance in the endoplasmic reticulum stress-related response induced by pro-apoptotic aminosteroid RM-133

The aminosteroid derivative RM-133 is an effective anticancer molecule for which proof of concept has been achieved in several mouse xenograph models (HL-60, MCF-7, PANC-1 and OVCAR-3). To promote this new family of molecules toward a clinical phase 1 trial, the mechanism of action governing the anticancer properties of the representative candidate RM-133 needs to be characterized. In vitro experiments were first used to determine that RM-133 causes apoptosis in cancer cells. Then, using proteomic and transcriptomic experiments, RM-133 cytotoxicity was proven to be achieved via the endoplasmic reticulum (ER)-related apoptosis, which characterizes RM-133 as an endoplasmic reticulum stress aggravator (ERSA) anticancer drug. Furthermore, an shRNA-genome-wide screening has permitted to identify the steroidogenic acute regulator-related lipid transfer protein 5 (STARD5) as a major player in the RM-133 ER-related apoptosis mechanism, which was validated by an in vitro binding experiment. Altogether, the results presented herein suggest that RM-133 provokes a disturbance of cholesterol homeostasis via the implication of STARD5, which delivers an ERSA molecule to the ER. These results will be a springboard for RM-133 in its path toward clinical use.