Tumor-bearing Animals Research Articles

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

Anti-gastric cancer effects of Z Ajoene and its molecular mechanisms

Objective: To investigate the effects of Z Ajoene on gastric cancer cell MGC-803 and its molecular mechanisms. Methods: The gastric cancer cells MGC-803 were treated with 0, 1, 5, 25 and 125 μmol/L Z Ajoene for 24 h, 48 h and 72 h, each with 3 replicate wells. The proliferation activity of MGC-803 cells was analyzed by MTS method, mitochondrial membrane potential was analyzed after JC-1 staining, nuclear type was observed after Hoechst 33342 staining, cytotoxicity was detected by LDH release method, and the apoptosis level and cell cycle were analyzed with flow cytometry. RT-qPCR and Western blot methods were used to evaluate the expression levels of P53, Caspase-3, RAS, ERK, BCL-2, AKT, mTOR and PI3K genes. At the same time, 4-week-old male BALB/C mice were randomly divided into 5 groups, 20 per group, and were subcutaneously inoculated with gastric cancer cell MGC-803 in the groin. Two days later, each group was injected with Z Ajoene at the doses of 0, 1, 5, 25 and 125 μmol/L, 0.1 ml/time, and was injected every other day. On the 20th day of the first injection of tumor cells, 10 mice in each group were killed, the tumor tissues were taken out and weighed. The survival period of the remaining mice was recorded and the effects of Z Ajoene on the growth and survival period of gastric cancer in tumor-bearing mice were observed. Results: After Z Ajoene treatment, the proliferation activity of MGC-803 cells was significantly inhibited and the apoptosis rate was significantly increased(P＜0.01). The transcription and expression levels of p53, Caspase-3 and BAX genes were significantly increased, while the transcription and expression levels of RAS, ERK1, BCL-2, AKT, mTOR and PI3K genes were decreased markedly(P＜0.01). The tumor inhibition experiments showed that the growth of the tumor could be inhibited and the survival time of the tumor-bearing animals could be greatly prolonged after Z Ajoene treatment(P＜0.01). Conclusion: Z Ajoene has therapeutic effects on gastric cancer, can inhibit the proliferation of gastric cancer cells and induce them apoptosis by regulating the expression of PI3K-AKT-mTOR and RAS-RAF-MEK-ERK signal pathways.

Nanocurcumin-Loaded UCNPs for Cancer Theranostics: Physicochemical Properties, In Vitro Toxicity, and In Vivo Imaging Studies.

Formulation of promising anticancer herbal drug curcumin as a nanoscale-sized curcumin (nanocurcumin) improved its delivery to cells and organisms both in vitro and in vivo. We report on coupling nanocurcumin with upconversion nanoparticles (UCNPs) using Poly (lactic-co-glycolic Acid) (PLGA) to endow visualisation in the near-infrared transparency window. Nanocurcumin was prepared by solvent-antisolvent method. NaYF4:Yb,Er (UCNP1) and NaYF4:Yb,Tm (UCNP2) nanoparticles were synthesised by reverse microemulsion method and then functionalized it with PLGA to form UCNP-PLGA nanocarrier followed up by loading with the solvent-antisolvent process synthesized herbal nanocurcumin. The UCNP samples were extensively characterised with XRD, Raman, FTIR, DSC, TGA, UV-VIS-NIR spectrophotometer, Upconversion spectrofluorometer, HRSEM, EDAX and Zeta Potential analyses. UCNP1-PLGA-nanocurcumin exhibited emission at 520, 540, 660 nm and UCNP2-PLGA-nanocurmin showed emission at 480 and 800 nm spectral bands. UCNP-PLGA-nanocurcumin incubated with rat glioblastoma cells demonstrated moderate cytotoxicity, 60–80% cell viability at 0.12–0.02 mg/mL marginally suitable for therapeutic applications. The cytotoxicity of UCNPs evaluated in tumour spheroids models confirmed UCNP-PLGA-nanocurcumin therapeutic potential. As-synthesised curcumin-loaded nanocomplexes were administered in tumour-bearing laboratory animals (Lewis lung cancer model) and showed adequate contrast to enable in vivo and ex vivo study of UCNP-PLGA-nanocurcumin bio distribution in organs, with dominant distribution in the liver and lungs. Our studies demonstrate promise of nanocurcumin-loaded upconversion nanoparticles for theranostics applications.

The Ability of the Nitric Oxide Synthases Inhibitor T1023 to Selectively Protect the Non-Malignant Tissues.

Previously, we showed that a nitric oxide synthase (NOS) inhibitor, compound T1023, induces transient hypoxia and prevents acute radiation syndrome (ARS) in mice. Significant efficacy (according to various tests, dose modifying factor (DMF)—1.6–1.9 against H-ARS/G-ARS) and safety in radioprotective doses (1/5–1/4 LD10) became the reason for testing its ability to prevent complications of tumor radiation therapy (RT). Research methods included studying T1023 effects on skin acute radiation reactions (RSR) in rats and mice without tumors and in tumor-bearing animals. The effects were evaluated using clinical, morphological and histological techniques as well as RTOG classification. T1023 administration prior to irradiation significantly limited the severity of acute RSR. This was due to a decrease in radiation alteration of the skin and underlying tissues, and the preservation of the functional activity of cell populations that are critical in the pathogenesis of radiation burn. The DMF values for T1023 for skin protection were 1.4–1.7. Moreover, its radioprotective effect was fully selective to normal tissues in RT models of solid tumors—T1023 reduced the severity of acute RSR and did not modify the antitumor effects of γ-radiation. The results indicate that T1023 can selectively protect the non-malignant tissues against γ-radiation due to hypoxic mechanism of action and potentiate opportunities of NOS inhibitors in RT complications prevention.

Use of FVB Myc-CaP cells as an immune competent, androgen receptor positive, mouse model of prostate cancer bone metastasis

Prostate cancer (PCa) metastasis research has been hamstrung by lack of animal models that closely resemble the disease present in most patients – that metastasize to bone, are dependent on the androgen receptor (AR), and grow in an immune competent host. Here, we adapt the Myc-CaP cell line for use as a PCa androgen dependent, immune competent bone metastases model and characterize the metastases. After injection into the left cardiac ventricle of syngeneic FVB/NJ mice, these cells formed bone metastases in the majority of animals; easily visible on H&E sections and confirmed by immunohistochemistry for Ar and epithelial cell adhesion molecule. Mediastinal tumors were also observed. We also labeled Myc-CaP cells with tdTomato, and confirmed the presence of cancer cells in bone by flow cytometry. To adapt the model to a bone predominant metastasis pattern and further examine the bone phenotype, we labeled the cells with luciferase, injected in the tibia and observed tumor formation only in tibia with a mixed osteolytic/osteoblastic phenotype. The presence of Myc-CaP tumors significantly increased tibia bone volume as compared to sham injected controls. The osteoclast marker, TRAcP-5b was not significantly changed in plasma from tibial tumor bearing animals vs. sham animals. However, conditioned media from Myc-CaP cells stimulated osteoclast formation in vitro from FVB/NJ mouse bone marrow. Overall, Myc-CaP cells injected in the left ventricle or tibia of syngeneic mice recapitulate key aspects of human metastatic PCa.

Targeted Delivery of Liposomal Temozolomide Enhanced Anti-Glioblastoma Efficacy through Ultrasound-Mediated Blood-Brain Barrier Opening.

Glioblastoma (GBM) is the commonest form of primary brain tumor in the central nervous system, with median survival below 15 months and only a 25% two-year survival rate for patients. One of the major clinical challenges in treating GBM is the presence of the blood–brain barrier (BBB), which greatly limits the availability of therapeutic drugs to the tumor. Ultrasound-mediated BBB opening provides a promising approach to help deliver drugs to brain tumors. The use of temozolomide (TMZ) in the clinical treatment of GBM has been shown to be able to increase survival in patients with GBM, but this improvement is still trivial. In this study, we developed a liposomal temozolomide formulation (TMZ-lipo) and locally delivered these nanoparticles into GBM through ultrasound-mediated BBB opening technology, significantly suppressing tumor growth and prolonging tumor-bearing animal survival. No significant side effects were observed in comparison with control rats. Our study provides a novel strategy to improve the efficacy of TMZ against GBM.

High Power Electromagnetic Waves Exposure of Healthy and Tumor Bearing Mice: Assessment of Effects on Mice Growth, Behavior, Tumor Growth, and Vessel Permeabilization.

High power radiofrequencies may transiently or permanently disrupt the functioning of electronic devices, but their effect on living systems remains unknown. With the aim to evaluate the safety and biological effects of narrow-band and wide-band high-power electromagnetic (HPEM) waves, we studied their effects upon exposure of healthy and tumor-bearing mice. In field experiments, the exposure to 1.5 GHz narrow-band electromagnetic fields with the incident amplitude peak value level in the range of 40 kV/m and 150 MHz wide-band electric fields with the amplitude peak value in the range of 200 kV/m, did not alter healthy and tumor-bearing animals’ growth, nor it had any impact on cutaneous murine tumors’ growth. While we did not observe any noticeable behavioral changes in mice during the exposure to narrow-band signals when wide-band HPEM signals were applied, mice could behave in a similar way as they respond to loud noise signals: namely, if a mouse was exploring the cage prior to signal application, it returned to companion mates when wide-band HPEM signals were applied. Moreover, the effect of wide-band signals was assessed on normal blood vessels permeability in real-time in dorsal-chamber-bearing mice exposed in a pilot study using wide-band signal applicators. Our pilot study conducted within the applicator and performed at the laboratory scale suggests that the exposure to wide-band signals with the amplitude of 47.5 kV/m does not result in increased vessel permeability.

Effects induced by a 50 HZ electromagnetic field and doxorubicin on Walker-256 carcinosarcoma growth and hepatic redox state in rats

ABSTRACT We compare the effects of an extremely low-frequency electromagnetic field (EMF) with the chemotherapeutic agent doxorubicin (DOX) on tumor growth and the hepatic redox state in Walker-256 carcinosarcoma-bearing rats. Animals were divided into five groups with one control (no tumor) and four tumor-bearing groups: no treatment, DOX, DOX combined with EMF and EMF. While DOX and DOX + EMF provided greater inhibition of tumor growth, treatment with EMF alone resulted in some level of antitumor effect (p < .05). Superoxide dismutase, catalase activity and glutathione content were significantly decreased in the liver of tumor-bearing animals as compared with the control group (p < .05). The decreases in antioxidant defenses accompanied histological findings of suspected liver damage. However, hepatic levels of thiobarbituric acid reactive substances, an indicator of lipid peroxidation, were three times lower in EMF and DOX + EMF groups than in no treatment and DOX (p < .05). EMF and DOX + EMF showed significantly lower activity of serum ALT than DOX alone (p < .05). These results indicate that EMF treatment can inhibit tumor growth, causing less pronounced oxidative stress damage to the liver. Therefore, EMF can be used as a therapeutic strategy to influence the hepatic redox state and combat cancer with reduced side-effects.

Alpha-Fetoprotein- and CD40Ligand-Expressing Dendritic Cells for Immunotherapy of Hepatocellular Carcinoma.

Simple SummaryIn first clinical trials, vaccinations against tumor-associated antigens (TAA), such as Alpha-Fetoprotein (AFP) using antigen presenting cells, such as dendritic cells (DC), failed to achieve effective immune responses towards hepatocellular carcinoma (HCC). CD40Ligand is a potent immune checkpoint, which can increase the antitumoral immune response of DC. In this study, a subcutaneous vaccination with DCs, which were transduced with AFP-coding adenoviruses and an intratumoral treatment with DCs, which were transduced with CD40L-coding adenoviruses, induced an antitumoral immune response and led to complete remissions and long-term survival in 62% of mice with established HCC. Combined strategy causes rapid and profound changes in the tumor environment with enhanced Th1-cytokine expression, strong tumor infiltration of cytotoxic T lymphocytes and DC, and higher tumor apoptosis, leading to effective tumor regression of HCC. Thus, intratumoral CD40L co-stimulation represents a promising tool for improving tumor-antigen DC-based immunotherapy of HCC.Dendritic cells (DC) as professional antigen presenting cells are able to prime T-cells against the tumor-associated antigen α-fetoprotein (AFP) for immunotherapy of hepatocellular carcinoma (HCC). However, a strong immunosuppressive tumor environment limits their efficacy in patients. The co-stimulation with CD40Ligand (CD40L) is critical in the maturation of DC and T-cell priming. In this study, the impact of intratumoral (i.t.) CD40L-expressing DC to improve vaccination with murine (m)AFP-transduced DC (Ad-mAFP-DC) was analyzed in subcutaneous (s.c.) and orthotopic murine HCC. Murine DC were adenovirally transduced with Ad-mAFP or Ad-CD40L. Hepa129-mAFP-cells were injected into the right flank or the liver of C3H-mice to induce subcutaneous (s.c.) and orthotopic HCC. For treatments, 106 Ad-mAFP-transduced DC were inoculated s.c. followed by 106 CD40L-expressing DC injected intratumorally (i.t.). S.c. inoculation with Ad-mAFP-transduced DC, as vaccine, induced a delay of tumor-growth of AFP-positive HCC compared to controls. When s.c.-inoculation of Ad-mAFP-DC was combined with i.t.-application of Ad-CD40L-DC synergistic antitumoral effects were observed and complete remissions and long-term survival in 62% of tumor-bearing animals were achieved. Analysis of the tumor environment at different time points revealed that s.c.-vaccination with Ad-mAFP-DC seems to stimulate tumor-specific effector cells, allowing an earlier recruitment of effector T-cells and a Th1 shift within the tumors. After i.t. co-stimulation with Ad-CD40L-DC, production of Th1-cytokines was strongly increased and accompanied by a robust tumor infiltration of mature DC, activated CD4+-, CD8+-T-cells as well as reduction of regulatory T-cells. Moreover, Ad-CD40L-DC induced tumor cell apoptosis. Intratumoral co-stimulation with CD40L-expressing DC significantly improves vaccination with Ad-mAFP-DC in pre-established HCC in vivo. Combined therapy caused an early and strong Th1-shift in the tumor environment as well as higher tumor apoptosis, leading to synergistic tumor regression of HCC. Thus, CD40L co-stimulation represents a promising tool for improving DC-based immunotherapy of HCC.

Abstract 928: Glypican-3 targeted thorium-227 alpha therapy reduces tumor burden in an orthotopic xenograft model of hepatocellular carcinoma

Abstract The purpose of this study is to develop a thorium-227 (227Th) antibody radioimmunoconjugate targeting glypican-3 (GPC3) and to test its therapeutic efficacy in a hepatocellular carcinoma (HCC) orthotopic xenograft model. GPC3 targeting antibody (αGPC3) was conjugated to bifunctional chelator p-SCN-Bn-H4octapa (octapa), and αGPC3-octapa binding affinity for GPC3 was evaluated by flow cytometry. 227Th radiolabeling of this conjugate was optimized, and in vitro stability of 227Th-αGPC3-octapa was evaluated in PBS with and without free radical scavenging agent over 14 days. For in vivo evaluation, an orthotopic xenograft model was generated by hepatic subcapsular injection of human HCC HepG2 cells. In vivo biodistribution was assessed in blood, tumor and organs at 1, 7 and 21 days after tail vein injection of 227Th-αGPC3-octapa (500 KBq/kg). To test therapeutic efficacy, tumor-bearing animals were injected with 227Th-αGPC3-octapa (250 kBq/kg or 500 kBq/kg) and compared to an irrelevant control antibody, 227Th-αBHV1-octapa (500 kBq/kg), and to a no-treatment control. Tumor burden was assessed with serial serum alpha-fetoprotein (AFP) measurements, a marker of tumor burden validated in this model. Toxicity to 227Th-αGPC3-octapa was measured by serum comprehensive metabolic panel obtained 21 days after injection. GPC3 binding affinity was highest after conjugation of αGPC3 with 10 equivalents of octapa. The protein recovery from the conjugation process was &amp;gt;85%, and mass spectral analysis indicated an average of 3.3 octapa moieties per molecule of αGPC3-octapa. After two weeks, &amp;gt;98% of αGPC3-octapa and αBHV1-octapa had 227Th bound in the presence of scavenging agent. αGPC3-octapa maintained high affinity for GPC3 as measured by flow cytometry, indicating the octapa conjugation reaction did not alter immunoreactivity of αGPC3. In vivo, 227Th-αGPC3-octapa accumulated in the tumor over time and cleared from normal tissues with a %ID/g of &amp;lt;5% by 21 days after injection. Significant antitumor activity was observed after treatment with 227Th-αGPC3-octapa (500 kBq/kg) with mean AFP level of 10,696 ± 19,754ng/mL compared to 308,175 ± 362,372ng/mL and 145,154 ± 166,780ng/mL in the 227Th-αBHV1-octapa and no-treatment control group, respectively. Reductions in serum AFP was observed in all but one tumor-bearing mouse after 500kBq/kg 227Th-αGPC3-octapa therapy. Organ-specific toxicity was not observed after treatment with 227Th-αGPC3-octapa (500 KBq/kg) compared to no-treatment control. In conclusion, we report the development of a GPC3 targeted thorium conjugate and demonstrate its in vivo therapeutic efficacy in a murine orthotopic xenograft model of hepatocellular carcinoma. Citation Format: Kevin P. Labadie, Donald K. Hamlin, Aimee Kenoyer, Sara K. Daniel, Alan F. Utria, Andrew D. Ludwig, Heidi L. Kenerson, Delphine L. Chen, Johnnie Orozco, Raymond S. Yeung, Lily Li, Chris Orvig, Yawen Li, D Scott Wilbur, James O. Park. Glypican-3 targeted thorium-227 alpha therapy reduces tumor burden in an orthotopic xenograft model of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 928.

Pulsed focused ultrasound can improve the anti-cancer effects of immune checkpoint inhibitors in murine pancreatic cancer.

Pulsed high-intensity focused ultrasound (pHIFU) uses acoustic pressure to physically disrupt tumours. The aim of this study was to investigate whether pHIFU can be used in combination with immune checkpoint inhibitors (ICIs) to enhance survival of tumour-bearing animals. Murine orthotopic pancreatic KPC tumours were exposed both to a grid of pHIFU lesions (peak negative pressure = 17 MPa, frequency = 1.5 MHz, duty cycle = 1%, 1 pulse s−1, duration = 25 s) and to anti-CTLA-4/anti-PD-1 antibodies. Acoustic cavitation was detected using a weakly focused passive sensor. Tumour dimensions were measured with B-mode ultrasound before treatment and with callipers post-mortem. Immune cell subtypes were quantified with immunohistochemistry and flow cytometry. pHIFU treatment of pancreatic tumours resulted in detectable acoustic cavitation and increased infiltration of CD8+ T cells in the tumours of pHIFU and pHIFU + ICI-treated subjects compared with sham-exposed subjects. Survival of subjects treated with pHIFU + ICI was extended relative to both control untreated subjects and those treated with either pHIFU or ICI alone. Subjects treated with pHIFU + ICI had increased levels of CD8+IFNγ+ T cells, increased ratios of CD8+IFNγ+ to CD3+CD4+FoxP3+ and CD11b+Ly6G+ cells, and decreased CD11chigh cells in their tumours compared with controls. These results provide evidence that pHIFU combined with ICI may have potential for use in pancreatic cancer therapy.

Abstract 2997: Novel orthotopic patient-derived xenograft mouse models of hepatoblastoma that replicate tumor heterogeneity, chemoresistance, and refractory disease

Abstract Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells. Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; and RNA sequencing (RNA-seq). Cells derived from tumors were grown in vitro and used for high throughput drug screening of candidate agents. Tumors were serially passaged in animals for further in vivo drug testing of novel targeted agents. Results: Nine patient-derived xenograft (PDX) models were generated that represent low- and high-risk tumors, treatment refractory cases, and relapsed tumors. Passaging of these models showed consistent implantation rates at or above 80% with tumors detectable in 2 to 4 weeks. Eight of nine models secrete human serum AFP. All models mimic gene expression and histological patterns of their primary tumor counterparts as well as identical STR DNA profiles. The models also show gene expression consistent with an HB2/high-risk profile according to the Sumazin HB expression signature. Interestingly, two models represent unique sub-clones of a very aggressive HB relapse with different AFP secretion and transcriptomic expression. The nine models also demonstrate a range of DNA mutations with three or four mutations per tumor; all variants present in the original clinical samples were conserved in the PDX models. Drug screening of tumor cells support the efficacy of novel targeted agents and indicate that these tumors are resistant to frontline standard chemotherapy, cisplatin and doxorubicin. More extensive in vivo testing showed the efficacy of a dual MDM2/MDM4 inhibitor (ALRN-6924), a cyclin dependent kinase inhibitor (dinaciclib), and a histone deacetylase inhibitor (panobinostat), and use of an orthotopic animal model also revealed drug toxicities associated with compromised liver function. Conclusions: These novel orthotopic PDX models of HB fully recapitulate the primary tumors and represent a platform for clinically relevant drug screening and testing. Further studies of the sub-clones of disease that grow in the animals will yield information about particularly aggressive HBs. Citation Format: Sarah Elizabeth Woodfield, Roma H. Patel, Samuel R. Larson, Andrew Badachhape, Rohit K. Srivastava, Aayushi P. Shah, Brandon Mistretta, Barry Zorman, Kevin Fisher, Ilavarasi Gandhi, Jacquelyn Reuther, Richard S. Whitlock, Aryana M. Ibarra, Sakuni Rankothgedera, Kimberly R. Holloway, Stephen F. Sarabia, Martin Urbicain, Andras Heczey, Dolores Lopez-Terrada, Angshumoy Roy, Preethi Gunaratne, Pavel Sumazin, Sanjeev A. Vasudevan. Novel orthotopic patient-derived xenograft mouse models of hepatoblastoma that replicate tumor heterogeneity, chemoresistance, and refractory disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2997.

Abstract 1174: Avenue to personalized targeted therapy for hepatoblastoma

Abstract Introduction: Hepatoblastoma (HB) is the most frequent liver malignancy of childhood with an annual incidence of 1.5 per million and a survival rate less than 50% for high-risk, relapse, and treatment refractory patients. The overall goal of this study is to assess the therapeutic efficacies and anti-cancer mechanisms of novel drugs with HB through a drug sensitivity testing pipeline utilizing multiple novel, clinically relevant preclinical patient-derived cell line (PDCL) and patient-derived xenograft (PDX) models. Methods: A patient derived xenograft (PDX) mouse model was generated with intrahepatic implantation of tumor thrombus from a non-metastatic stage 3 patient who recurred after transplantation. A stable PDCL was grown from the murine tumor, and 60 targeted agents were tested for efficacy with the PDCL by using CellTiter-Glo Luminescent Viability assays. Anti-oncogenic effects of the most efficacious agent, dinaciclib (cyclin-dependent kinase (CDK) inhibitor), were further assayed in vitro with HB cell lines (HepG2, HepT1, Huh6, HB17) with phenotypic assays for cell toxicity (MTT assay), proliferation (CCK-8 assay), and anchorage independent colony formation (soft agar assay). Immunoblotting assays were used to assess changes in drug targets and induction of apoptosis (PARP cleavage) with cell lines. Dinaciclib was also tested in vivo in our orthotopic PDX models of high-risk HB. The drug was administered three times a week by intraperitoneal injection (20 mg/kg, 100 µL) for 3 weeks. Magnetic resonance imaging (MRI) and Alpha-fetoprotein (AFP) ELISA were done to evaluate tumor growth throughout the study as tumor-bearing animals show expression of human serum AFP. Tumor weights and volumes, as well as immunohistochemistry for H&amp;E, Ki-67, and TUNEL, were also used to assess effects of drug in vivo. Results: The agent that showed the lowest IC50 value out of 60 tested agents was the CDK inhibitor, dinaciclib (0.7 µM). Dinaciclib suppressed cell proliferation and viability in a dose-dependent manner in all HB cell lines tested with IC50 values in the low micromolar range (HepG2 3.75 µM; HepT1 0.007 µM; Huh-6 1.42 µM; HB17 0.72 µM). Immunoblotting with dinaciclib-treated cells showed induction of PARP cleavage, indicating apoptosis, along with decreased protein expression of CDK1, 2, 5, and 9. Dinaciclib also showed significant in vivo inhibition of tumor growth compared to placebo (relative tumor volume p = 0.03, tumor weight p = 0.02). Conclusions: Drug screening of a novel HB PDCL identified an agent, dinaciclib, that shows preclinical efficacy with refractory disease. Further in vitro and in vivo validation confirmed that dinaciclib is a promising therapeutic agent for the treatment of HB. Such a personalized medicine pipeline of development of PDCLs and testing them with targeted agents will lead to the identification of new drugs that can be used with patients that do not respond to standard therapies. Citation Format: Rohit Kumar Srivastava, Roma Patel, Richard S. Whitlock, Samuel R. Larson, Andrew Badachhape, Sarah E. Woodfield, Sanjeev A. Vasudevan. Avenue to personalized targeted therapy for hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1174.

Abstract 1235: Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive breast cancer with shorter overall survival compared to other breast cancer types. One of the six molecularly-classified TNBC subtypes is the luminal androgen receptor subtype (LAR), which overexpresses androgen receptor (AR) and is dependent on AR for its growth. About 10-20% of TNBCs belong to the LAR subtype. Competitive AR antagonists, enzalutamide and bicalutamide, were effective in preclinical models of LAR TNBC and in clinical trials. This led us to hypothesize that potent selective AR degraders (SARDs), due to their ability to inhibit and degrade the AR, could provide a novel therapeutic approach for the treatment of LAR subtype of TNBC. Description: Western blots, cell line proliferation assays, and gene expression analyses were performed to evaluate novel small molecule SARDs. LAR TNBC cell lines and patient-derived xenografts (PDX) were utilized for in vivo evaluation of the SARDs. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally for four weeks with vehicle, SARD UT-34, SARD UT-105, enzalutamide, or bicalutamide. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. Summary: SARDs bind to the N-terminus of the AR and have been characterized in preclinical advanced prostate cancer models. In this study, the SARDs were evaluated in preclinical models of LAR TNBC. Western blot for AR in LAR MDA-MB-453 cells demonstrated degradation of the AR protein by SARDs at low micromolar concentrations. Gene expression studies showed a complete inhibition of androgen-induced AR target gene transcription by the SARDs. Androgen-induced proliferation of MDA-MB-453 cells was inhibited by SARDs. MDA-MB-453 cells implanted subcutaneously in NOD SCID Gamma female mice grew robustly to 100-300 mm3 in 15-20 days. Treatment of tumor-bearing animals with the SARDs completely inhibited or regressed the tumors. Conclusion: These results support the findings that AR is the driver of MDA-MB-453 cell and tumor growth. SARDs with their unique mechanism of action may provide a new therapeutic option to women affected by the LAR subtype of TNBC. Disclosure: This work was partially supported by Oncternal Therapeutics and by an NCI supplement award to R01 (CA229164S1 to author RN). The SARD program has been licensed to Oncternal Therapeutics, Inc. by the University of Tennessee Research Foundation. Author RN is a consultant to Oncternal Therapeutics. Citation Format: Sarah Asemota, Kirsten Young Young, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Dong-Jin Hwang, Yali He, James B. Breitmeyer, Gunnar F. Kaufmann, Duane D. Miller, Ramesh Narayanan. Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1235.

MyD88/CD40 Enhanced CD19-Specific CAR-T Cells Maintain Therapeutic Efficacy Following Resolution of Cytokine-Related Toxicity Using Inducible Caspase-9

Introduction: The efficacy of chimeric antigen receptor-modified T cells (CAR-T) targeting CD19+ leukemias and lymphomas is dependent on their in vivo expansion following adoptive transfer. Additional genetic augmentations to improve CAR-T expansion may improve therapeutic efficacy but risks increasing CAR-T toxicity. Here, we demonstrate that a highly active CD19-specific CAR-T cell constitutively expressing a MyD88/CD40 (MC) costimulatory fusion protein and the inducible caspase-9 (iC9) safety switch, is effective at eliminating tumors but induces acute cytokine-related toxicity in animal models. This toxicity, however, can be resolved by administration of rimiducid to induce partial CAR-T apoptosis preserving long-term anti-tumor effects.Methods: T cells were activated with anti-CD3/CD28 antibodies and transduced with a retrovirus encoding iC9, a first-generation CAR (with CD3ζ) targeting CD19, and a constitutively active MC costimulatory protein (SFG-iC9-CD19.ζ-MC). In vitro coculture assays were used to measure specific killing of CD19+ tumor cells (Daudi and Raji) and cytokine production. To assess anti-tumor efficacy, immunodeficient NSG mice were injected i.v. with 5x105 Raji-EGFPluciferase (EGFPluc) or 1x106 Daudi-EGFPluc tumor cells followed by i.v. administration of variable doses (1x106 to 1x107) non-transduced (NT) or iC9-CD19.ζ-MC-modified T cells on days 4 to 10 after tumor engraftment. Tumor growth was measured by in vivo imaging. CAR-T cell toxicity was assessed by weight loss and physical appearance. In initial experiments, CAR-T toxicity was resolved by i.p. administration of 0.5 mg/kg rimiducid after 15% weight loss. Serum cytokines were measured before and after rimiducid treatment. In addition, mice were treated with neutralizing antibodies to hIL-6, hIFN-γ and hTNF-α (100 ug i.p. twice weekly). Further, iC9-CD19.ζ-MC-modified T cells were sorted into CD4+ and CD8+ T cell fractions to assess the impact of cytokine production from CAR-T cells on animal health. Mice treated with iC9-CD19.ζ-MC-modified T cells that also received rimiducid to resolve toxicity were subsequently rechallenged with tumors to measure persistence of residual CAR-T cells, and at the end of the experiment were sacrificed and the spleens analyzed for the presence of human T cells and functional CAR-T activity using in vitro assays.Results: T cells were efficiently transduced (40-85%) with the iC9-CD19.ζ-MC retrovirus. In vitro assays showed that these CAR-T cells eliminated CD19+ Raji and Daudi tumor cells at low (1:20) effector-to-target (E:T) ratios in 7 day coculture assays, and produced high levels of IL-2 (669±324 pg/ml), IFN-γ (6715±2541 pg/ml), IL-6 (753±398 pg/ml) and TNF-α (2668±1867 pg/ml). NSG mice engrafted with CD19+ EGFPluc-expressing Raji or Daudi tumor cells were treated with variable doses of iC9-CD19.ζ-MC-modified T cells which were capable of eliminating tumor by day 21. However, acute toxicity (>15% body weight reduction) was observed in a CAR-T cell dose dependent manner occuring day 7 to 14 post-CAR-T injection. Systemic administration of 0.5 mg/kg rimiducid rapidly resolved toxicity and mice regained weight within 3-5 days. Serum cytokine concentrations of hIL-6 and hIFN-γ decreased to background levels within 24 hours post-rimiducid treatment. All mice treated with iC9-CD19.ζ-MC-modified T cells demonstrated long-term (>100 days) control of tumor growth and rejected CD19+ when rechallenged at day 150. Use of neutralizing antibodies against hIL-6, hIFN-γ and hTNF-α showed that blockade of hTNF-α resulted a significant reduction in weight loss. Further, iC9-CD19.ζ-MC-modified T cells were fractionated into CD4+ and CD8+ and measured for cytokine production, showing that CD4+ CAR-T cells produced higher levels of IL-2 (60-fold), IFN-γ (4-fold), IL-6 (37-fold) and TNF-α (10-fold) compared to CD8+ CAR-T cells. Furthermore, treatment of tumor-bearing animals with CD8+ selected iC9-CD19.ζ-MC-modified T cells reduced toxicity and the requirement of iC9 activation, while maintaining anti-tumor efficacy.Conclusions: Enhanced CAR-T cell activity can raise the risk of T cell toxicity. Constitutively active MC costimulation provides robust CAR-T cell proliferation and anti-tumor activity, but also increase cytokine-related toxicity. iC9 can be used to resolve toxicity without affecting long-term efficacy of CAR-T treatment in animals. DisclosuresFoster:Bellicum Pharmaceuticals: Employment, Other: stockholders . Lin:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Khalil:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Consultancy, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders .

Therapeutic Targeting of Interferon Regulatory Factor 4 with Next Generation Antisense Oligonucleotides Produces Robust In Vivo Antitumor Activity in Preclinical Models of Multiple Myeloma

Despite significant recent advances in therapeutic options, multiple myeloma (MM) remains an incurable disease, and novel agents targeting novel molecular pathways with activity in advanced patients are urgently needed. Interferon Regulatory Factor 4 (IRF4) is a transcription factor involved in immune responses in normal B and T cells, and is strongly implicated in the development of hematological malignancies, especially MM. Although recent publications have pointed to IRF4 as a driver oncogene in the progression of MM, it is considered an intractable target by conventional therapeutic approaches.Here we describe an antisense oligonucleotide (ASO) approach to selectively target IRF4. Administration of next generation human IRF4 ASOs via ‘free-uptake’ (without transfection) to a large panel of human MM cell lines resulted in a dose-dependent IRF4 reduction with concomitant induction of apoptosis. ASO-mediated IRF4 depletion also decreased the levels of c-Myc, another key oncogene in MM. MM cells were sensitive to even modest depletion of IRF4 (<50%), suggesting the strong dependence of MM cells on IRF4 for their survival. IRF4 ASOs also enhanced the sensitivity of the tumor cells to the treatment of IMiD (lenalidomide), proteasome inhibitor (Bortezomib) and BTK inhibitor (Ibrutinib) in MM cell, including KMS-11, JJN3 and MM1.R. In addition, Bortezomib-resistant clones of KMS-11 cells established from a long-term exposure to the drug remained sensitive to IRF4 inhibition. Importantly, in two different subcutaneously implanted MM tumor models, systemic administration of 2 independent human IRF4 ASOs depleted IRF4 protein levels in tumors and inhibited the growth of tumors compared to control treatment. Moreover, in a clinically relevant MM mouse model where human MM cells accumulate in the mouse bone marrow, treatment with human IRF4 ASOs substantially prolonged the survival of the tumor-bearing animals along with specific downregulation of human IRF4 and its downstream gene c-Myc. Finally, systematic treatment of mice with mouse-selective IRF4 ASOs at 50 mg/kg, twice a week for 4 weeks was well tolerated in normal mice, which is consistent with the lack of phenotypes observed in IRF4 heterozygous animals. Taken together, these results suggest that MM cells rely heavily on the survival pathway mediated by IRF4 and that selective knockdown of IRF4 by next generation ASOs is an attractive therapeutic strategy for the treatment of patients with advanced treatment-refractory MM. DisclosuresZhou:Ionis Pharmaceuticals: Employment, Equity Ownership. Schmidt:Ionis Pharmaceuticals: Employment. Kim:Ionis Pharmaceuticals: Employment. MacLeod:Ionis Pharmaceuticals: Employment.

A Review of Auraptene as an Anticancer Agent

Auraptene is a bioactive monoterpene coumarin isolated from Citrus aurantium and Aegle marmelos that belong to the Rutaceae family. Auraptene can modulate intracellular signaling pathways that control cell growth, inflammation and apoptosis and can exert pharmacological properties such as anti-bacterial, anti-fungal, antileishmania and anti-oxidant activity. Auraptene had inhibitory and chemo-preventive effects on the proliferation, tumorigenesis and growth of several cancer cell lines through increase in the activity of glutathione S-transferase, formation of DNA adducts and reduction of the number of aberrant crypt foci. Auraptene exhibits anticancer effects via targeting different cell signaling pathways such as cytokines, genes modulating cellular proliferation, growth factors, transcription factors and apoptosis. The present review is a detailed survey of scientific researches on the cytotoxicity and anticancer activity of Auraptene on cancer cells and tumor bearing animals.

RARE-08. POTENTIAL NEW THERAPIES FOR DIFFUSE INTRINSIC PONTINE GLIOMAS IDENTIFIED THROUGH HIGH THROUGHPUT DRUG SCREENING

Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most devastating of all brain tumors. There are no effective treatments, hence almost all children will die of their tumor within 12-months. There is an urgent need for novel effective therapies for this aggressive tumor. We performed a high-throughput drug screen with over 3,570 biologically active, clinically approved compounds against a panel of neurosphere-forming DIPG cells. We identified 7 compounds - auranofin, fenretinide, ivermectin, lanatoside, parthenolide, SAHA and mefloquine - that were confirmed to have potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells. Using cytotoxicity and clonogenic assays, we found that these drugs were able to inhibit DIPG-neurosphere proliferation and colony formation in vitro. To determine whether the in vitro efficacy could be replicated in vivo, we tested the activity of each of these compounds in an orthotopic DIPG model. Of the agents tested, fenretinide, auranofin and SAHA were the most active anti-tumor agents, significantly enhancing the survival of tumor bearing animals. Mechanistic studies showed fenretinide enhancing apoptotic cell death of DIPG cells via inhibition of PDGFRa transcription and downregulation of the PI3K/AKT/MTOR pathway. We therefore examined the therapeutic efficacy of fenretinide using a second orthotopic model with PDGFRa amplification. We used two different fenretinide formulations which were found to enhance survival. Fenretinide is clinically available with safety data in children. Validation of the activity of Fenretinide in PDGFRa-amplified or overexpressed DIPGs will lead to the development of a clinical trial, allowing the advancement of fenretinide as potentially the first active therapy for DIPG.

EPEN-03. ZFTA/C11ORF95 FUSIONS DRIVE SUPRATENTORIAL EPENDYMOMA VIA SHARED ONCOGENIC MECHANISMS

The majority of supratentorial ependymomas (ST-EPN) are driven by fusion genes between RELA and zinc finger translocation associated, ZFTA, previously named C11orf95. Apart from fusions with a portion of the Hippo effector YAP1, which affects a small group of infant patients, the oncogenic mechanism of remaining ST-EPNs remains unclear. Aiming at refining the molecular classification of ST-EPNs, we have analyzed methylation profiles, RNA and DNA sequencing results as well as clinical data in a cohort of 613 ST-EPNs. An unbiased approach revealed distinct methylation clusters composed of tumors with ependymal but also various other histological features containing alternative translocations that shared ZFTA as a partner gene. Tumors within these additional clusters were characterized by fusions of ZFTA to numerous fusion partners different from RELA, e.g. MAML2, MAML3, NCOA2 and SS18, implying a general role of ZFTA in tumorigenesis of ST-EPN. Indeed, the transforming capacity of newly identified fusion genes was validated using an electroporation-based in vivo gene transfer technology in mice. All fusion genes themselves were sufficient to drive malignant transformation in the developing cerebral cortex and resulting tumors faithfully recapitulated molecular characteristics of their human counterparts. We found that both, the partner gene and the zinc finger DNA binding domain of ZFTA, were essential to exert tumorigenesis. Together with two additional studies, we performed a comprehensive analysis across datasets to derive a 93 gene signature of ZFTA-RELA-driven tumors, in which the Sonic Hedgehog effector gene GLI2 was identified as a promising downstream target. Subsequent co-expression of ZFTA:RELA and a dominant negative form of Gli2 indeed hampered tumorigenesis. Targeting GLI2 with arsenic trioxide caused extended survival of tumor-bearing animals, indicating GLI2 as a critical regulator of ZFTA fusion-positive tumorigenesis as well as a potential therapeutic vulnerability in these tumors.

HGG-14. ACT001 – A PROMISING THERAPEUTIC FOR DIFFUSE INTRINSIC PONTINE GLIOMAS

Diffuse Intrinsic Pontine Gliomas (DIPGs) are a subset of Diffuse Midline Gliomas (DMG) and are the most devastating of all brain tumors. There are no effective treatments and all children die of their tumor within 12-months. We performed a high-throughput drug screen with 3,570 biologically active, clinically approved compounds against a panel of DIPG cultures. Parthenolide, a compound derived from the herb T.parthenium, was found to be one of the most effective drugs tested, demonstrating significant anti-tumor activity. However, parthenolide also affected healthy cell viability and showed no in-vivo efficacy. ACT001 is a novel agent in clinical development that is a fumarate salt form of dimethylamino-micheliolide, which is semi-synthesized from parthenolide. ACT001 is blood-brain-barrier permeable and exerts an anti-tumor effect via inhibition of NF-κB and STAT3 pathways. ACT001 demonstrated potent anti-tumor activity against a panel of DIPG-neurospheres, with minimal effect on normal cells and inhibited colony formation in-vitro. To determine whether this activity could be replicated in-vivo, we tested ACT001 in a DIPG-orthotopic model. ACT001 was well tolerated and significantly improved survival of tumor-bearing animals, extending survival by 33% in ACT001 treated mice. We have initiated a Phase 1 paediatric trial of ACT001 for children with relapsed/refractory solid or CNS tumors, with an expansion cohort planned for patients with DIPG/DMG. Eleven patients have been enrolled, and the dose escalated from dose level-1 at 188mg/m2 bd to dose level-4 at 700mg/m2 bd. To date, no dose limiting or Grade 3/4 toxicities have been observed. At the highest dose level, clinical activity has been demonstrated in two patients, one with DIPG with a reduction in tumor burden, and another with DMG with H3K27M mutation with an objective radiographic and clinical response. These combined preclinical and clinical results suggest that ACT001 is an active novel therapy for patients with DIPG/DMG.