NOD Scid Gamma Mice Research Articles

3JC48-3: Novel MYC inhibition in advanced prostate cancer.

181 Background: The proto-oncogene c-Myc is a transcription factor that is upregulated in several human cancers. Despite its key role, therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MAX to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. Methods: We used NOD SCID gamma mice (NGS mice) that are immunodeficient, non-obese diabetic laboratory mice (Jackson Laboratories, Cat #J000077451) carrying patient-derived xenograft (PDX) following implantation of grade IV adenocarcinoma of the prostate from a patient into the flanks of each mouse. For maximum tolerable dose studies, we used C57BL/6 wild type mice (Jackson Laboratories, Cat #J000664). We performed a maximum dose tolerability study with the drug 3JC48-3 up to 100 mg/kg to determine limiting toxicity. We monitored the rate of growth for 2 weeks and measured the final volume of the tumors. Immunoprecipitation studies were performed using the prostate cancer cell line, PC-3, that were treated with the drug 3JC48-3 at different doses (0, 5, 10, and 20uM) for 24 hours and subsequently lysed. Results: We found 3JC48-3 decreases prostate cancer growth and viability in a dose-dependent fashion in vitro. We confirmed the inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. Conclusions: Our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability in association with upregulation of both PrKD1 expression and kinase activity. In addition, 3JC48-3 is well tolerated in prostate cancer PDX mice models and may be a candidate drug for further preclinical development.

Abstract P2-13-24: Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer

Abstract Background.Targeting HER2 gene amplification is one of the great achievements in oncology resulting in the use of a wide array of anti-HER2 agents in the clinic. Unfortunately, 20% of patients still relapse with secondary organ metastasis and are currently incurable. While only 1.6% of primary non-HER2-amplified ER+ breast cancers harbor HER2 mutations, 6-10% of all metastatic breast cancers harbor HER2 mutations, suggesting their causal role in contributing to metastasis. The clinical value of HER2 activating mutations is being tested with the pan-HER tyrosine kinase inhibitor neratinib in phase II clinical trials (NCT01670877, NCT01953926, and NCT02465060). To date, neratinib has elicited only modest responses in ER+ breast cancer, often rapidly followed by progression. This study characterizes HER2-mutant induced resistance to endocrine therapy or neratinib induced resistance and resulting metastasis, in order to determine a more effective rational therapeutic approach for treating ER+ HER2-mutant breast cancer. Methods.HER2 mutation frequency and its impact on patient outcome was determined using METABRIC primary breast cancer (BC) and MSK IMPACT metastatic BC ER+ sequencing studies. Effects of estrogen (E2), fulvestrant, or neratinib on cell growth and HER2 signaling were examined on ER+/ILC cells (MM134) stably expressing HER2/WT, HER2/S310F, and HER2/L755S. Cell growth was measured using CellTiter-Glo and HER2 signaling was analyzed by western blot analysis. Additionally, the effect of these ER+/ILC and IDC HER2 mutations on tumor growth and endocrine or neratinib treatment resistance was determined using fat pad injections and MIND xenografts in NOD-scid gamma mice. Results.We searched ER+ sequencing datasets and identified HER2 mutations that are highly enriched in ER+ ILC as compared to ER+ IDC. These activating HER2 mutations in ER+ ILC are associated with early relapse and poor overall survival. Moreover, we are finding that ILC patients harboring the recurrent HER2/L755S mutation have worse overall survival compared to non-mutant HER2 ILC. MM134 cells expressing HER2/S310F and HER2/L755S show increased cell growth, strongly activated autophosphorylation of HER2, and increased downstream signaling (pMAPK and pAKT) as compared to cells expressing HER2/WT upon treatment with fulvestrant (1μM). Three clinically relevant in vivo models including ILC HER2/L755S Mammary INtraDuctal (MIND) xenografts, IDC HER2/L755S fat pad xenografts, and IDC HCI-003 (an ER+ patient-derived xenograft (PDX) naturally harboring the exon 20 activating HER2G778_P780 dup) exhibit fulvestrant and neratinib resistance and lung and ovary metastases. In addition, we find that HER2 mutations induced mTOR signaling. In contrast, however, the pan-HER drug poziotinib does potently inhibit tumor growth and organ-specific metastasis and perturbs mTOR activation in these models. Conclusion.We demonstrate that clinically associated HER2 mutations drive endocrine therapy or neratinib resistance and poor patient outcome in ER+ patients. Our data propose the use of the irreversible pan-HER TKI poziotinib for treating endocrine therapy or neratinib refractory ER+ HER2-mutant metastatic breast cancer. Citation Format: Shyam. M Kavuri, Rashi Kalra, Ching Hui Chen, Junkai Wang, Ahmad Bin Salam, Lacey Dobrolecki, Alaina Lewis, Christina Sallas, Clayton Yates, Carolina Gutierrez, Balasubramanyam Karanam, Meenakshi Anurag, Bora Lim, Matthew Ellis. Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ HER2 mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-24.

Understanding Cell Lines, Patient-Derived Xenograft and Genetically Engineered Mouse Models Used to Study Cutaneous T-Cell Lymphoma.

Cutaneous T cell lymphoma (CTCL) is a spectrum of lymphoproliferative disorders caused by the infiltration of malignant T cells into the skin. The most common variants of CTCL include mycosis fungoides (MF), Sézary syndrome (SS) and CD30+ Lymphoproliferative disorders (CD30+ LPDs). CD30+ LPDs include primary cutaneous anaplastic large cell lymphoma (pcALCL), lymphomatoid papulosis (LyP) and borderline CD30+ LPD. The frequency of MF, SS and CD30+ LPDs is ~40–50%, <5% and ~10–25%, respectively. Despite recent advances, CTCL remains challenging to diagnose. The mechanism of CTCL carcinogenesis still remains to be fully elucidated. Hence, experiments in patient-derived cell lines and xenografts/genetically engineered mouse models (GEMMs) are critical to advance our understanding of disease pathogenesis. To enable this, understanding the intricacies and limitations of each individual model system is highly important. Presently, 11 immortalized patient-derived cell lines and different xenograft/GEMMs are being used to study the pathogenesis of CTCL and evaluate the therapeutic efficacy of various treatment modalities prior to clinical trials. Gene expression studies, and the karyotyping analyses of cell lines demonstrated that the molecular profile of SeAx, Sez4, SZ4, H9 and Hut78 is consistent with SS origin; MyLa and HH resemble the molecular profile of advanced MF, while Mac2A and PB2B represent CD30+ LPDs. Molecular analysis of the other two frequently used Human T-Cell Lymphotropic Virus-1 (HTLV-1)+ cell lines, MJ and Hut102, were found to have characteristics of Adult T-cell Leukemia/Lymphoma (ATLL). Studies in mouse models demonstrated that xenograft tumors could be grown using MyLa, HH, H9, Hut78, PB2B and SZ4 cells in NSG (NOD Scid gamma mouse) mice, while several additional experimental GEMMs were established to study the pathogenesis, effect of drugs and inflammatory cytokines in CTCL. The current review summarizes cell lines and xenograft/GEMMs used to study and understand the etiology and heterogeneity of CTCL.

Modulated TRPC1 Expression Predicts Sensitivity of Breast Cancer to Doxorubicin and Magnetic Field Therapy: Segue Towards a Precision Medicine Approach.

Chemotherapy is the mainstream treatment modality for invasive breast cancer. Unfortunately, chemotherapy-associated adverse events can result in early termination of treatment. Paradoxical effects of chemotherapy are also sometimes observed, whereby prolonged exposure to high doses of chemotherapeutic agents results in malignant states resistant to chemotherapy. In this study, potential synergism between doxorubicin (DOX) and pulsed electromagnetic field (PEMF) therapy was investigated in: 1) MCF-7 and MDA-MB-231 cells in vitro; 2) MCF-7 tumors implanted onto a chicken chorioallantoic membrane (CAM) and; 3) human patient-derived and MCF-7 and MDA-MB-231 breast cancer xenografts implanted into NOD-SCID gamma (NSG) mice. In vivo, synergism was observed in patient-derived and breast cancer cell line xenograft mouse models, wherein PEMF exposure and DOX administration individually reduced tumor size and increased apoptosis and could be augmented by combined treatments. In the CAM xenograft model, DOX and PEMF exposure also synergistically reduced tumor size as well as reduced Transient Receptor Potential Canonical 1 (TRPC1) channel expression. In vitro, PEMF exposure alone impaired the survival of MCF-7 and MDA-MB-231 cells, but not that of non-malignant MCF10A breast cells; the selective vulnerability of breast cancer cells to PEMF exposure was corroborated in human tumor biopsy samples. Stable overexpression of TRPC1 enhanced the vulnerability of MCF-7 cells to both DOX and PEMF exposure and promoted proliferation, whereas TRPC1 genetic silencing reduced sensitivity to both DOX and PEMF treatments and mitigated proliferation. Chronic exposure to DOX depressed TRPC1 expression, proliferation, and responses to both PEMF exposure and DOX in a manner that was reversible upon removal of DOX. TRPC1 channel overexpression and silencing positively correlated with markers of epithelial-mesenchymal transition (EMT), including SLUG, SNAIL, VIMENTIN, and E-CADHERIN, indicating increased and decreased EMT, respectively. Finally, PEMF exposure was shown to attenuate the invasiveness of MCF-7 cells in correlation with TRPC1 expression. We thus demonstrate that the expression levels of TRPC1 consistently predicted breast cancer sensitivity to DOX and PEMF interventions and positively correlated to EMT status, providing an initial rationale for the use of PEMF-based therapies as an adjuvant to DOX chemotherapy for the treatment of breast cancers characterized by elevated TRPC1 expression levels.

B7-H3 Targeted CAR T Cell Immunotherapy for Primary and Metastatic/Multi-focal Intrahepatic Cholangiocarcinoma

Introduction: The lack of effective therapy for intrahepatic cholangiocarcinoma (ICC) has prompted us to develop a novel immunotherapy using T cells engineered with a chimeric antigen receptor (CAR T cells). The aim of this preliminary study is to demonstrate that B7-H3 CAR T cells are safe and effective in eradicating ICC cells orthotopically grafted in NSG (NOD scid gamma) mice and in prolonging their survival. Method: Primary tumor was established in 15 mice by orthotopic grafting of human ICC cells ICC3-GFP-Luc. Then mice were randomized into 3 groups: treatment group injected through the tail vein with B7-H3 CAR T cells (n=5), intratumorally injected (n=5) and control group (n=5), left untreated. The multi-focal/metastatic mice model was established by tail vein injection of the same cell line in 8 mice. Then mice were randomized into 2 groups: treatment group, injected through the tail vein with B7-H3 CAR T cells (n=4) and control group (n=4), left untreated. Tumor size and development of metastasis were monitored weekly by bioluminescent imaging (BLI). Results: While tail vein injected B7-H3 CAR T cells eradicated primary tumor in all the mice, the intratumorally administered B7-H3 CART cells only reduce the growth of the tumor. As far as mice with multifocal/metastatic disease is concerned, B7-H3 CART cells completely eradicate the tumor in all the mice and prolong their survival. Conclusions: B7-H3 CAR T cell therapy is effective in eradicating both primary and multi- focal disease and ICC metastases established in NSG mice and in prolonging their survival.

Modelling Graft-Versus-Host Disease in Mice Using Human Peripheral Blood Mononuclear Cells.

Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. Graft-versus-host disease (GvHD) is a significant complication of allogeneic hematopoietic stem cell transplantation. In order to develop new therapeutic approaches, there is a need to recapitulate GvHD effects in pre-clinical, in vivo systems, such as mouse and humanized mouse models. In humanized mouse models of GvHD, mice are reconstituted with human immune cells, which become activated by xenogeneic (xeno) stimuli, causing a multi-system disorder known as xenoGvHD. Testing the ability of new therapies to prevent or delay the development of xenoGvHD is often used as pre-clinical, proof-of-concept data, creating the need for standardized methodology to induce, monitor, and report xenoGvHD. Here, we describe detailed methods for how to induce xenoGvHD by injecting human peripheral blood mononuclear cells into immunodeficient NOD SCID gamma mice. We provide comprehensive details on methods for human T cell preparation and injection, mouse monitoring, data collection, interpretation, and reporting. Additionally, we provide an example of the potential utility of the xenoGvHD model to assess the biological activity of a regulatory T-cell therapy. Use of this protocol will allow better standardization of this model and comparison of datasets across different studies. This protocol was validated in: Sci Transl Med (2020), DOI: 10.1126/scitranslmed.aaz3866 Graphical abstract.

Employing Novel Porcine Models of Subcutaneous Pancreatic Cancer to Evaluate Oncological Therapies.

Immunocompromised mice are commonly utilized to study pancreatic cancer and other malignancies. The ability to xenograft tumors in either subcutaneous or orthotopic locations provides a robust model to study diverse biological features of human malignancies. However, there is a dire need for large animal models that better recapitulate human anatomy in terms of size and physiology. These models will be critical for biomedical device development, surgical optimization, and drug discovery. Here, we describe the generation and application of immunocompromised pigs lacking RAG2 and IL2RG as a novel model for human xenograft studies. These SCID-like pigs closely resemble NOD scid gamma mice and are receptive to human tumor tissue, cell lines, and organoid xenografts. However, due to their immunocompromised nature, these immunocompromised animals require housing and maintenance under germfree conditions. In this protocol, we describe the use of these pigs in a subcutaneous tumor injection study with human PANC1 cells. The tumors demonstrate a steady, linear growth curve, reaching 1.0cm within 30days post injection. The model described here is focused on subcutaneous injections behind the ear. However, it is readily adaptable for other locations and additional human cell types.

Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

Development of Anti-CD99scFvs for the Treatment of Acute Myeloid Leukemia

CD99 has gained much attention in recent years as a novel therapeutic target in hematological malignancies, due to its upregulation in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). We have recently shown that targeting CD99 with a knockdown approach or with commercial antibodies results in antileukemia activity in AML cells. We have also developed anti-CD99 nanoparticles and demonstrated excellent in vitro and in vivo antileukemia activity. Here we report the development of human single chain variable fragment targeting CD99 (anti-CD99 scFv) and the preclinical activity in AML cells and in AML xenograft mouse model. The anti-CD99 scFv was developed by inserting CD99scFv DNA sequence into the pFUSE vector, encoding pATL103-CD99 scFvs. To produce anti-CD99 scFv, the plasmids were transfected in Expi293 (HEK293T) cells. Then the medium was harvested and underwent two cycles of dialysis. To determine product purity, proteins were analyzed on SDS-PAGE gel stained with Coomassie blue. For each 240ml cell culture media with recombinant CD99 plasmids, we obtained about 1-2mg anti-CD99 scFv. The binding affinity of CD99 scFv to CD99 surface protein was assessed in 293T cells (CD99 null cells) which exhibited no binding and in MOLM-13 cells and MV4-11 (CD99 positive AML cells) which demonstrated strong binding. Treatment with 5uM of anti-CD99 scFv significantly reduced cell viability in both leukemic cell line MOLM-13 and MV4-11 (MOLM-13: 35.14%, P= 0.008; MV4-11: 30.17%, P=0.002) and primary AML patient blasts (29.37%, P=0.048) compared with control cells. Colony forming assay showed that anti-CD99 scFv treated AML blasts exhibited less number of colonies compared with control cells (plating efficiency (PE): 0.035% vs 0.12%). We also established the in vivo antileukemia activity of anti-CD99 scFv using MOLM-13 cells (FLT3-ITD positive AML cells) NOD scid gamma (NSG) xenograft mouse model. MOLM-13 (2.5x10^6) cells were engrafted into NSG mice via IV tail injection (N=4 mice per group). Mice were treated with PBS (group 1) or 4mg/kg of anti-CD99 scFV on days 10, 14, 18 and 22 post cell engraftment. Mice were euthanized on day 24 and levels of leukemia engraftment were assessed by flow cytometry measurements of huCD45 staining of cells collected from the bone marrow and the peripheral blood and compared between the two groups. Mice treated with four doses of 4mg/kg CD99scFv demonstrated significant reduction in leukemia engraftment in the bone marrow assessed by flow cytometry measurements of huCD45 staining compared with the PBS mice group (huCD45%: 39.7 vs 56, P = 0.0017). In conclusion, we report the development of anti-CD99 single chain variable fragments for the treatment of AML. Our study demonstrates good binding affinity and specificity and a promising preclinical antileukemia activity both in AML cells and in xenograft mouse model. DisclosuresYaghmour: Jazz: Consultancy, Honoraria; Astellas: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

Palbociclib and Ponatinib Suppress Acute Myeloid Leukemia in Patient-Derived Xenograft

Aims: Palbociclib, a breast cancer approved CDK4/6 inhibitor, and ponatinib, a BCR/ABL1 inhibitor with a multi-kinase activity approved for chronic myeloid and acute lymphoid leukemia, were previously shown to be effective in vitro against acute myeloid leukemia (AML). Here, we aimed to test this effect in a patient-derived xenograft model.Methods: Two newly diagnosed AMLs (AML #1: myelomonocytic AML, intermediate cytogenetic risk; AML #2: AML with myelodysplasia-related changes, poor cytogenetic risk) were xenotransplanted into NOD SCID gamma mice. Treatment was initiated at detection of approximately 5-20% hCD45+ cells in mouse peripheral blood (PB). Palbociclib, ponatinib, vehicle, and venetoclax as a comparative treatment, were administered orally for 3 weeks, 5 days per week. Chemotherapy (cytarabine+doxorubicine, AraC/Dox) 5+3 regimen served as a positive control. Azacitidine served as another comparative drug and was administered subcutaneously, five days per week in 3 cycles - 1 week on, 1 week off.Results: Significant reduction of disease burden and prolongation of overall survival (OS) were seen with palbociclib and the reference treatment venetoclax in both AMLs, and with chemotherapy in AML#1 (Fig. 1). Ponatinib prolonged OS in AML#1 but failed to provide reduction of disease burden in PB. Interestingly, azacitidine induced the longest remission (<1% hCD45+ cells in PB), for almost 10 weeks, but only in 2/4 mice for AML#2. Treatment toxicity manifested by weight decrease was only seen with chemotherapy (AML#1: 24% weight loss, p = 0.0001; AML#2: 19% loss, p = 0.04), and was also accompanied with early mouse mortality in 3/4 mice for AML#2. None of the tested treatments lead to complete AML eradication and a gradual relapse was seen in PB. The AML infiltration was higher in bone marrow than in spleen at final analysis at relapse (AML#1, 83 vs 73% of hCD45+ cells, mean, p = 0.02; AML#2, 95 vs 65%, p = 0.0001). No changes in AML phenotype were observed between treated and vehicle mice in case of AML#1, except for azacitidine which decreased monocyte (SSCdim, CD45high; 94 vs 96%, mean, p = 0.03), CD34+ (2 vs 5%, p = 0.01) and primitive CD34+CD38- cell (0.1 vs 0.3%, p = 0.005) percentages. For AML#2, blast (SSClow, CD45dim) percentage compared to vehicle (70%) was decreased by chemotherapy (24%, p = 0.01) and increased by venetoclax (80%, p = 0.03) and azacitidine (87%, p = 0.03); monocytes (6% for vehicle) were decreased by chemotherapy (1%, p = 0.004), venetoclax (3%, p = 0.01), and azacitidine (0%, p = 0.008); CD34+ cells were increased by venetoclax (59 vs 39%, p = 0.02).Summary: Palbociclib, and partially ponatinib, demonstrated AML suppression in vivo. This encourages further investigation of their efficacy in different AML subtypes and in combination with other drugs.Funding: Supported by MUNI/A/1595/2020. [Display omitted] DisclosuresMayer: AOP Orphan Pharmaceuticals: Research Funding.

4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence...

In vivo quantitative assessment of therapeutic response to bortezomib therapy in disseminated animal models of multiple myeloma with [18F]FDG and [64Cu]Cu-LLP2A PET

BackgroundMultiple myeloma (MM) is a disease of cancerous plasma cells in the bone marrow. Imaging-based timely determination of therapeutic response is critical for improving outcomes in MM patients. Very late antigen-4 (VLA4, CD49d/CD29) is overexpressed in MM cells. Here, we evaluated [18F]FDG and VLA4 targeted [64Cu]Cu-LLP2A for quantitative PET imaging in disseminated MM models of variable VLA4 expression, following bortezomib therapy.MethodsIn vitro and ex vivo VLA4 expression was evaluated by flow cytometry. Human MM cells, MM.1S-CG and U266-CG (C: luciferase and G: green fluorescent protein), were injected intravenously in NOD-SCID gamma mice. Tumor progression was monitored by bioluminescence imaging (BLI). Treatment group received bortezomib (1 mg/kg, twice/week) intraperitoneally. All cohorts (treated, untreated and no tumor) were longitudinally imaged with [18F]FDG (7.4–8.0 MBq) and [64Cu]Cu-LLP2A (2–3 MBq; Molar Activity: 44.14 ± 1.40 MBq/nmol) PET, respectively.ResultsFlow cytometry confirmed high expression of CD49d in U266 cells (> 99%) and moderate expression in MM.1S cells (~ 52%). BLI showed decrease in total body flux in treated mice. In MM.1S-CG untreated versus treated mice, [64Cu]Cu-LLP2A localized with a significantly higher SUVmean in spine (0.58 versus 0.31, p < 0.01) and femur (0.72 versus 0.39, p < 0.05) at week 4 post-tumor inoculation. There was a four-fold higher uptake of [64Cu]Cu-LLP2A (SUVmean) in untreated U266-CG mice compared to treated mice at 3 weeks post-treatment. Compared to [64Cu]Cu-LLP2A, [18F]FDG PET detected treatment-related changes at later time points.Conclusion[64Cu]Cu-LLP2A is a promising tracer for timely in vivo assessment of therapeutic response in disseminated models of MM.

An Improved Animal Model of Multiple Myeloma Bone Disease.

Simple SummaryMultiple myeloma is a plasma cell cancer involving bone destruction and is considered an incurable disease despite significant improvements in therapeutic strategies. During myeloma progression, over 90% of patients develop a bone disease that causes patient injury and death. There are limited animal models available to demonstrate multiple myeloma bone disease (MMBD). The current study identifies and validates the newly developed MMBD models with uniformity of tumor burden and severe bone lesions. This model will help study the biology of MMBD and serve as a valuable tool for screening therapeutic candidates by monitoring their response to disease progression.Multiple myeloma (MM) is a plasma cell malignancy that causes an accumulation of terminally differentiated monoclonal plasma cells in the bone marrow, accompanied by multiple myeloma bone disease (MMBD). MM animal models have been developed and enable to interrogate the mechanism of MM tumorigenesis. However, these models demonstrate little or no evidence of MMBD. We try to establish the MMBD model with severe bone lesions and easily accessible MM progression. 1 × 106 luciferase-expressing 5TGM1 cells were injected into 8–12 week-old NOD SCID gamma mouse (NSG) and C57BL/KaLwRij mouse via the tail vein. Myeloma progression was assessed weekly via in vivo bioluminescence (BL) imaging using IVIS-200. The spine and femur/tibia were extracted and scanned by the micro-computer tomography for bone histo-morphometric analyses at the postmortem. The median survivals were 56 days in NSG while 44.5 days in C57BL/KaLwRij agreed with the BL imaging results. Histomorphic and DEXA analyses demonstrated that NSG mice have severe bone resorption that occurred at the lumbar spine but no significance at the femur compared to C57BL/KaLwRij mice. Based on these, we conclude that the systemic 5TGM1 injected NSG mouse slowly progresses myeloma and develops more severe MMBD than the C57BL/KaLwRij model.

Establishment and Characterization of Feline Mammary Tumor Patient-Derived Xenograft Model.

Simple SummaryFeline mammary tumor (FMT) is a relatively commonly diagnosed neoplasm in the cat. Development of new veterinary cancer therapies is limited by the shortage of in vivo models that reproduce tumor microenvironment and metastatic progression. In this study, FMT patient-derived xenografts (PDX) model were established and primary cell line isolated from orthotopic PDX. The tumor grafts conserve original tumor essential features, including distant metastasis. FMT-PDX represents an available resource for bridging the biology of FMT with preclinical studies of FMT in cats.Feline mammary tumor is a relatively commonly diagnosed neoplasm in the cat. Development of new veterinary cancer therapies is limited by the shortage of in vivo models that reproduce tumor microenvironment and metastatic progression. Four feline mammary tumor orthotopic patient-derived xenograft model (PDX) successfully established in NOD-SCID gamma (NSG) mice. The overall success rate of PDX establishment was 36% (4/11). Histological, immunohistochemical, and short tandem repeat analysis showed a remarkable similarity between patient’s tumor and xenograft. The tumor grafts conserve original tumor essential features, including distant metastasis. Primary FMT-1807 cell line isolated from FMT-1807PDX tumor tissue. Tumorigenicity of FMT-1807 cells expanded from PDX was assessed by orthotopic injection into NSG mice. Mice yielded tumors which preserve the lung and liver metastasis ability. This work provides a platform for FMT translational investigation.

E2F3 drives the epithelial-to-mesenchymal transition, cell invasion, and metastasis in breast cancer.

E2F3 is a transcription factor that may initiate tumorigenesis if overexpressed. Previously, we demonstrated that E2F3 mRNA is overexpressed in breast cancer and that E2F3 overexpression results in centrosome amplification and unregulated mitosis, which can promote aneuploidy and chromosome instability to initiate and sustain tumors. Further, we demonstrated that E2F3 leads to overexpression of the mitotic regulator Shugoshin-1, which until recently had unknown roles in cancer. This study aims to evaluate the roles of E2F3 and Shugoshin-1 in breast cancer metastatic potential. Here we demonstrated that E2F3 and Shugoshin-1 silencing leads to reduced cell invasion and migration in two mesenchymal triple-negative breast cancer (TNBC) cell lines (MDA-MB-231 and Hs578t). Moreover, E2F3 and Shugoshin-1 modulate the expression of epithelial-to-mesenchymal transition-associated genes such as Snail, E-Cadherin, and multiple matrix metalloproteinases. Furthermore, E2F3 depletion leads to reductions in tumor growth and metastasis in NOD-scid Gamma mice. Results from this study suggest a key role for E2F3 and a novel role for Shugoshin-1 in metastatic progression. These results can further help in the improvement of TNBC targeted therapies by interfering with pathways that intersect with the E2F3 and Shugoshin-1 signaling pathways.

A Humanized Anti-GPC3 Antibody for Immuno-Positron Emission Tomography Imaging of Orthotopic Mouse Model of Patient-Derived Hepatocellular Carcinoma Xenografts.

Simple SummaryLiver cancer, the majority of which is hepatocellular carcinoma, is a typically fatal adult liver malignancy. It is hard to detect in the early stages of the cancer, and therefore patients are often diagnosed at the advanced stages, when treatment options become more limited and survival outcomes are poor. To improve early detection, and therefore treatment and prognosis of liver cancer patients, we have developed an imaging probe for positron emission tomography, targeting a protein, glypican-3, which is specifically expressed at high levels in liver cancer cells. Our probe consists of the 89Zr radioisotope conjugated to a humanized monoclonal antibody against glypican-3, and it demonstrates specific ability to detect patient-derived liver cancer xenografts in a mouse model. With a high tumor to normal liver contrast, we believe this imaging probe can provide a useful tool in the early diagnosis and timely medical intervention for liver cancer patients.Glypican-3 (GPC3) is an attractive diagnostic marker for hepatocellular carcinoma (HCC). We previously reported the potential of an 89Zr-labeled murine anti-GPC3 antibody (clone 1G12) for immunoPET imaging of HCC in orthotopic patient-derived xenograft (PDX) mouse models. We now humanized the murine antibody by complementarity determining region (CDR) grafting, to allow its clinical translation for human use. The engineered humanized anti-GPC3 antibody, clone H3K3, retained comparable binding affinity and specificity to human GPC3. H3K3 was conjugated with desferrioxamine (Df) and radiolabeled with 89Zr to produce the PET/CT tracer 89Zr-Df-H3K3. When injected into GPC3-expressing orthotopic HCC PDX in NOD SCID Gamma (NSG) mice, 89Zr-Df-H3K3 showed specific high uptake into the orthotopic PDX and minimal, non-specific uptake into the non-tumor bearing liver. Specificity was demonstrated by significantly higher uptake of 89Zr-Df-H3K3 into the non-blocked PDX mice, compared with the blocked PDX mice (which received prior injection of 100 mg of unlabeled H3K3). Region of interest (ROI) analysis showed that the PDX/non-tumor liver ratio was highest (mean ± SD: 3.4 ± 0.31) at 168 h post injection; this ratio was consistent with biodistribution studies at the same time point. Thus, our humanized anti-GPC3 antibody, H3K3, shows encouraging potential for use as an immunoPET tracer for diagnostic imaging of HCC patients.

Abstract 1448: Combination therapy of anti-sense oligonucleotide targeting TGF-beta2 (TASO) and IL-2 (Proleukin) has anti-cancer effect in solid cancer

Abstract Background: TGF-Β (transforming growth factor-beta) is an essential cytokine for tumor proliferation and metastasis. The expression of TGF-Β correlates with malignancy of various cancers and involves immunosuppression and angiogenesis of a tumor. IL-2 is a major cytokine to proliferate T cells and NK cells which are major players of cancer immunity. However, the toxicity of high dose IL-2 limits its use in cancer therapy. Combination treatment of TGF-Β inhibitor and IL-2 would have an anti-tumor effect by immune cells through diminishing immunosuppression by TGF-Β and enforcement of immune cells by IL-2. Trabedersen is an anti-sense oligonucleotide targeting human TGF-Β mRNA. It is shown that Trabedersen is well tolerable in cancer patients and effective reagent to treat pancreatic cancer, melanoma, and glioblastoma. Proleukin is the only approved IL-2 reagent to treat Renal cell carcinoma and Melanoma. Methods: Trabedersen and Proleukin activated human PBMC (Peripheral Blood Mononuclear Cell) are treated to several solid cancer cell lines, such as Breast cancer, Pancreatic cancer, Melanoma, Lung cancer, and Colon cancer to see the cytotoxicity effect of combination therapy of Trabedersen and Proleukin. NSG mouse (NOD Scid Gamma mouse, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) which are humanized by human PBMC engraftment and the tumor growth of Melanoma and TNBC (Triple Negative Breast Cancer cell) cell line are monitored. Results: The combination treatment of Trabedersen and low dose Proleukin decreased cancer cell viability in vitro experiment in solid cancer cell lines. Melanoma and TNBC tumor growth was delayed in humanized NSG mouse model by Trabedersen and low dose IL-2 combination therapy and tumor growth delay was statistically significant to Trabedersen alone or IL-2 alone group. Tumor infiltrating lymphocyte population was increased in Trabedersen treated group and FoxP3+ regulatory T cell population in blood and tumor microenvironment was decreased by treatment of Trabedersen. Conclusion: TGF-Β inhibitor (Trabedersen) and low dose IL-2 (Proleukin) combination treatment is expected to be an effective regimen in solid cancer treatment than individual treatment by alteration of tumor environment. Modulation of the dose of Proleukin expects to help reduce the toxicity of IL-2 and increase the anti-cancer effect by combination with Trabedersen. Citation Format: Jun-Eui Park, Jihye Koo, Hong-Kyu Lee, Tae Hun Kim. Combination therapy of anti-sense oligonucleotide targeting TGF-beta2 (TASO) and IL-2 (Proleukin) has anti-cancer effect in solid 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 1448.

Abstract 1066: Pre-clinical combination of a PARP inhibitor, talazoparib, and carboplatin in triple-negative breast cancer

Abstract Background: PARP inhibitors, such as talazoparib, demonstrated an improvement in progression-free survival as monotherapy in germline BRCA1/2-mutated (BRCA-MUT) HER2-negative locally advanced/metastatic breast cancer patients. BRCA-MUT tumors constitute 15-20% of triple-negative breast cancers (TNBCs). TNBCs are an aggressive subtype and lack overexpression of hormone receptors and HER2. Here, we evaluate the impact of the combination of talazoparib and a chemotherapeutic agent, carboplatin, to better understand which TNBCs will benefit from this combination, and the manner in which different sequencing strategies can impact primary tumor growth and distant metastasis development. Methods: We performed a 10-day chemosensitivity assay using 7 BRCA-MUT and BRCA wild-type TNBC cell lines. We tested 9 concentrations of talazoparib (Pfizer), and carboplatin (Selleckchem) each. Post-treatment, we used automated high-content imaging with Operetta (Perkin Elmer). We calculated IC50 values for talazoparib alone, CI (Combination Index) values, the DNA damage response (product of the percentage of cells positive for 53BP1 and mean 53BP1 foci per cell), and the apoptotic index (percentage of cells positive for cleaved-PARP). We also used an orthotopic xenograft model of MDAMB231 in NSG (NOD scid gamma) mice, with 8-14 mice in each treatment group. We treated mice with carboplatin (C) (35 mg/kg intraperitoneally) in combination with talazoparib (T) (0.03 mg/kg oral gavage) using 2 dosing strategies: a) concomitant administration of C + T; and b) T first, followed by C three days later, each compared to vehicle control. Results: We found a synergistic effect of the combination of talazoparib and carboplatin (CI &amp;lt; 1) in all 7 cell lines, with the greatest synergy (CI&amp;lt; 0.65) identified in three cell lines that were PARPi (PARP inhibitor)-resistant, namely HCC1143, MDAMB231, and Hs578T. For these three cell lines, we found that the mean 53BP1 product score increased by 7-16 fold in combination versus talazoparib alone at 0.2 µM, and the apoptotic index also increased by 4-26 fold at the same conditions. Concomitant administration of talazoparib and carboplatin in the MDAMB231 xenograft resulted in a 53.1% inhibition in primary tumor volume in comparison to control (P=0.0004), whereas sequential administration resulted in a 69.2% primary tumor volume inhibition (P&amp;lt;0.0001). The talazoparib first combination approach also resulted in a 53.9% decrease in lung micrometastasis (P=0.0003). Conclusion: The combination of talazoparib and carboplatin demonstrated a synergistic effect in 7 TNBC cell lines. We found greater DNA damage and cell death amongst PARPi-resistant cell lines and at lower concentrations of talazoparib when combined with carboplatin. In-vivo, sequential administration of talazoparib and carboplatin was the most effective approach to inhibit primary tumor growth. Citation Format: Alexia Cotte, Michèle Beniey, Takrima Haque, Nelly Béchir, Audrey Hubert, Korotoum W. Diallo, Danh Tran-Thanh, Saima N. Hassan. Pre-clinical combination of a PARP inhibitor, talazoparib, and carboplatin in 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 1066.

Abstract 1004: Defining stearoyl-CoA desaturase 1 as a molecular therapeutic target against cholangiocarcinoma

Abstract Cancer cells increase lipid metabolism by up-regulation lipogenic enzymes such as stearoyl-CoA desaturase 1 (SCD1). It is the rate-limiting enzyme in the de novo synthesis of fatty acids (FAs), catalyzing the conversion and biosynthesis of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). We developed SSI-4, a novel highly specific SCD1 inhibitor. The anti-tumor activity ofSSI-4 was examined against different cholangiocarcinoma (CCA) models that included human and mouseCCA cell lines, CCA patient-derived xenografts (PDX) NOD SCID gamma (NSG) mouse models, and CCA PDX derived organoids. Using cell proliferation assay, we examined twelve CCA cell lines and 4 were very sensitive to SSI-4 (1-40 nM IC50). The remaining CCA cell lines showed moderate to no sensitivity to SSI-4. Studies at the protein level have shown that SCD1 is present in both sensitive and non-sensitive cells. Thus, SCD1 must be present in sensitive cells but is not predictive of response to an SCD1 inhibitor. In an effort to identify biomarkers predicting response to therapy, we performed assays, such as lipid, lactate, or energy phenotype which identified elevated lactate and unsaturated fatty acid levels as well as a glycolytic energy phenotype. In vivo, CCA PDX models have demonstrated that SSI-4strongly inhibited tumor growth. Immunohistochemical analysis of CCA tumors revealed elevated glycolytic genes in sensitive cells and thus, a potential predictive biomarker. We are currently pursuing a better understanding of the relationship between glycolysis and fatty acid synthesis/metabolism. We are also testing novel combination therapy of SSI-4 with the standard of care and other drugs to develop novel treatments for CCA. Using CCA PDX derived organoids showed synergistic antitumor activity of SSI-4in combination with cancer standard of care drugs such as gemcitabine and cisplatin. Collectively, we expect to develop a biomarker-driven selection of CCA patients sensitive to SCD1 inhibitors and novel SSI-4 combination therapy leading to improved patient survival. Citation Format: Justyna J. Gleba, Laura A. Marlow, Erin E. Miller, James L. Miller, Aylin Alasonyalilar-Demirer, Yi Guo, Kabir Mody, Lewis R. Roberts, Steven R. Alberts, Mark J. Truty, Tushar C. Patel, John A. Copland. Defining stearoyl-CoA desaturase 1 as a molecular therapeutic target against cholangiocarcinoma [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 1004.

Abstract 1899: Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting

Abstract Activating missense mutations within RAS oncogenes are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here, we employ multi-omic approaches to characterize optimal HLA class I-restricted mKRAS epitopes. Using in silico epitope prediction, we identify candidate mKRAS G12 epitopes with high predicted binding affinity to prevalent HLA class I alleles. We utilize biochemical approaches to identify mKRAS epitopes with high measured binding affinity and stability to HLA-A*03:01, -A*11:01 and -B*07:02. Furthermore, we provide proteomic validation of epitope processing and presentation by these alleles. We report mKRAS G12V and G12R epitopes are immunogenic as evidenced by the isolation of mKRAS G12V- and G12R-specific TCRαβ from healthy donors. The transfer of mKRAS-TCRs to primary CD8+ T cells serves as sensitive probes to validate mKRAS epitope processing and presentation by tumor cells and confers cytotoxicity against mKRAS human tumor cell lines of various histologies. The kinetics of lytic activity correlate with measured TCR avidity and mKRAS/HLA class I complex abundance expressed on the tumor cell surface. Finally, the adoptive transfer of mKRAS-TCR engineered primary CD8+ T cells to NOD scid gamma mice leads to tumor eradication in an orthotopic mKRAS lung cancer model. This study validates mKRAS as a bona fide tumor neoantigen and supports strategies to identify recurrent driver alterations for immunological targeting. Based on this work, clinical studies targeting mKRAS in cancer patients using vaccines and adoptive T cell therapy are under active investigation. Citation Format: Adham S. Bear, Tatiana Blanchard, Mark H. O'Hara, John Scholler, Robert H. Vonderheide, Gerald P. Linette, Beatriz M. Carreno. Biochemical and functional characterization of mutant KRAS epitopes validates this oncoprotein for immunological targeting [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 1899.