Renal Cell Carcinoma Xenograft Models Research Articles

Abstract LB122: JNJ-87890387, a novel ENPP3 bispecific T-cell redirector (ENPP3xCD3) with tumor selectivity through targeting apical surface antigens

Abstract Clinical success of T-cell redirection therapy for solid tumors has been limited potentially due to a lack of target specificity against cancer, leading to on-target off-tumor toxicity at sub-efficacious doses. ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3) is a transmembrane protein that was identified with the help of an artificial intelligence machine learning algorithm. ENPP3 is characterized by apically restricted expression in most normal tissues and elevated, depolarized expression in several solid tumors, thus potentially providing tumor selectivity. ENPP3 expression exhibits a high prevalence in several solid tumors including renal cell carcinoma (RCC) - clear cell (93%) and papillary (78%), lung adenocarcinoma (50%), endometrioid uterine (53%) and ovarian (47%) cancers, colorectal carcinoma (51%), and hepatocellular carcinoma (HCC, 47%). JNJ-87890387 is a fully human bispecific antibody with high affinity and specific binding to ENPP3 on tumor cells and lower affinity binding to cluster of differentiation (CD)3 on T-cells. In vitro, JNJ-87890387 demonstrated potent (pM) T-cell activation and cytotoxicity against tumor cell lines with different levels of endogenous ENPP3 membrane expression, and absence of killing against cells lacking ENPP3 expression, confirming JNJ-87890387 specificity. In vivo, JNJ-87890387 demonstrated ENPP3-expression-dependent potent anti-tumor activity in multiple cell line-derived and patient tumor-derived RCC and HCC xenograft models with complete tumor regressions, and evidence of dose-dependent CD4+ and CD8+ T cell infiltration into the tumors. Preclinical safety assessment in the cynomolgus monkey with a tool molecule using the therapeutic ENPP3 binding arm but with a cynomolgus CD3 cross-reactive arm only identified toxicities consistent with the modality. In summary, these data support JNJ-87890387’s clinical development with an ongoing first-in-human Phase I study to evaluate the safety and preliminary anti-tumor activity in advanced-stage solid tumors with a high prevalence of ENPP3 expression. Citation Format: Smruthi Vijayaraghavan, Pankaj Seth, Annmarie Winkis, Kristen Chevalier, Alexa Marthaler, Katrin Sproesser, Vince Torti, Nirav Shah, Eilyn Lacy, Anna-Lena Frisk, Heather Deutsch, Gerald Chu, Michael Sharp, Jonathan Pabalan, Cholpon Tilegenova, Katherine Thorton, Josh Lauring, Bethany Mattson, Ken Tian, Jacqueline Kinyamu-Akunda, Laurie Lenox, Wan Cheung Cheung, Sheri Moores, Sylvie Laquerre. JNJ-87890387, a novel ENPP3 bispecific T-cell redirector (ENPP3xCD3) with tumor selectivity through targeting apical surface antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB122.

Therapeutic Efficacy of YM155 to Regulate an Epigenetic Enzyme in Major Subtypes of RCC.

Renal cell carcinoma (RCC) is the most common type of kidney cancer and includes more than 10 subtypes. Compared to the intensively investigated clear cell RCC (ccRCC), the underlying mechanisms and treatment options of other subtypes, including papillary RCC (pRCC) and chromogenic RCC (chRCC), are limited. In this study, we analyzed the public databases for ccRCC, pRCC, and chRCC and found that BIRC5 was commonly overexpressed in a large cohort of pRCC and chRCC patients as well as ccRCC and was closely related to the progression of RCCs. We investigated the potential of BIRC5 as a therapeutic target for these three types of RCCs. Loss and gain of function studies showed the critical role of BIRC5 in cancer growth. YM155, a BIRC5 inhibitor, induced a potent tumor-suppressive effect in the three types of RCC cells and xenograft models. To determine the mechanism underlying the anti-tumor effects of YM155, we examined epigenetic modifications in the BIRC5 promoter and found that histone H3 lysine 27 acetylation (H3K27Ac) was highly enriched on the promoter region of BIRC5. Chromatin-immunoprecipitation analysis revealed that H3K27Ac enrichment was significantly decreased by YM155. Immunohistochemistry of xenografted tissue showed that overexpression of BIRC5 plays an important role in malignancy in RCC. Furthermore, high expression of P300 was significantly associated with the progression of RCC. Our findings demonstrate the P300-H3K27Ac-BIRC5 cascade in three types of RCC and provide a therapeutic path for future research on RCC.

Abstract B026: Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma

Abstract Purpose: Although renal cell carcinoma (RCC) is relatively more resistant to radiation therapy (RT) compared to other cancer indications, recent advances in single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) made it possible to achieve better outcome and reduce treatment related toxicities. In addition, with studies demonstrating that certain therapeutic agents including chemotherapies can act as radiosensitizers, the idea of combining radiotherapy with those agents are being actively pursued. Among them, mammalian target of rapamycin (mTOR) inhibitor everolimus (E) and survivin inhibitor YM155 (Y) are of special interest since they target multiple RT-resistance mechanisms simultaneously. Additionally, YM155 has been shown to help in overcoming resistance to mTOR inhibitors in various cancer including RCC. Hence, the present study aims to evaluate the efficacy of a tumor-targeted liposomal formulation combining everolimus and YM155 (EY-L) in sensitizing RCC tumors towards radiation. Experimental Design: Previously we developed a tumor-targeted liposomal formulation and used it successfully to deliver multiple drugs for the treatment of RCC in both immune-deficient and immune-competent mouse models. Here we developed a similar formulation combining everolimus and YM155 (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further evaluated the efficacy of EY-L in sensitizing RCC cell lines and tumors towards radiation in vitro using clonogenic assay and in vivo in RCC xenografts in immunodeficient mice and murine RCC models in immunocompetent mice. Results: EY-L (2 mg/kg E, 1.4 mg/kg Y, twice a week intravenous administration) was better than E-L or Y-L, the corresponding single drug loaded formulations in containing the primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. Furthermore, EY-L leads to significantly higher sensitization of RCC cells towards radiation in vitro than E-L or Y-L. Finally, EY-L was able to sensitize RCC tumors towards radiation therapy in both RCC xenograft and murine RCC model. Conclusion: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy using in vitro clonogenic assay and in vivo radiosensitization studies in multiple RCC tumor models. Citation Format: Krishnendu Pal, Vijay S. Madamsetty, Hari K. Rachamala, Shamit K. Dutta, Enfeng Wang, Debabrata Mukhopadhyay. Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B026.

PiRNA-1742 promotes renal cell carcinoma malignancy by regulating USP8 stability through binding to hnRNPU and thereby inhibiting MUC12 ubiquitination

Accumulating studies have confirmed that PIWI-interacting RNAs (piRNAs) are considered epigenetic effectors in cancer. We performed piRNA microarray expression analysis on renal cell carcinoma (RCC) tumor tissues and paired normal tissues and performed a series of in vivo and in vitro experiments to explore piRNAs associated with RCC progression and investigate their functional mechanisms. We found that piR-1742 was highly expressed in RCC tumors and that patients with high piR-1742 expression had a poor prognosis. Inhibition of piR-1742 significantly reduced tumor growth in RCC xenograft and organoid models. Mechanistically, piRNA-1742 regulates the stability of USP8 mRNA by binding directly to hnRNPU, which acts as a deubiquitinating enzyme that inhibits the ubiquitination of MUC12 and promotes the development of malignant RCC. Subsequently, nanotherapeutic systems loaded with piRNA-1742 inhibitors were found to effectively inhibit the metastasis and growth of RCC in vivo. Therefore, this study highlights the functional importance of piRNA-related ubiquitination in RCC and demonstrates the development of a related nanotherapeutic system, possibly contributing to the development of therapeutic approaches for RCC.

Abstract 2898: CAT-248, an allogeneic CD70-directed CAR-NK cell therapy effectively controls CD70-positive tumor xenografts

Abstract Engineered, off-the-shelf, allogeneic natural killer (NK) cell therapy is an attractive approach for targeting solid tumors, given their emerging clinical safety, efficacy, and intrinsic anti-tumor recognition and activity. However, improvements to support persistence and maintain durable anti-tumor activity within the tumor microenvironment may be necessary to achieve meaningful clinical efficacy. Here we describe the preclinical activity of CAT-248, a CD70-directed CAR-NK cell therapy, engineered using the TcBuster™ Transposon System (Bio-Techne) multiplexed with CRISPR/Cas9 editing. CD70 is highly expressed in many tumor types while normal tissue expression is restricted to a subset of activated immune cells. CAT-248 is an allogeneic, healthy donor peripheral blood-derived NK cell product designed for durable efficacy against CD70 expressing tumors. CAT-248 is engineered to express CD70 CAR, interleukin 15 (IL-15), and transforming growth factor β (TGFβ) dominant negative receptor (DNR). In addition, CAT-248 includes CRISPR/Cas9 knockout of CD70 to mitigate fratricide due to endogenous CD70 expression in activated NK cells. IL-15 enhances persistence of CAT-248 to enable durable efficacy, and TGFβ DNR enables CAT-248 to maintain high activity in TGFβ-enriched and immunosuppressive solid tumor microenvironments. CAT-248 is manufactured using transposon-based engineering which enables stable integration of the three transgenes and CRISPR/Cas9 knockout of CD70 in a single electroporation step, resulting in 40-80% CAR expression and 80-90% knockout of CD70 in CAT-248 NK cells. CAT-248 activity was characterized across a panel of in vitro assays to evaluate the function of CD70 CAR, TGFβ DNR, and IL-15 transgenes. CD70-directed cytotoxicity was assessed against a panel of tumor cell lines with a broad range of CD70 expression. In vitro, CAT-248 cells demonstrated both CAR-dependent cytotoxicity and over 2-fold greater secretion of effector cytokines IFNγ and TNFα than control NK cells. TGFβ DNR effectively prevented TGFβ-induced SMAD phosphorylation and TGFβ-induced downregulation of DNAM-1, an NK cell activating receptor. IL-15 secretion enabled in vitro NK cell expansion over a 9-day time course without exogenous cytokine support. To confirm cytolytic activity in vivo, CAT-248 cells were administered therapeutically in a 786-O CD70+ renal cell carcinoma xenograft model. CAT-248 cells effectively controlled tumor, demonstrating >99% reduction in tumor burden relative to control NK cells (p<0.01). Further, CAT-248 cells demonstrated significant in vivo persistence beyond 4 weeks post-dosing in peripheral blood. Overall, the results demonstrate the potential for CAT-248 as a novel off-the-shelf, cryopreserved, allogeneic NK cell therapy for CD70-positive renal cell carcinoma and other solid tumor malignancies. Citation Format: Bashar Hamza, Eugene Choi, Dominic Picarella, Finola Moore, Angela Nunez, Henry Moreno, Marilyn Marques, Alexia Barandiaran, Meghan Walsh, Kisha Pradhan, Krista Daniel, Kelly Becker, Jennifer Johnson, Karl Malakian, Keith H. Wong, Mark A. Omobono, Charlotte Franco, Andres Alvarez, Jenna Nguyen, Billy Blanco, Priya Khurana, Beatriz Marques, Adriana Rivera, Michele McAuliffe, Jae-Woong Chang, Joshua Krueger, Beau R. Webber, Branden S. Moriarity, Joseph Gold, Cherry Thomas, Vipin Suri. CAT-248, an allogeneic CD70-directed CAR-NK cell therapy effectively controls CD70-positive tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2898.

Overexpressed lncRNA FTX promotes the cell viability, proliferation, migration and invasion of renal cell carcinoma via FTX/miR‑4429/UBE2C axis.

The present study aimed to explore the role of long non-coding (lnc)RNA FTX and ubiquitin-conjugating enzyme E2C (UBE2C) in promoting the progression of renal cell carcinoma (RCC) and the underlying regulatory mechanism. Relative levels of lncRNA FTX, UBE2C, AKT, CDK1 and CDK6 in RCC cell lines were detected by reverse transcription-quantitative (RT-q). Expression levels of UBE2C, phosphorylated (p)-AKT/AKT, p-CDK1/CDK1 and p-CDK6/CDK6 in RCC and paracancerous specimens and RCC cells were measured by western blot or immunohistochemistry assay. In addition, the proliferative rate, cell viability, cell cycle progression, migratory rate and invasive rate of RCC cells overexpressing lncRNA FTX by lentivirus transfection were determined by a series of functional experiments, including the colony formation assay, MTT assay, flow cytometry, Transwell assay and wound healing assay. The targeted binding relationship in the lncRNA FTX/miR-4429/UBE2C axis was validated by dual-luciferase reporter assay. By intervening microRNA (miR)-4492 and UBE2C by the transfection of miR-4429-mimics or short interfering UBE2C-2, the regulatory effect of lncRNA FTX/miR-4429/UBE2C axis on the progression of RCC was evaluated. Finally, a xenograft model of RCC in nude mice was established by subcutaneous implantation, thus evaluating the in vivo function of lncRNA FTX in the progression of RCC. The results showed that lncRNA FTX and UBE2C were upregulated in RCC specimens and cell lines. The overexpression of lncRNA FTX in RCC cells upregulated UBE2C. In addition, the overexpression of lncRNA FTX promoted the cell viability and proliferative, migratory and invasive capacities of RCC cells and accelerated the cell cycle progression. A dual-luciferase reporter assay validated that lncRNA FTX exerted the miRNA sponge effect on miR-4429, which was bound to UBE2C 3′UTR. Knockdown of UBE2C effectively reversed the regulatory effects of overexpressed lncRNA FTX on the abovementioned phenotypes of RCC cells. In the xenograft model of RCC, the mice implanted with RCC cells overexpressing lncRNA FTX showed a larger tumor size and higher tumor weight than those of controls, while the in vivo knockdown of UBE2C significantly reduced the size of RCC lesions, indicating the reversed cancer-promoting effect of lncRNA FTX. Overall, the present study showed that lncRNA FTX was upregulated in RCC and could significantly promote the proliferative, migratory and invasive capacities, enhancing the viability and accelerating the cell cycle progression of RCC cells by exerting the miRNA sponge effect on miR-4429 and thus upregulating UBE2C. lncRNA FTX and UBE2C are potential molecular biomarkers and therapeutic targets of RCC.

Abstract 2649: Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers

Abstract Purpose: Pro-angiogenic and immune cells expressing chemokine receptor CXCR4 traffic along concentration gradients of the chemokine ligand CXCL12, which disseminates from stromal niches. The CXCR4/CXCL12 axis is hijacked by various cancer types characterized by dramatic CXCR4 and/or CXCL12 upregulation. This chemokine network misregulation causes hyperactivation of CXCR4-mediated processes, leading to (1) excessive stimulation of protective tumor-stromal interactions, (2) metastatic expansion of CXCR4+ cancer cells to CXCL12-rich sites, and (3) intratumoral infiltration of CXCR4+ immunosuppressive cells. Accordingly, CXCR4 antagonists have significant therapeutic potential against cancer. While clinical CXCR4 inhibitors are severely limited by suboptimal pharmacokinetic (PK) properties and off-target activities, several of our tetrahydroisoquinoline-based CXCR4 antagonists exhibit superior selectivity and PK profiles to these clinical comparators (e.g., X4P-001). Methods: To test the hypothesis that improved PK profiles would translate to enhanced efficacy, our lead CXCR4 antagonist EMU-116 was compared to X4P-001 head-to-head in three mouse models of genitourinary cancers. First, human 786.0 renal cell carcinoma (RCC) xenograft-bearing female nude mice were treated p.o., q.d. with vehicle, axitinib (30 mg/kg), X4P-001 (100 mg/kg), EMU-116 (3, 10, or 30 mg/kg), X4P + axitinib, or EMU-116 + axitinib. Subcutaneous tumor volume was monitored using calipers. Second, nude male mice bearing intratibial, luciferase-expressing human PC-3 prostate cancer xenografts were treated with vehicle (p.o., q.d.), docetaxel (10 mg/kg i.p. weekly), X4P-001 (10 or 30 mg/kg p.o., q.d.), EMU-116 (10 or 30 mg/kg p.o., q.d.), X4P + axitinib, or EMU + axitinib. Intratibial tumor volume was monitored via luminescence imaging. Third, syngeneic RENCA RCC tumor-bearing female Balb/c mice were treated p.o., q.d. with vehicle, X4P-001 (30 mg/kg), or EMU-116 (30 mg/kg). Subcutaneous tumor volume was monitored using calipers, and immune cell subsets in bone marrow, blood, tumor, and tumor-draining lymph nodes were quantified via flow cytometry. Results: In the RCC xenograft model in combination with axitinib, EMU-116 (3 or 10 mg/kg) was equally effective at decreasing tumor burden compared to X4P-001 (100 mg/kg), whereas EMU-116 (30 mg/kg) was more effective. In the bone metastatic prostate cancer xenograft model, EMU-116 was as or more effective than X4P-001 when paired with docetaxel. In the syngeneic RCC model, EMU-116 mobilized T cells more effectively than X4P-001. Conclusion: EMU-116, at the same or lower dose, was more efficacious than X4P-001 in these models. These results highlight EMU-116 as a clinical candidate with significant therapeutic potential to synergize with chemotherapeutics, targeted therapies, and immuno-oncology agents. Citation Format: Eric J. Miller, Carrie Q. Sun, Petra Gregorova, Edgars Jecs, Yesim Altas Tahirovic, Robert J. Wilson, Huy H. Nguyen, Savita K. Sharma, Perry Bartsch, Zachary Sticher, Levi Moellering, Priscilla Davidson, Ryan Jajosky, Michael D'Erasmo, Manohar Saindane, Zafer Sahin, Nicholas S. Akins, Alexander A. Kolykhalov, Lawrence Wilson, Rebecca S. Arnold, John A. Petros, Haydn Kissick, Lingjie Xu, Yi Jiang, Dennis C. Liotta. Orally bioavailable small molecule CXCR4 antagonists with enhanced efficacy in mouse models of genitourinary cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2649.

Abstract 95: In vitro, in vivo and molecular effects of triptolide and minnelide in renal cell carcinoma

Abstract Background: The worldwide incidence of Renal Cell Carcinoma (RCC) is increasing. Although new therapies have improved outcomes in advanced RCC, most patients eventually fail treatment and succumb to this devastating disease. Systemic therapy strategies have focused on TKI and immunotherapy, alone or in combination. Triptolide, a diterpenoid epoxide extracted from Tripterygium wilfordii Hook f (TWHf), has potent antitumor activity in multiple cancer models. Few studies have focused on the potential of triptolide as a novel therapeutic option in RCC. Objective: the objectives of this study are to characterize the in vitro and in vivo effects of Triptolide (T) and its water-soluble form, Minnelide (M), in models of renal cell carcinoma. Methods: The in vitro effects of triptolide (T) on human and murine RCC (786-0, A498, Caki-1, ACHN, and RENCA) cell proliferation was assessed using cell count and xCelligence assays at 24, 48, 72 and 96h. Molecular and mechanistic characterization of triptolide’s effects in 786-0 cells were analyzed by the (Reverse Phase Protein Array) and validated by western blot analysis. The in vivo effects (tumor progression and survival) of M were assessed in nude mice bearing 786-0 tumors (8x106 cells per mouse). M was administered to tumor-bearing mice at two different doses (0.21mg/kg and 0.42mg/kg daily), intraperitoneally, for 21 days. Correlative studies to explain the in vivo effect of M and the correlation with the molecular changes in vitro seen are being performed and will be shown in the poster. Results: T significantly inhibited, in a dose-response manner, cell proliferation in all human and murine renal cancer cell lines, with IC50 ranging between 12.5 and 25 nM). T was associated with significant negative modulation of proliferation, cell cycle, survival, increased apoptosis, and ER stress pathways in 786-0 cells, as demonstrated by RPPA analysis and validated by western Blot of selected pathway proteins, such as apoptosis (PARP cleavage and Caspase 3 activation), ER stress induction (pEIF2a, CHOP), and down-regulation of survival and proliferation pathways (pAKT). In vivo, M was associated with significant antitumor effects in 786-0 xenografts, with complete responses in the majority of mice while on treatment. These effects were associated with significant prolongation in overall survival in M treated vs. control mice. No significant toxicity or treatment-related deaths were observed. Conclusion: Our results have shown for the first time the potent in vitro and in vivo antitumor effects of T in RCC and the molecular changes associated with these effects. The profound antitumor effects in the aggressive 786-0 RCC xenograft model are highly encouraging and warrant further preclinical studies and potential clinical trials of M this devastating disease. Correlative tumor studies to understand the mechanisms of M in vivo antitumor effects are underway and will be presented at the meeting. Citation Format: Valery A. Chavez, Floritza Bustamante, Abner Murray, Ashok Saluja, Jaime Merchan. In vitro, in vivo and molecular effects of triptolide and minnelide in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 95.

Patient-Derived Renal Cell Carcinoma Xenografts Capture Tumor Genetic Profiles and Aggressive Behaviors

BACKGROUND: Patient-derived xenografts (PDX) have emerged as one of the most promising model systems to study cancer biology and to develop new antineoplastic drugs. Renal cell carcinoma (RCC) represents up to 90% of all kidney tumors, exhibits aggressive behavior, and has a propensity for metastasis. At diagnosis, 30% of patients with RCC have metastases, while up to 50% of those with localized disease treated with curative protocols experience recurrence. OBJECTIVE: This study aimed to establish an RCC PDX platform to identify novel clinical and molecular biomarkers of recurrence risk in order to facilitate precision medicine. METHODS: Tumor samples were obtained from surgical specimens of 87 RCC patients; fragments were implanted in immunodeficient NOD/SCID/gamma (NSG) mice. 17 Fragments were implanted subcutaneously in an initial group while a second group of 70 samples were implanted orthotopically in the subcapsular space. RESULTS: A total of 19 PDX developed only after orthotopic implantation, and included 15 cases of clear cell RCC subtype, 3 cases of papillary subtype, and 1 unclassifiable tumor. 1 PDX of clear cell RCC recapitulated the phenotype of vena caval tumor thrombus extension that had been diagnosed in the source patient. PDX characterization by immunohistochemistry and targeted sequencing indicated that all PDXs preserved RCC identity and major molecular alterations. Moreover, the capacity of tumor engraftment was a strong prognostic indicator for patients with locally advanced disease. CONCLUSION: Taken together, these results suggest that the orthotopic xenograft model of RCC represents a suitable tool to study RCC biology, identify biomarkers, and to test therapeutic candidates.

Preclinical activity of cobimetinib alone or in combination with chemotherapy and targeted therapies in renal cell carcinoma.

Background: The poor outcome of advanced renal cell carcinoma (RCC) necessitates new treatments. Cobimetinib is a MEK inhibitor and approved for the treatment of melanoma. This work investigated the efficacy of cobimetinib alone and incombination with anti-RCC drugs. Methods: Proliferation and apoptosis assays were performed, and combination index was analyzed on RCC cell lines(CaKi-2, 786-O, A-704, ACHN andA489) and xenograft models. Immunoblotting analysis was conducted to investigate the MAPK pathway. Results: Cobimetinib was active against RCC cells, with IC50 at 0.006-0.8μM, and acted synergistically with standard-of-care therapy. Cobimetinib at nontoxic doses prevented tumor formation, inhibited tumor growthand enhanced efficacy of 5-fluorouracil, sorafenib and sunitinibvia suppressing Raf/MEK/ERK, leading to MAPK pathway inhibition. Conclusion: Our findings demonstrate the potent anti-RCC activity of cobimetinib and its synergism with RCC standard-of-care drugs, and confirm the underlying mechanism of the action of cobimetinib.

Discovery of 2-amino-3-amido-5-aryl-pyridines as highly potent, orally bioavailable, and efficacious PERK kinase inhibitors

The protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is one of the three endoplasmic reticulum (ER) transmembrane sensors of the unfolded protein response (UPR) that regulates protein synthesis, alleviates cellular ER stress and has been implicated in tumorigenesis and prolonged cancer cell survival. In this study, we report a series of 2-amino-3-amido-5-aryl-pyridines that we have identified as potent, selective, and orally bioavailable PERK inhibitors. Amongst the series studied herein, compound (28) a (R)-2-Amino-5-(4-(2-(3,5-difluorophenyl)-2-hydroxyacetamido)-2-ethylphenyl)-N-isopropylnicotinamide has demonstrated potent biochemical and cellular activity, robust pharmacokinetics and 70% oral bioavailability in mice. Given these data, this compound (28) was studied in the 786-O renal cell carcinoma xenograft model. We observed dose-dependent, statistically significant tumor growth inhibition, supporting the use of this tool compound in additional mechanistic studies.

Adoptive cell therapy of patient-derived renal cell carcinoma xenograft model with IL-15-induced γδT cells.

Adoptive transfer of γδ T cells is an attractive approach for cell-based immunotherapy in treatment of renal cell carcinoma (RCC). Interleukin-15 (IL-15) is the key physiological cytokine that regulates γδ T cell differentiation, proliferation and survival. In this work, we determined that IL-15 have the capacity to enhance the anti-tumoral functions of γδ T cells. IL-15 can induce the upregulation of cytotoxicity-associated molecules on the γδ T cell surface, incite γδ T cell proliferation and decrease apoptosis. Moreover, the enhanced cytotoxicity of IL-15-induced γδ T cell was dependent on the interaction of NKG2D and MICA. Most importantly, we found that IL-15-induced γδ T cells effectively suppressed the tumor growth in vivo and prolonged the survival time of RCC-bearing patient‑derived xenograft (PDX) mice. These results are important for the prospective use of γδ T cells in clinical practice when designing novel cell-based immunotherapies against RCC.

Cytotoxic T Lymphocytes Regenerated from iPS Cells Have Therapeutic Efficacy in a Patient-Derived Xenograft Solid Tumor Model.

SummaryCurrent adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors.

Ultrasound localization microscopy of renal tumor xenografts in chicken embryo is correlated to hypoxia

Ultrasound localization microscopy (ULM) permits the reconstruction of super-resolved microvascular images at clinically relevant penetration depths, which can be potentially leveraged to provide non-invasive quantitative measures of tissue hemodynamics and hypoxic status. We demonstrate that ULM microbubble data processing methods, applied to images acquired with a Verasonics Vantage 256 system, can provide a non-invasive imaging surrogate biomarker of tissue oxygenation status. This technique was applied to evaluate the microvascular structure, vascular perfusion, and hypoxia of a renal cell carcinoma xenograft model grown in the chorioallantoic membrane of chicken embryos. Histological microvascular density was significantly correlated to ULM measures of intervessel distance (R = −0.92, CI95 = [−0.99,−0.42], p = 0.01). The Distance Metric, a measure of vascular tortuosity, was found to be significantly correlated to hypoxyprobe quantifications (R = 0.86, CI95 = [0.17, 0.99], p = 0.03). ULM, by providing non-invasive in vivo microvascular structural information, has the potential to be a crucial clinical imaging modality for the diagnosis and therapy monitoring of solid tumors.

SUB-PHENOTYPE ANALYSES OF BIPOLAR DISORDER IN DATA FROM THE PGC BIPOLAR DISORDER WORKING GROUP

Current adoptive T cell therapies conducted in an autologous setting are costly, time consuming, and depend on the quality of the patient's T cells. To address these issues, we developed a strategy in which cytotoxic T lymphocytes (CTLs) are regenerated from iPSCs that were originally derived from T cells and succeeded in regenerating CTLs specific for the WT1 antigen, which exhibited therapeutic efficacy in a xenograft model of leukemia. In this study, we extended our strategy to solid tumors. The regenerated WT1-specific CTLs had a strong therapeutic effect in orthotopic xenograft model using a renal cell carcinoma (RCC) cell line. To make our method more generally applicable, we developed an allogeneic approach by transducing HLA-haplotype homozygous iPSCs with WT1-specific TCR α/β genes that had been tested clinically. The regenerated CTLs antigen-specifically suppressed tumor growth in a patient-derived xenograft model of RCC, demonstrating the feasibility of our strategy against solid tumors.

Downregulation of Human DAB2IP Gene Expression in Renal Cell Carcinoma Results in Resistance to Ionizing Radiation.

Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.

ERK Inhibitor Enhances Everolimus Efficacy through the Attenuation of dNTP Pools in Renal Cell Carcinoma

The clinical efficiency of everolimus, an mammalian target of rapamycin (mTOR) inhibitor, is palliative as sequential or second-line therapy for renal cell carcinoma (RCC). However, the limited response of everolimus in RCC remains uncertain. In the present study, everolimus-resistant RCC models were established to understand the mechanisms and to seek combination approaches. Consequently, the activation of ERK was found to contribute toward everolimus-acquired resistance and poor prognosis in patients with RCC. In addition, the efficacy and mechanism of combination treatment underlying RCC using everolimus and ERK inhibitors was investigated. The ERK inhibitor in combination with everolimus synergistically inhibited the proliferation of RCC cells by arresting the cell cycle in the G1 phase. The combination treatment markedly attenuated the deoxyribonucleoside triphosphate (dNTP) pools by downregulating the mRNA expression of RRM1 and RRM2 through E2F1. The overexpression of E2F1 or supplementation of dNTP rescued the anti-proliferation activity of the everolimus-SCH772984 combination. The antitumor efficacy of combination therapy was reiterated in RCC xenograft models. Thus, the current findings provided evidence that the everolimus-ERK inhibitor combination is a preclinical therapeutic strategy for RCC.

Synergistic Activity of Paclitaxel, Sorafenib, and Radiation Therapy in advanced Renal Cell Carcinoma and Breast Cancer

Advanced cancer has been shown to be associated with a higher percentage of epigenetic changes than with genetic mutations. Preclinical models have shown that the combination of paclitaxel, sorafenib, and radiation therapy (RT) plays a crucial role in renal cell carcinoma (RCC) and breast cancer. This study aimed to investigate the involvement of mitochondrial cytochrome c–dependent apoptosis in the mechanism of action of a combination of paclitaxel, sorafenib, and RT in RCC and breast cancer. RCC and breast cancer cell lines were exposed to paclitaxel and sorafenib alone or combined in the presence of radiation, and cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The synergistic anticancer effects of the combination therapy on cell cycle and intracellular signaling pathways were estimated using flow cytometry and immunoblot analysis. RCC and breast cancer cell line xenograft models were used to examine the antitumor activity in vivo. Our results suggest that paclitaxel, sorafenib, and RT synergistically decreased the viability of RCC and breast cancer cells and significantly induced their apoptosis, as shown by caspase-3 cleavage. Paclitaxel, sorafenib, and radiation cotreatment reduced antiapoptotic factor levels in these cells and, thereby, significantly reduced the tumor volume of RCC and breast cancer cell xenografts. The current study suggests that paclitaxel, sorafenib, and radiation cotreatment was more effective than cotreatment with paclitaxel or sorafenib and radiation. These findings may offer a new therapeutic approach to RCC and breast cancer.

Abstract 12: Antitumor activity of lenvatinib in combination with everolimus or an anti-PD1 antibody in preclinical RCC models

Abstract Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. Lenvatinib plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase3 clinical study of lenvatinib in combination with everolimus or pembrolizumab for metastatic RCC patients is in progress. However, the detailed mechanisms underlying the efficacy of these combination treatments remain to be elucidated. In this study, we investigated the antitumor activities of lenvatinib in combination with everolimus or anti-PD1 mAb in preclinical RCC models. We examined antitumor activities in four human RCC (A-498, VMRC-RCW, Caki-1, and ACHN) xenograft models orally treated with lenvatinib, everolimus, and their combination. Expression of Ki67 and phosphorylation of S6 in RCC xenograft models were analyzed by immunofluorescence (IF) staining. Inhibitory effects of lenvatinib and everolimus on the phosphorylation of S6K (T389 and T421/S424), and S6 (S235/S236) were analyzed by western blot in vitro. We also examined antitumor activity of combination treatment of lenvatinib and anti-PD-1 mAb in RAG murine RCC model. The combination of lenvatinib with everolimus showed greater antitumor activity than that of either mono-treatment in all of 4 RCC xenograft models we examined and led to tumor regression in three out of 4 models (A-498, Caki-1, and ACHN). IF imaging showed expression of Ki67 was further suppressed with the combination treatment compared to lenvatinib and everolimus mono-treatments. Phospho-S6 (p-S6) signal were localized in perivascular regions of control group. Lenvatinib mono-treatment decreased p-S6 in the region apart from vessels, but some staining of p-S6 in perivascular regions were remained. The combination treatment further suppressed p-S6 staining regardless of the regions. In RAG syngeneic model, the combination treatment of lenvatinib at 10 mg/kg and anti-PD-1 mAb showed tumor shrinkage and tumors were regressed to nonpalpable sizes in 3 of 6 mice, although lenvatinib and anti-PD-1 mAb mono-treatments only slowed growths of RAG tumor. Our results indicate that combination of lenvatinib and everolimus enhances the inhibitory effects of mTOR signaling and tumor proliferation and showed greater antitumor activities. Combination of lenvatinib and anti-PD-1 mAb showed greater antitumor activity including tumor shrinkage, which was not demonstrated by each monotherapy in the murine RCC syngeneic model. These preclinical results provide one of the mechanisms of combinational effect of lenvatinib and everolimus, or PD-1 blockade in RCC. Citation Format: Yusuke Adachi, Takayuki Kimura, Masahiro Matsuki, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of lenvatinib in combination with everolimus or an anti-PD1 antibody in preclinical RCC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 12.

Mevalonate pathway blockage enhances the efficacy of mTOR inhibitors with the activation of retinoblastoma protein in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common malignancy of kidney and remains largely intractable once it recurs after resection. mTOR inhibitors have been one of the mainstays used against recurrent RCC; however, there has been a major problem of the resistance to mTOR inhibitors, and thus new combination treatments with mTOR inhibitors are required. We here retrospectively showed that regular use of antilipidemic drug statins could provide a longer progression free survival (PFS) in RCC patients prescribed with an mTOR inhibitor everolimus than without statins (median PFS, 7.5 months vs. 3.2 months, respectively; hazard ratio, 0.52; 95% CI, 0.22–1.11). In order to give a rationale for this finding, we used RCC cell lines and showed the combinatorial effects of an mTOR inhibitor with statins induced a robust activation of retinoblastoma protein, whose mechanisms were involved in statins-mediated hindrance of KRAS or Rac1 protein prenylation. Finally, statins treatment also enhanced the efficacy of an mTOR inhibitor in RCC xenograft models. Thus, we provide molecular and (pre)clinical data showing that statins use could be a drug repositioning for RCC patients to enhance the efficacy of mTOR inhibitors.