Preclinical Models Of Renal Cell Carcinoma Research Articles

Venetoclax Synergizes Sunitinib in Renal Cell Carcincoma through Inhibition of Bcl-2.

More effective treatment options for patients with renal cell carcinoma (RCC) are needed, in particular advanced RCC. Sunitinib, a multitarget tyrosine kinase inhibitor, is a first-line treatment of metastatic RCC. However, the management of sunitinib-induced adverse events and resistance is complex. In hematological malignancies, effective targeting of anti-apoptotic proteins such as Bcl-2 has been achieved, but limited progress has been made in solid tumors. This work systematically investigated the therapeutic potential of the combination of sunitinib and venetoclax, a Bcl-2 inhibitor, in preclinical RCC models. Quantitative analysis of drug interactions was performed. Cell viability was examined after drug treatment or Bcl-2 siRNA depletion. RCC xenograft mouse model was applied to validate the efficacy of sunitinib and venetoclax. A strong synergistic interaction between sunitinib and venetoclax was observed across a range of different dose levels in all tested RCC cell lines. Sequential treatment studies show that the sequential addition of venetoclax and then sunitinib is superior to concurrent treatment and the sequential addition of sunitinib and then venetoclax in decreasing RCC cell viability. The sensitivity of RCC cell lines to venetoclax treatment negatively correlates with their Bcl-2 levels. Specific depletion of Bcl-2 mimics the synergistic effects of venetoclax with sunitinib. Treatment of mice implanted with high Bcl-2-expressing RCC cells reveals that a combination of venetoclax and sunitinib at a non-toxic dose displays complete regression of tumor growth throughout the whole duration of treatment. Our work demonstrates that inhibiting Bcl-2 by venetoclax synergistically enhances sunitinib's efficacy in RCC. Venetoclax holds great potential as a viable option for clinical use.

A modified IL-18 drug in combination with CTLA-4 blockade enhances anti-tumor efficacy in preclinical models of renal cell carcinoma

Abstract Background Cytokine-based drugs are currently being explored as alternative cancer immunotherapies. While the cytokine interleukin-18 (IL-18) has immunostimulatory effects, it is negatively regulated by a secreted high-affinity binding protein, IL-18BP, that functions as an immune checkpoint that limits IL-18’s efficacy as a cancer therapeutic. A modified version of IL-18, termed “decoy-resistant” or DR-18, that can avoid trapping by IL-18BP while still maintaining its immune signaling potential, has recently been developed. DR-18 has shown promising preclinical activity in melanoma and colorectal murine models, including potential synergy with anti-PD-1 therapy, and is currently in Phase I trials. In this study, we aim to test the efficacy and determine the cellular mechanism of action of DR-18 in combination with immune checkpoint inhibitors (ICIs) in immunocompetent preclinical models of renal cell carcinoma (RCC), with the goal of establishing the basis for testing these combinations in early phase clinical trials. Methods We engrafted tumors subcutaneously using two different syngeneic, immunocompetent murine RCC models: Renca and RAG. Mice were treated with single-agent DR-18 and combinations of DR-18 with single- and dual-agent anti-PD-1 and anti-CTLA-4. Tumor growth and survival were monitored. In the Renca model, plasma was collected at early time-points and cytokine/chemokine levels were profiled using a 31-plex discovery assay. Single-cell RNA and TCR sequencing was also performed on Renca tumors. Additionally, immune cell depletion studies were conducted in the Renca model with antibodies targeting CD8, CD4, NK cells, and interferon-gamma. Results In the Renca model, DR-18 monotherapy modestly inhibited tumor growth and prolonged survival. The effects were comparable to single- and dual-agent ICIs. Adding PD-1 blockade to DR-18 did not enhance efficacy whereas the addition of anti-CTLA-4 to DR-18 significantly increased anti-tumor effects. Triple-therapy (DR-18 plus anti-PD-1 plus anti-CTLA-4) did not further inhibit tumor growth or prolong survival compared to the doublet (DR-18 plus anti-CTLA-4). The RAG model was more sensitive to ICIs but produced similar results, again showing modest anti-tumor activity of single-agent DR-18 and enhanced benefit of combining with anti-CTLA-4 but not anti-PD-1. Cytokine/chemokine profiling revealed significantly elevated levels of IP-10 (CXCL10) and MIG (CXCL9) after one cycle of DR-18 plus anti-CTLA-4 compared to control and single-agent treatments, suggesting that these chemokines may be key early mediators of the anti-tumor immune response. Single-cell transcriptomic analysis demonstrated changes in intra-tumoral T cell, macrophage, and granulocyte populations with DR-18 plus anti-CTLA-4 relative to other regimens, including enrichment of CD8+ precursor and terminally exhausted T cells and a neutrophil population associated with interferon signaling. Additionally, single-cell TCR analysis showed a reduction in intra-tumoral clonotype diversity with DR-18 plus anti-CTLA-4 compared to other treatments. Immune cell depletion studies identified CD8+ T cells, NK cells, and interferon-gamma, but not CD4+ T cells, as equally required for efficacy of DR-18 plus anti-CTLA-4. Conclusions In this study, we identify DR-18, an IL-18-based drug engineered with resistance to a secreted decoy-receptor protein, in combination with anti-CTLA-4 as having enhanced anti-tumor activity in preclinical models of RCC. This regimen was associated with a more pro-inflammatory immune microenvironment. Further investigation is ongoing to elucidate the cellular mechanism of action of this regimen more fully and lay the groundwork for clinical testing of DR-18-based combination therapy in RCC. In the future, testing novel partner agents outside of anti-PD-1/CTLA-4 and using RCC models of ICI-resistance could be particularly informative and clinically relevant.

Abstract 2242: Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway

Abstract Lenvatinib mesilate (LEN) 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. LEN plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase1b/2 clinical trial of LEN in combination with pembrolizumab with selected solid tumors including RCC and Phase3 study of LEN in combination with everolimus or pembrolizumab for metastatic RCC patients are in progress. In this study, we investigated the antitumor and immunomodulatory activities of LEN alone and in combination with anti-PD1 Ab and explored the mechanism of action underlying the activities of LEN and PD1 blockade in preclinical syngeneic RCC models. We examined antitumor activity of combination treatment of LEN at 10 mg/kg (po, qd) and anti-PD1 Ab at 10 mg/kg (ip, twice weekly) in the murine subcutaneous RCC model using RAG cell line. Immune cell population analyses in tumor tissues were performed by flow cytometer. Immune-related gene expression profile in tumor tissues or cultured cells was analyzed by qPCR and RNA seq. Effects on FGFR and IFN gamma (IFNγ) signaling pathways and their downstream molecules in cultured cells were analyzed by western blot. In the murine syngeneic RAG tumor model, the combination treatment of LEN and anti-PD1 Ab showed tumor shrinkage, and some of tumors were regressed to nonpalpable sizes with long duration of response. LEN alone and in combination with anti-PD1 Ab significantly decreased the population of tumor associated macrophage in tumor tissues by FCM analysis. Gene expression analysis for immune cell responses in tumor tissues revealed the upregulation of IFNγ and its downstream genes including PD-L1 with combination of LEN and anti-PD1 Ab. Western blot analysis elucidated that FGFR signaling pathway inhibited the activation of IFNγ signaling pathway and the induction of PD-L1 expression in murine RCC cells, and LEN-treatment reactivated the IFNγ signaling pathway by inhibition of FGFR signaling. Combination of LEN and anti-PD1 Ab showed greater antitumor activity including tumor shrinkage with long duration of response in the murine RCC model. Modulation of immune microenvironment by LEN and combination of LEN with PD1 Ab may underlie the strong antitumor activity in preclinical RCC model. Inhibitory activity of LEN against FGFR reactivated the IFNγ pathway and PD-L1 expression suppressed by activation of FGFR signaling pathway, and this inhibitory activity of LEN may potentiate the antitumor activity of combination treatments with anti-PD1 Ab. These preclinical results provide one of the unique mechanisms of combinational effect of LEN with PD1 blockade in the preclinical RCC model. Citation Format: Yusuke Adachi, Kenji Ichikawa, Yu Kato, Yasuhiro Funahashi. Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2242.

CANTATA: Randomized, international, double-blind study of CB-839 plus cabozantinib versus cabozantinib plus placebo in patients with metastatic renal cell carcinoma.

TPS682 Background: Glutamine metabolism is upregulated in renal cell carcinoma (RCC) and important for RCC tumor cell proliferation and survival. CB-839 is a first-in-clinic, potent, oral inhibitor of the mitochondrial enzyme glutaminase (GLS), which controls a critical step in tumor cell metabolism of glutamine. CB-839 demonstrated synergistic anti-tumor activity when combined with cabozantinib, a VEGFR2/MET/AXL inhibitor, in preclinical RCC models. In a phase 1 study cohort, CB-839 plus cabozantinib as 2L+ therapy showed encouraging safety and efficacy results, with 50% overall response rate (ORR; RECIST v1.1) and 100% disease control rate in 10 patients with clear cell advanced/metastatic RCC (mRCC). A randomized, double-blind study comparing CB-839 plus cabozantinib vs. cabozantinib plus placebo has been initiated in patients with clear cell mRCC. Methods: In this ongoing international, randomized, double-blind, multi-center study, enrollment is planned for ~300 patients with clear cell mRCC. To be eligible, patients should have received 1-2 prior lines of systemic therapy for mRCC including ≥1 anti-angiogenic therapy or the combination of nivolumab + ipilimumab, have KPS ≥70%, measurable disease (RECIST v1.1), and no prior cabozantinib (or other MET inhibitor). Patients are randomized 1:1 to receive either CB-839 (800 mg twice daily per oral [PO] route) plus cabozantinib (60 mg daily PO) or cabozantinib plus placebo in 28-day cycles until disease progression or unacceptable toxicity. Patients are stratified by prior PD-1/PD-L1 inhibitor therapy and by IMDC prognostic risk group (favorable vs. intermediate vs. poor). The primary endpoint is progression-free survival (PFS) per RECIST v1.1, determined by blinded independent radiology review. Secondary endpoints are investigator-assessed PFS and overall survival. Safety, response per RECIST, and quality of life are also assessed. Findings of this randomized, international clinical trial will inform the efficacy and safety profile of CB-839, a first-in-clinic metabolic inhibitor, in combination with cabozantinib in patients with mRCC. Clinical trial information: NCT03428217.

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.

Establishment of patient-derived renal cell carcinoma (RCC) models based on orthotopic xenografts (PDX) and cancer stem cell (CSC) isolation to provide prognostic and predictive information.

e16055 Background: The introduction of VEGF/VEGFR- and mTOR, and immune checkpoint-targeted agents has radically changed metastatic RCC treatment; however, predictive biomarkers are still lacking and the potential for curative impact of such treatments in earlier disease stages is hotly debated. To identify potential diagnostic, prognostic, and predictive biomarkers, we developed patient-derived preclinical RCC models based on PDX obtained from freshly dissociated cancer tissues and combining isolation of cell populations endowed with self renewal and multi-lineage differentiation abilities (CSC). Methods: 182 surgical RCC specimens (clear cell, n = 110; papillary type I/II, n = 23, chromophobe/oncocytoma, n = 26, other, n = 23; stage I-III, n = 124, stage IV, n = 28, n/a n = 30) were collected; tumor spheroids were obtained and characterized in 57 cases, using a stem-cell isolating medium supplemented with EGF/b-FGF; 30 cancer samples were orthotopically injected in immunocompromised mice and were able to engraft in 67% of cases (G3-G4 cases more frequently than G1-G2 cases, p = 0.04). Results: PDX tumors recapitulated histological appearance, sarcomatoid features when they were present, and tumor heterogeneity of their human counterpart; interestingly, PDX engraftment was significantly correlated with shorter DFS in the corresponding patient population (p = 0.12). We conducted proteomic analysis by Reverse Phase Protein Array (RPPA) in 21 patient-derived CSC models in vitro: a panel of established (HIF, VEGFR, mTOR) and novel (EGFR(Y1148)), AKT, PI3K) oncogenic signals were deregulated in our isolated cell populations; molecular endpoints were correlated with grading and with angiogenesis and mTOR pathways (p < 0.05). Data analysis is ongoing to ascertain whether specific RPPA-signatures can potentially complement clinical prognostic information and suggest candidate biomarkers and potential targets for therapy. Conclusions: In the era of personalized therapy, the combined use of PDX and RPPA from tumor specimens may be very useful for drug testing and patient stratification in RCC.

Targeting of tumor growth and angiogenesis underlies the enhanced antitumor activity of lenvatinib in combination with everolimus.

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A‐498, Caki‐1, and Caki‐2). In particular, the combination led to tumor regression in the A‐498 and Caki‐1 models. In the A‐498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti‐angiogenic effects. The anti‐angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki‐1 xenografts, in which fibroblast growth factor (FGF)‐driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)‐activated, and synergistic activity against FGF‐activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR‐S6K‐S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP‐1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti‐angiogenic activity in human RCC xenograft models.

Abstract 3753: Potent anti-tumor activity of mTOR kinase inhibitor in combination with anti-angiogenic agents in preclinical models of renal cell cancer

Abstract Background: Multiple agents that inhibit angiogenesis and tumor cell growth via the VEGF and mTOR (TORC1) pathways have been approved for locally advanced or metastatic renal cell carcinoma (RCC) in recent years. Despite this progress, improving overall survival remains a significant clinical challenge for the treatment of this cancer type. The tumorigenesis of certain types of RCC is dominated by genes that play critical role in hypoxia response, including stimulation of neo-angiogenesis. Anti-angiogenic therapies target the tumor microenvironment but provide little direct impact to the tumor cell, allowing the potential for disease progression despite treatment. Approval of allosteric partial TORC1 inhibitors in RCC stimulated interest in pursuing TORC1/2 inhibitors, that block mTOR signaling more potently and comprehensively, alone and in combination with anti-angiogenic agents in preclinical models of RCC. Results: INK128 is an orally bioavailable, potent and selective ATP site mTOR kinase inhibitor (TORC1/2) that has entered clinical development. INK128 inhibited the phosphorylation of S6 and 4EBP1, downstream substrates of TORC1, and AKT and NRDG1, downstream substrate of TORC2 in RCC cell lines. TORC1/2 pathway inhibition correlated with potent and complete blockade of cell proliferation. In vivo, daily treatment of INK128 as single agent suppressed tumor growth in multiple mouse RCC xenograft models. Pharmacodynamic analysis demonstrated that INK128 inhibits both TORC1 and TORC2 pathways in vivo. INK128 did not alter the tumor-associated microvasculature density despite significant decrease of VEGF supply by lowering the capacity of protein translation of the tumor cells. This prompted us to explore the potential of the combination of INK128 with anti-angiogenic agents to maximize anti-tumor activity by targeting both tumor microenvironment and tumor cells directly. Co-treatment of INK128 with sorafenib resulted in much enhanced activity in RCC tumor models but did not achieve sufficient tolerability. We next employed an alternating dosing strategy of INK128 and sorafenib to improve tolerability. An optimized dose/schedule sequence of both agents caused sustained inhibition of tumor-associated angiogenesis, suppression of mTOR pathway resulting into marked blockade of tumor growth with significant induction of apoptosis and acceptable tolerability. Conclusion: The sequential, yet potent blockade of both mTOR and VEGF pathways obtained by combining INK128 with sorafenib acts synergistically in inducing cell death and tumor regression in preclinical renal cancer models. Adaptive combination of receptor tyrosine kinase-based anti-angiogenic agents with mTOR kinase inhibitors currently in development may offer a safe and effective therapeutic strategy for renal cancer patients who fail to respond to standard of care therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3753. doi:1538-7445.AM2012-3753

Valproic acid blocks adhesion of renal cell carcinoma cells to endothelium and extracellular matrix

Treatment strategies for metastatic renal cell carcinoma (RCC) have been limited due to chemotherapy and radiotherapy resistance. The development of targeted drugs has now opened novel therapeutic options. In the present study, anti-tumoral properties of the histone deacetylase inhibitor valproic acid (VPA) were tested in vitro and in vivo on pre-clinical RCC models. RCC cell lines Caki-1, KTC-26 or A498 were treated with various concentrations of VPA to evaluate tumour cell adhesion to vascular endothelial cells or to immobilized extracellular matrix proteins. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. VPA was also combined with low dosed interferon-alpha (IFN-alpha) and the efficacy of the combination therapy, as opposed to VPA monotherapy, was compared. VPA significantly and dose-dependently prevented tumour cell attachment to endothelium or matrix proteins, accompanied by elevated histones H3 and H4 acetylation. VPA altered integrin-alpha and -beta subtype expression, in particular alpha(3), alpha(5) and beta(3), and blocked integrin-dependent signalling. In vivo, VPA significantly inhibited the growth of Caki-1 in subcutaneous xenografts with the 200 mg/kg being superior to the 400 mg/kg dosing schedule. VPA-IFN-alpha combination markedly enhanced the effects of VPA on RCC adhesion, and in vivo tumour growth was further reduced by the 400 mg/kg but not by the 200 mg/kg VPA dosing schedule. VPA profoundly blocked the interaction of RCC cells with endothelium and extracellular matrix and reduced tumour growth in vivo. Therefore, VPA should be considered an attractive candidate for clinical trials.