CXCR4 inhibition modulates the tumor microenvironment and retards the growth of B16-OVA melanoma and Renca tumors.

To determine whether blockade of the chemokine receptor CXCR4 might alter the tumor microenvironment and inhibit tumor growth, we tested the efficacy of the CXCR4 antagonist X4-136 as a single agent and in combination with various immune checkpoint inhibitors in the syngeneic murine melanoma model B16-OVA. We also tested its activity alone and in combination with axitinib in the renal cancer model Renca. We found that X4-136 exhibited potent single agent antitumor activity in the B16-OVA model that was additive to that of an anti-PDL1 antibody. The antitumor activities were associated with a reduction in the number of immunosuppressive regulatory T cells and myeloid-derived suppressor cells and an increase in the number of tumor-specific CD8/perforin cells in the tumor-microenvironment. Apart from these immune effects, X4-136 alone and in combination with checkpoint inhibitors inhibited the Akt/FOXO-3a cell survival pathway in vitro and in vivo, suggesting that it might have antitumor activity independent of its effects on immune cell trafficking. Similar effects on tumor growth and cytotoxic T lymphocytes infiltration were observed in the Renca model. These studies show that the effects of CXCR4 blockade on immune cell trafficking might serve as a useful adjunct to immune checkpoint inhibitors and other therapies in the treatment of cancer.

The tumor microenvironment in renal cell cancer.

PURPOSE OF REVIEW: In addition to the provision of nutrients and growth factors that facilitate tumor cell proliferation and metastasis, the tumor microenvironment (MEV) restricts immune surveillance of tumor-associated antigens and limits the efficacy of immune checkpoint inhibitors, tumor vaccines, and other immune therapies. This review will focus on the immunosuppressive mechanisms operative within the tumor MVE of renal cell carcinoma. RECENT FINDINGS: Several of the immunosuppressive mechanisms within the tumor MEV have been identified and are potentially druggable. Clinical trials with agents that target several of these inhibitory pathways are currently underway. SUMMARY: Although renal cell carcinoma is one of several tumor types responsive to immune checkpoint inhibitors, the effectiveness of these agents is likely to be limited by the various tumor-infiltrating bone marrow-derived myeloid cells that comprise the MEV. Several strategies to combat the recruitment of these cells into tumor tissue or to neutralize their immunosuppressive function have shown encouraging results in animal tumor models and clinical trials.

First-in-Class ERK1/2 Inhibitor Ulixertinib (BVD-523) in Patients with MAPK Mutant Advanced Solid Tumors: Results of a Phase I Dose-Escalation and Expansion Study.

Ulixertinib (BVD-523) is an ERK1/2 kinase inhibitor with potent preclinical activity in BRAF- and RAS-mutant cell lines. In this multicenter phase I trial (NCT01781429), 135 patients were enrolled to an accelerated 3 + 3 dose-escalation cohort and six distinct dose-expansion cohorts. Dose escalation included 27 patients, dosed from 10 to 900 mg twice daily and established the recommended phase II dose (RP2D) of 600 mg twice daily. Ulixertinib exposure was dose proportional to the RP2D, which provided near-complete inhibition of ERK activity in whole blood. In the 108-patient expansion cohort, 32% of patients required dose reduction. The most common treatment-related adverse events were diarrhea (48%), fatigue (42%), nausea (41%), and dermatitis acneiform (31%). Partial responses were seen in 3 of 18 (17%) patients dosed at or above maximum tolerated dose and in 11 of 81 (14%) evaluable patients in dose expansion. Responses occurred in patients with NRAS-, BRAF V600-, and non-V600 BRAF-mutant solid tumors.Significance: Here, we describe the first-in-human dose-escalation study of an ERK1/2 inhibitor for the treatment of patients with advanced solid tumors. Ulixertinib has an acceptable safety profile with favorable pharmacokinetics and has shown early evidence of clinical activity in NRAS- and BRAF V600- and non-V600-mutant solid-tumor malignancies. Cancer Discov; 8(2); 184-95. ©2017 AACR.See related commentary by Smalley and Smalley, p. 140This article is highlighted in the In This Issue feature, p. 127.

FCGR Polymorphisms Influence Response to IL2 in Metastatic Renal Cell Carcinoma.

Purpose: Fc-gamma receptors (FCGRs) are expressed on immune cells, bind to antibodies, and trigger antibody-induced cell-mediated antitumor responses when tumor-reactive antibodies are present. The affinity of the FCGR/antibody interaction is variable and dependent upon FCGR polymorphisms. Prior studies of patients with cancer treated with immunotherapy indicate that FCGR polymorphisms can influence antitumor response for certain immunotherapies that act via therapeutically administered mAbs or via endogenous tumor-reactive antibodies induced from tumor antigen vaccines. The previously published "SELECT" trial of high-dose aldesleukin (HD-IL2) for metastatic renal cell carcinoma resulted in an objective response rate of 25%. We evaluated the patients in this SELECT trial to determine whether higher-affinity FCGR polymorphisms are associated with outcome.Experimental Design: SNPs in FCGR2A, FCGR3A, and FCGR2C were analyzed, individually and in combination, for associations between genotype and clinical outcome.Results: When higher-affinity genotypes for FCGR2A, FCGR3A, and FCGR2C were considered together, they were associated with significantly increased tumor shrinkage and prolonged survival in response to HD-IL2.Conclusions: Although associations of higher-affinity FCGR genotype with clinical outcome have been demonstrated with mAb therapy and with idiotype vaccines, to our knowledge, this is the first study to show associations of FCGR genotypes with outcome following HD-IL2 treatment. We hypothesize that endogenous antitumor antibodies may engage immune cells through their FCGRs, and HD-IL2 may enhance antibody-induced tumor destruction, or antibody-enhanced tumor antigen presentation, via augmented activation of innate or adaptive immune responses; this FCGR-mediated immune activity would be augmented through immunologically favorable FCGRs. Clin Cancer Res; 23(9); 2159-68. ©2016 AACR.

Novel drugs that target the metabolic reprogramming in renal cell cancer.

Molecular profiling studies of tumor tissue from patients with clear cell renal cell cancer (ccRCC) have revealed extensive metabolic reprogramming in this disease. Associations were found between metabolic reprogramming, histopathologic Fuhrman grade, and overall survival of patients. Large-scale genomics, proteomics, and metabolomic analyses have been performed to identify the molecular players in this process. Genes involved in glycolysis, the pentose phosphate pathway, glutamine metabolism, and lipogenesis were found to be upregulated in renal cell cancer (RCC) specimens as compared to normal tissue. Preclinical research indicates that mutations in VHL, FBP1, and the PI3K-AKT-mTOR pathway drives aerobic glycolysis through transcriptional activation of the hypoxia-inducible factors (HIF). Mechanistic studies revealed glutamine as an important source for de novo fatty acid synthesis through reductive carboxylation. Amplification of MYC drives reductive carboxylation. In this review, we present a detailed overview of the metabolic changes in RCC in conjunction with potential novel therapeutics. We discuss preclinical studies that have investigated targeted agents that interfere with various aspects of tumor cell metabolism and emphasize their impact specifically on glycolysis, lipogenesis, and tumor growth. Furthermore, we describe a number of phase 1 and 2 clinical trials that have been conducted with these agents.

Inhibition of ALK1 signaling with dalantercept combined with VEGFR TKI leads to tumor stasis in renal cell carcinoma.

Treatment of metastatic renal cell carcinoma (mRCC) with agents that block signaling through vascular endothelial growth factor receptor 2 (VEGFR2) induces disease regression or stabilization in some patients; however, these responses tend to be short-lived. Therefore, development of combination therapies that can extend the efficacy of VEGFR antagonists in mRCC remains a priority.We studied murine xenograft models of RCC that become refractory to treatment with the VEGFR tyrosine kinase inhibitor (TKI) sunitinib. Dalantercept is a novel antagonist of Activin receptor-like kinase 1 (ALK1)/Bone morphogenetic protein (BMP) 9 signaling. Dalantercept inhibited growth in the murine A498 xenograft model which correlated with hyperdilation of the tumor vasculature and an increase in tumor hypoxia. When combined with sunitinib, dalantercept induced tumor necrosis and prevented tumor regrowth and revascularization typically seen with sunitinib monotherapy in two RCC models. Combination therapy led to significant downregulation of angiogenic genes as well as downregulation of endothelial specific gene expression particularly of the Notch signaling pathway. We demonstrate that simultaneous targeting of molecules that control distinct phases of angiogenesis, such as ALK1 and VEGFR, is a valid strategy for treatment of mRCC. At the molecular level, combination therapy leads to downregulation of Notch signaling.

Sunitinib activates Axl signaling in renal cell cancer.

Mass spectrometry-based phosphoproteomics provides a unique unbiased approach to evaluate signaling network in cancer cells. The tyrosine kinase inhibitor sunitinib is registered as treatment for patients with renal cell cancer (RCC). We investigated the effect of sunitinib on tyrosine phosphorylation in RCC tumor cells to get more insight in its mechanism of action and thereby to find potential leads for combination treatment strategies. Sunitinib inhibitory concentrations of proliferation (IC50) of 786-O, 769-p and A498 RCC cells were determined by MTT-assays. Global tyrosine phosphorylation was measured by LC-MS/MS after immunoprecipitation with the antiphosphotyrosine antibody p-TYR-100. Phosphoproteomic profiling of 786-O cells yielded 1519 phosphopeptides, corresponding to 675 unique proteins including 57 different phosphorylated protein kinases. Compared to control, incubation with sunitinib at its IC50 of 2 µM resulted in downregulation of 86 phosphopeptides including CDK5, DYRK3, DYRK4, G6PD, PKM and LDH-A, while 94 phosphopeptides including Axl, FAK, EPHA2 and p38α were upregulated. Axl- (y702), FAK- (y576) and p38α (y182) upregulation was confirmed by Western Blot in 786-O and A498 cells. Subsequent proliferation assays revealed that inhibition of Axl with a small molecule inhibitor (R428) sensitized 786-O RCC cells and immortalized endothelial cells to sunitinib up to 3 fold. In conclusion, incubation with sunitinib of RCC cells causes significant upregulation of multiple phosphopeptides including Axl. Simultaneous inhibition of Axl improves the antitumor activity of sunitinib. We envision that evaluation of phosphoproteomic changes by TKI treatment enables identification of new targets for combination treatment strategies.

A Phase I Trial of Bortezomib and Sorafenib in Advanced Malignant Melanoma.

LESSONS LEARNED: This study is a rare example of effective doses of both targeted agents being both administered and tolerated.This combination should not be used in melanoma. BACKGROUND: Sorafenib and bortezomib affect BCL family member expression. We previously demonstrated that bortezomib augmented sorafenib-mediated cytotoxicity in melanoma cell lines in vitro. We aimed to combine sorafenib 400 mg b.i.d. with increasing doses of weekly bortezomib. METHODS: Patients with metastatic melanoma were enrolled in dose-escalation cohorts to determine the maximum tolerated dose (MTD) of sorafenib (twice daily) in combination with bortezomib (weekly for 3 of 4 weeks). The MTD was defined as the highest dose level at which less than 33% of patients exhibited a dose-limiting toxicity (DLT). Efficacy, as measured by 6-month progression-free survival and response rate per RECIST, was documented. RESULTS: Eleven patients were enrolled at three dose levels. DLTs (fatigue and rash) were seen in two of three patients at the highest dose level. Five patients were enrolled for sorafenib 400 mg b.i.d. and bortezomib 1.0 mg/m(2) weekly for 3 of every 4 weeks; none had DLTs, and this dose level was defined as the MTD. Of 10 evaluable patients, no responses were seen. Two of 11 patients (18%) remained progression free for longer than 6 months. CONCLUSION: The combination of sorafenib and bortezomib is safe but not active in patients with melanoma.

Anti-S1P Antibody as a Novel Therapeutic Strategy for VEGFR TKI-Resistant Renal Cancer.

PURPOSE: VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC. EXPERIMENTAL DESIGN: In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC. RESULTS: Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI. CONCLUSIONS: Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy.

The high-dose aldesleukin "select" trial: a trial to prospectively validate predictive models of response to treatment in patients with metastatic renal cell carcinoma.

PURPOSE: High-dose aldesleukin (HD IL2) received FDA approval for the treatment of metastatic renal cell carcinoma (MRCC) in 1992, producing a 14% objective response rate (ORR) and durable remissions. Retrospective studies suggested that clinical and pathologic features could predict for benefit. The Cytokine Working Group conducted this prospective trial to validate proposed predictive markers of response to HD IL2. EXPERIMENTAL DESIGN: Standard HD IL2 was administered to prospectively evaluate whether the ORR of patients with mRCC with "good" predictive pathologic features based on an "integrated selection" model [ISM (e.g., clear-cell histology subclassification and carbonic anhydrase-9 (CA-9) IHC staining] was significantly higher than the ORR of a historical, unselected population. Archived tumor was collected for pathologic analysis including tumor programmed death-ligand 1 (PD-L1) expression. RESULTS: One hundred and twenty eligible patients were enrolled between June 11 and September 7; 70% were Memorial Sloan Kettering Cancer Center (New York, NY) intermediate risk, 96% had clear cell RCC, and 99% had prior nephrectomy. The independently assessed ORR was 25% (30/120, 95% CI, 17.5%-33.7%, P = 0.0014; 3 complete responses, 27 partial responses) and was higher than a historical ORR. Thirteen patients (11%) remained progression free at 3 years and the median overall survival was 42.8 months. ORR was not statistically different by ISM classification ("good-risk" 23% vs. "poor-risk" 30%; P = 0.39). ORR was positively associated with tumor PD-L1 expression (P = 0.01) by IHC. CONCLUSIONS: In this prospective, biomarker validation study, HD IL2 produced durable remissions and prolonged survival in both "good" and "poor-risk" patients. The proposed ISM was unable to improve the selection criteria. Novel markers (e.g., tumor PD L1 expression) appeared useful, but require independent validation.

Predictive biomarkers in renal cell cancer: insights in drug resistance mechanisms.

INTRODUCTION: VEGF-targeted therapy is currently the first line treatment for patients with metastatic clear cell renal cell carcinoma (ccRCC), but most patients either display primary (intrinsic) resistance or acquire drug resistance. In recent years multiple mechanisms of resistance to VEGF-targeted therapy emerged from preclinical research, but it is currently unknown to what extent these drug resistance modalities play a role in the clinic. Here we reviewed the current literature on biomarkers that predict treatment outcome in patients with ccRCC to gain insight in clinical drug resistance mechanisms. METHODS: A search syntax was compiled by combining different synonyms of "biomarker" AND "renal" AND "cancer". MEDLINE was accessed through PubMed, where this syntax was entered and used to search titles and abstracts of publications. Articles were selected based on three criteria: (1) description of patients with clear cell RCC, (2) treatment with VEGF targeted therapy and (3) discussion of biomarkers that were studied for potential association with treatment response. RESULTS: The literature search was performed on March 4th 2014 and yielded 1882 articles. After carefully reading the titles and abstracts based on the three previously mentioned criteria, 103 publications were evaluated. Backward citation screening was performed on all eligible studies and revealed another 24 articles. This search revealed that (1) High glucose uptake and low contrast enhancement on PET- and CT-imaging before start of treatment may correlate with poor response to therapy, (2) Low dose intensity due to treatment intolerance is related to shorter progression free survival. (3) Acquired resistance appears to be associated with rebound vascularization based on both longitudinal monitoring of contrast enhancement by CT and blood vessel counts in tumor tissue, and (4) Based on plasma cytokine and single nucleotide polymorphism (SNP) studies, interleukin-8, VEGFR-3, FGFR2 and HGF/MET emerged as potential clinical markers for chemoresistance. CONCLUSION: Low dose intensity, specific tumor-imaging techniques and potential biological biomarkers may be predictive for response to VEGF-targeted therapy in ccRCC. Some of these plausible biomarkers may also provide more insight into the underlying mechanisms of resistance such as altered glucose metabolism and rapid rebound vascularization.

Clinical utility of a blood-based BRAF(V600E) mutation assay in melanoma.

BRAF inhibitors (BRAFi) have led to clinical benefit in patients with melanoma. The development of a blood-based assay to detect and quantify BRAF levels in these patients has diagnostic, prognostic, and predictive capabilities that could guide treatment decisions. Blood BRAF(V600E) detection and quantification were performed on samples from 128 patients with stage II (19), III (67), and IV (42) melanoma. Tissue BRAF analysis was performed in all patients with stage IV disease and in selected patients with stage II and III disease. Clinical outcomes were correlated to initial BRAF levels as well as BRAF level dynamics. Serial analysis was performed on 17 stage IV melanoma patients treated with BRAFi and compared with tumor measurements by RECIST. The assay was highly sensitive (96%) and specific (95%) in the stage IV setting, using a blood level of 4.8 pg as "positive." BRAF levels typically decreased following BRAFi. A subset of these patients (5) had an increase in BRAF(V600E) values 42 to 112 days before clinical or radiographic disease progression (PD). From 86 patients with resected, stage II or III melanoma, 39 had evidence of disease relapse (45.3%). Furthermore, BRAF mutation in the blood after surgical resection in these patients was not associated with a difference in relapse risk, although tissue BRAF status was only available for a subset of patients. In summary, we have developed a highly sensitive and specific, blood-based assay to detect BRAF(V600) mutation in patients with melanoma.

Clinical profiling of BCL-2 family members in the setting of BRAF inhibition offers a rationale for targeting de novo resistance using BH3 mimetics.

While response rates to BRAF inhibitiors (BRAFi) are high, disease progression emerges quickly. One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis. We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination. The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax) would improve outcome. We tested this hypothesis in cell lines and in mice. Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression. Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A. No significant changes were observed with BCL-2. Using reverse phase protein array (RPPA), significant increases in protein levels were found in BIM and BID. No changes in mRNA or protein correlated with response. Concurrent BRAF (PLX4720) and BCL2 (navitoclax) inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment. In xenograft models, navitoclax enhanced the efficacy of PLX4720. The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance. Trial registrations: ClinicalTrials.gov NCT01006980; ClinicalTrials.gov NCT01107418; ClinicalTrials.gov NCT01264380; ClinicalTrials.gov NCT01248936; ClinicalTrials.gov NCT00949702; ClinicalTrials.gov NCT01072175.

The role of angiopoietins as potential therapeutic targets in renal cell carcinoma.

Angiopoietin 2 (Ang2) is a secreted glycoprotein upregulated at sites of angiogenesis and has been implicated in cancer neovascularization. Recent studies have suggested efficacy of combined Ang and vascular endothelial growth factor receptor (VEGFR) inhibition for patients with metastatic renal cell carcinoma (mRCC). We measured Ang2 expression in human tissue and plasma, and tested the effect of dual Ang1/2 (trebananib; AMG386) or Ang2 alone (L1-7) inhibition with VEGFR inhibition on murine RCC growth and blood flow. Ang2 levels were higher in human tumors than normal tissues with RCC ranking highest for Ang2 expression across all tumor types tested. Plasma Ang2 was significantly higher in patients with mRCC compared to controls or patients with stage I disease. Plasma Ang2 decreased with sunitinib treatment and increased at time of disease progression. In the RCC mouse, dual Ang1/2 and Ang2 inhibition improved the activity of sunitinib. Combined Ang1/2 and VEGFR inhibition prevented the resumption of blood flow associated with sunitinib resistance. Thus, Ang2 inhibition, independent of Ang1 inhibition, improves the activity of sunitinib and plasma Ang2 increases in the setting of progression on sunitinib possibly contributing to resistance. Further, arterial spin-labeled perfusion magnetic resonance imaging might be a non-invasive marker of the antiangiogenic activity of Ang inhibitors.

Assaying for BRAF V600E in tissue and blood in melanoma.

The Braf(V600E) mutation has been detected in patients with metastatic melanoma, colon, thyroid, and other cancers. Studies suggested that tumors with this mutation are especially sensitive to BRAF inhibitors-hence the need to reliably determine the BRAF status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild-type BRAF alleles and thus may fail to detect the presence of mutant BRAF(V600E) tumors. We have developed a rapid, inexpensive method of BRAF analysis that reduces the contamination of wild-type BRAF sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild-type and mutant BRAF RNA at codon 600. Using this protocol, mutant BRAF can be detected in RNA from mixed populations with as few as 0.1 % BRAF(V600E) mutant containing cells.