Characterization of indoleamine-2,3-dioxygenase 1, tryptophan-2,3-dioxygenase, and Ido1/Tdo2 knockout mice.

Indoleamine-2,3-dioxygenase 1 (IDO1) and tryptophan-2,3-dioxygenase 2 (TDO2) degrade tryptophan (Trp) to kynurenine (Kyn), and these enzymes have promise as therapeutic targets. A comprehensive characterization of potential safety liabilities of IDO1 and TDO2 inhibitors using knockout (KO) mice has not been assessed, nor has the dual Ido1/Tdo2 KO been reported. Here we characterized male and female mice with KOs for Ido1, Tdo2, and Ido1/Tdo2 and compared findings to the wild type (WT) mouse strain, evaluated for 14 days, using metabolomics, transcriptional profiling, behavioral analysis, spleen immunophenotyping, comprehensive histopathological analysis, and serum clinical chemistry. Multiple metabolomic changes were seen in KO mice. For catabolism of Trp to Kyn and anthranilic acid, both substrates were decreased in liver of Tdo2 and dual KO mice. Metabolism of Trp to serotonin and its metabolites resulted in an increase in 5-Hydroxyindole-3-acetic acid in the Tdo2 and dual KO mice. Ido1 and dual KO mice displayed a Kyn reduction in plasma but not in liver. Nicotinamide synthesis and conversion of glucose to lactic acid were not impacted. A slight decrease in serum alkaline phosphatase was seen in all KOs, and small changes in liver gene expression of genes unrelated to tryptophan metabolism were observed. Regarding other parameters, no genotype-specific changes were observed. In summary, this work shows metabolomic pathway changes for metabolites downstream of tryptophan in these KO mice, and suggests that inhibition of the IDO1 and TDO2 enzymes would be well tolerated whether inhibited individually or in combination since no safety liabilities were uncovered.

Cortical-Bone Fragility--Insights from sFRP4 Deficiency in Pyle's Disease.

BACKGROUND: Cortical-bone fragility is a common feature in osteoporosis that is linked to nonvertebral fractures. Regulation of cortical-bone homeostasis has proved elusive. The study of genetic disorders of the skeleton can yield insights that fuel experimental therapeutic approaches to the treatment of rare disorders and common skeletal ailments. METHODS: We evaluated four patients with Pyle's disease, a genetic disorder that is characterized by cortical-bone thinning, limb deformity, and fractures; two patients were examined by means of exome sequencing, and two were examined by means of Sanger sequencing. After a candidate gene was identified, we generated a knockout mouse model that manifested the phenotype and studied the mechanisms responsible for altered bone architecture. RESULTS: In all affected patients, we found biallelic truncating mutations in SFRP4, the gene encoding secreted frizzled-related protein 4, a soluble Wnt inhibitor. Mice deficient in Sfrp4, like persons with Pyle's disease, have increased amounts of trabecular bone and unusually thin cortical bone, as a result of differential regulation of Wnt and bone morphogenetic protein (BMP) signaling in these two bone compartments. Treatment of Sfrp4-deficient mice with a soluble Bmp2 receptor (RAP-661) or with antibodies to sclerostin corrected the cortical-bone defect. CONCLUSIONS: Our study showed that Pyle's disease was caused by a deficiency of sFRP4, that cortical-bone and trabecular-bone homeostasis were governed by different mechanisms, and that sFRP4-mediated cross-regulation between Wnt and BMP signaling was critical for achieving proper cortical-bone thickness and stability. (Funded by the Swiss National Foundation and the National Institutes of Health.).

A soluble bone morphogenetic protein type IA receptor increases bone mass and bone strength.

Diseases such as osteoporosis are associated with reduced bone mass. Therapies to prevent bone loss exist, but there are few that stimulate bone formation and restore bone mass. Bone morphogenetic proteins (BMPs) are members of the TGFß superfamily, which act as pleiotropic regulators of skeletal organogenesis and bone homeostasis. Ablation of the BMPR1A receptor in osteoblasts increases bone mass, suggesting that inhibition of BMPR1A signaling may have therapeutic benefit. The aim of this study was to determine the skeletal effects of systemic administration of a soluble BMPR1A fusion protein (mBMPR1A-mFc) in vivo. mBMPR1A-mFc was shown to bind BMP2/4 specifically and with high affinity and prevent downstream signaling. mBMPR1A-mFc treatment of immature and mature mice increased bone mineral density, cortical thickness, trabecular bone volume, thickness and number, and decreased trabecular separation. The increase in bone mass was due to an early increase in osteoblast number and bone formation rate, mediated by a suppression of Dickkopf-1 expression. This was followed by a decrease in osteoclast number and eroded surface, which was associated with a decrease in receptor activator of NF-κB ligand (RANKL) production, an increase in osteoprotegerin expression, and a decrease in serum tartrate-resistant acid phosphatase (TRAP5b) concentration. mBMPR1A treatment also increased bone mass and strength in mice with bone loss due to estrogen deficiency. In conclusion, mBMPR1A-mFc stimulates osteoblastic bone formation and decreases bone resorption, which leads to an increase in bone mass, and offers a promising unique alternative for the treatment of bone-related disorders.

Discovery and Evaluation of TLR-Targeted Immune Agonists.

Toll-like receptor (TLR) activation converts immunologically inactive tumors into immunologically active tumors by activating tumor residing antigen-presenting cells and recruitment of cytotoxic T lymphocytes. Targeted immune agonists (TIAs) are antibody drug conjugates with small-molecule TLR agonist payloads. The mechanism of action of TIAs involves tumor antigen recognition, Fcγ-receptor-dependent phagocytosis, and TLR-mediated activation to drive tumor killing by myeloid cells. Several new low DAR anti-HER2 TIAs conjugated with novel TLR7 or dual-TLR7/8 agonists with cleavable and noncleavable linkers were synthesized and profiled. In vitro studies demonstrated that these TIAs activate myeloid cells only in the presence of antigen-expressing cancer cells. Evaluation in ELISpot-based assays confirmed the low immunogenicity of these constructs. Systemic administration of the novel TIAs in tumor-bearing mice resulted in tumor reduction at low doses. These results provide a strong rationale for further development of the TIAs as a novel class of immunotherapeutics.

Discovery of MK-1084: An Orally Bioavailable and Low-Dose KRASG12C Inhibitor.

Oncogenic mutations in the RAS gene account for 30% of all human tumors; more than 60% of which present as KRAS mutations at the hotspot codon 12. After decades of intense pursuit, a covalent inhibition strategy has enabled selective targeting of this previously "undruggable" target. Herein, we disclose our journey toward the discovery of MK-1084, an orally bioavailable and low-dose KRASG12C covalent inhibitor currently in phase I clinical trials (NCT05067283). We leveraged structure-based drug design to identify a macrocyclic core structure, and hypothesis-driven optimization of biopharmaceutical properties to further improve metabolic stability and tolerability.

Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth.

Endoglin (CD105), a transmembrane protein of the transforming growth factor ß superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26-359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

Oxetane Promise Delivered: Discovery of Long-Acting IDO1 Inhibitors Suitable for Q3W Oral or Parenteral Dosing.

3,3-Disubstituted oxetanes have been utilized as bioisosteres for gem-dimethyl and cyclobutane functionalities. We report the discovery of a novel class of oxetane indole-amine 2,3-dioxygenase (IDO1) inhibitors suitable for Q3W (once every 3 weeks) oral and parenteral dosing. A diamide class of IDO inhibitors was discovered through an automated ligand identification system (ALIS). Installation of an oxetane and fluorophenyl dramatically improved the potency. Identification of a biaryl moiety as an unconventional amide isostere addressed the metabolic liability of amide hydrolysis. Metabolism identification (Met-ID)-guided target design and the introduction of polarity resulted in the discovery of potent IDO inhibitors with excellent pharmacokinetic (PK) profiles in multiple species. To enable rapid synthesis of the key oxetane intermediate, a novel oxetane ring cyclization was also developed, as well as optimization of a literature route on kg scale. These IDO inhibitors may enable unambiguous proof-of-concept testing for the IDO1 inhibition mechanism for oncology.

Verteporfin-based photodynamic therapy overcomes gemcitabine insensitivity in a panel of pancreatic cancer cell lines.

BACKGROUND AND OBJECTIVE: Pancreatic cancer is notoriously difficult to treat and resistant to virtually all therapeutics including gemcitabine, the standard front line agent for palliative chemotherapy. Early clinical studies point to a potential role for photodynamic therapy (PDT) in the management of this deadly disease. Here we examine PDT with verteporfin for treatment of cells that are nonresponsive to gemcitabine and identify intracellular and extracellular factors that govern sensitivity to each modality. STUDY DESIGN: Using MTS we assess cytotoxicity of verteporfin-PDT in gemcitabine-treated nonresponsive populations from a panel of five pancreatic cancer cell lines representing a range of tumor histopathology and origin. We conduct Western blots for pro-/anti-apoptotic proteins bax and Bcl-XL to identify factors relevant to PDT and gemcitabine sensitivity. To examine the role of extracellular matrix influences we compare response to each modality in traditional cell culture conditions and cells grown on a laminin-rich basement membrane. RESULTS: All cell lines have gemcitabine nonresponsive populations (17-33%) at doses up to 1 mM while moderate total verteporfin PDT doses (1-6 µM J/cm2) produce nearly complete killing. Our data shows that cells that are nonresponsive to sustained gemcitabine incubation are sensitive to verteporfin PDT indicating that the latter is agnostic to gemcitabine sensitivity. Verteporfin-based PDT decreases Bcl-XL and increases the bax/Bcl-XL ratio toward a pro-apoptotic balance. Insensitivity to gemcitabine is increased in cells that are adherent to basement membrane relative to traditional tissue culture conditions. CONCLUSIONS: Collectively these results indicate the ability of verteporfin-based PDT to bypass intracellular and extracellular cues leading to gemcitabine resistance and point to the emerging role of this therapy for treatment of pancreatic cancer.

Utilization of Metabolite Identification and Structural Data to Guide Design of Low-Dose IDO1 Inhibitors.

Herein the discovery of potent IDO1 inhibitors with low predicted human dose is discussed. Metabolite identification (MetID) and structural data were used to strategically incorporate cyclopropane rings into this tetrahydronaphthyridine series of IDO1 inhibitors to improve their metabolic stability and potency. Enabling synthetic chemistry was developed to construct these unique fused cyclopropyl compounds, leading to inhibitors with improved pharmacokinetics and human whole blood potency and a predicted human oral dose as low as 9 mg once daily (QD).

Comprehensive investigation of T and B cell receptor repertoires in an MC38 tumor model following murine anti­PD­1 administration.

The MC38 (derived from carcinogen­induced colon adenocarcinoma) tumor model is sensitive to anti­programmed cell death­1 (anti-PD­1) treatment. However, there is no comprehensive description of the T and B cell receptor (TCR, BCR) repertoires of the MC38 tumor model following anti­PD­1 treatment, an improved understanding of which is highly important in the development of anti­PD­1 immunotherapy. The present study analyzed the TCR and BCR repertoires of three types of tissue, including tumor, spleen and tumor draining lymph node (DLN) from 20 MC38 syngeneic mice receiving murine anti­PD­1 (mDX400) treatment or mouse immunoglobulin G1 (mIgG1) control treatment. To obtain enough tissues for high­throughput sequencing, samples were collected on day 8 after the start of initial treatment. The usage frequencies of seven TCR ß chain (TRB) V genes and one TRBJ gene were significantly different between mDX400­ and mIgG1­group tumors. TCR repertoire diversity was significantly lower in mDX400­group tumors compared with mIgG1­group tumors, with the top 10 most frequent TCR clonotypes notably expanded in mDX400­group tumors. In addition, the proportion of high­frequency TCR clonotypes from mDX400­group tumors that were also present both in the DLN and spleen was significantly higher than that in mIgG1­group tumors. Among the highly expanded TCR clonotypes, one TCR clonotype was consistently expanded in >50% of the mDX400­group tumors compared with mIgG1­group tumors. Similarly, one BCR clonal family was highly expanded in >50% of mDX400­group tumor samples. The consistently expanded TCR and BCR clones were co­expanded in 29% of mDX400­group tumors. Moreover, mutation rates of immunoglobulin heavy chain sequences in the spleen within complementarity determining region 2 and framework region 3 were significantly higher in the mDX400 group than in the mIgG1 group. The findings of this study may contribute to an improved understanding of the molecular mechanisms of anti­PD­1 treatment.

Discovery of Potent and Orally Available Bicyclo[1.1.1]pentane-Derived Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitors.

Indoleamine-2,3-dioxygenase 1 (IDO1) inhibition and its combination with immune checkpoint inhibitors like pembrolizumab have drawn considerable attention from both academia and the pharmaceutical industry. Here, we describe the discovery of a novel class of highly potent IDO1 heme-displacing inhibitors featuring a unique bicyclo[1.1.1]pentane motif. Compound 1, evolving from an ALIS (automated ligand identification system) hit, exhibited excellent potency but lacked the desired pharmacokinetic profile due to extensive amide hydrolysis of the benzamide moiety. Replacing the central phenyl ring in 1 with a bicyclo[1.1.1]pentane bioisostere effectively circumvented the amide hydrolysis issue, resulting in the discovery of compound 2 with a favorable overall profile such as excellent potency, selectivity, pharmacokinetics, and a low predicted human dose.

In vivo optical molecular imaging of vascular endothelial growth factor for monitoring cancer treatment.

PURPOSE: Vascular endothelial growth factor (VEGF) expression is a critical component in tumor growth and metastasis. Capabilities to monitor VEGF expression in vivo can potentially serve as a useful tool for diagnosis, prognosis, treatment planning, monitoring, and research. Here, we present the first report of in vivo hyperspectral molecular imaging strategy capable of monitoring treatment-induced changes in VEGF expression. EXPERIMENTAL DESIGN: VEGF was targeted with an anti-VEGF antibody conjugated with a fluorescent dye and was imaged in vivo using a hyperspectral imaging system. The strategy was validated by quantitatively monitoring VEGF levels in three different tumors as well as following photodynamic treatment. Specificity of the molecular imaging strategy was tested using in vivo competition experiments and mathematically using a quantitative pharmacokinetic model. RESULTS: The molecular imaging strategy successfully imaged VEGF levels quantitatively in three different tumors and showed concordance with results from standard ELISA. Changes in tumoral VEGF concentration following photodynamic treatment and Avastin treatment were shown. Immunohistochemistry shows that (a) the VEGF-specific contrast agent labels both proteoglycan-bound and unbound VEGF in the extracellular space and (b) the bound VEGF is released from the extracellular matrix in response to photodynamic therapy. In vivo competition experiments and quantitative pharmacokinetic model-based analysis confirmed the high specificity of the imaging strategy. CONCLUSION: This first report of in vivo quantitative optical molecular imaging-based monitoring of a secreted cytokine in tumors may have implications in providing tools for mechanistic investigations as well as for improved treatment design and merits further investigation.

Discovery of Amino-cyclobutarene-derived Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitors for Cancer Immunotherapy.

Checkpoint inhibitors have demonstrated unprecedented efficacy and are evolving to become standard of care for certain types of cancers. However, low overall response rates often hamper the broad utility and potential of these breakthrough therapies. Combination therapy strategies are currently under intensive investigation in the clinic, including the combination of PD-1/PD-L1 agents with IDO1 inhibitors. Here, we report the discovery of a class of IDO1 heme-binding inhibitors featuring a unique amino-cyclobutarene motif, which was discovered through SBDD from a known and weakly active inhibitor. Subsequent optimization efforts focused on improving metabolic stability and were greatly accelerated by utilizing a robust SNAr reaction of a facile nitro-furazan intermediate to quickly explore different polar side chains. As a culmination of these efforts, compound 16 was identified and demonstrated a favorable overall profile with superior potency and selectivity. Extensive studies confirmed the chemical stability and drug-like properties of compound 16, rendering it a potential drug candidate.

A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer.

Therapy-induced stimulation of angiogenic molecules can promote tumor angiogenesis leading to enhanced tumor growth and cancer metastasis. Several standard and emerging therapies, such as radiation and photodynamic therapy (PDT), can induce angiogenic molecules, thus limiting their effectiveness. PDT is approved for the treatment of several cancers; however, its induction of vascular endothelial growth factor (VEGF) creates conditions favorable to enhanced tumor growth and metastasis, therefore mitigating its cytotoxic and antivascular effects. This is the first report showing that subcurative PDT in an orthotopic model of prostate cancer (LNCaP) increases not only VEGF secretion (2.1-fold) but also the fraction of animals with lymph node metastases. PDT followed by administration of an antiangiogenic agent, TNP-470, abolished this increase and reduced local tumor growth. On the other hand, administration of TNP-470 before PDT was less effective at local tumor control. In addition, animals in all groups, except in the PDT + TNP-470 group, had a weight loss of >3 g at the time of sacrifice; the weight of the animals in the PDT + TNP-470 group did not change. The significant reduction (P < 0.05) in tumor weight and volume observed between the PDT + TNP-470 group and the control group suggests that the combination of PDT and antiangiogenic treatment administered in the appropriate sequence was not only more effective at controlling local tumor growth and metastases but also reduced disease-related toxicities. Such molecular response-based combinations merit further investigations as they enhance both monotherapies and lead to improved treatment outcomes.

Mechanistic investigation and implications of photodynamic therapy induction of vascular endothelial growth factor in prostate cancer.

Photodynamic therapy (PDT) is now an approved therapeutic modality, and induction of vascular endothelial growth factor (VEGF) following subcurative PDT is of concern as VEGF may provide a survival stimulus to tumors. The processes that limit the efficacy of PDT warrant investigation so that mechanism-based interventions may be developed. This study investigates VEGF increase following subcurative PDT using the photosensitizer benzoporphyrin derivative (BPD) both in an in vitro and in an orthotopic model of prostate cancer using the human prostate cancer cell line LNCaP. The two subcurative doses used, 0.25 and 0.5 J/cm(2), mimicked subcurative PDT and elicited a 1.6- and 2.1-fold increase, respectively, in secreted VEGF 24 hours following PDT. Intracellular VEGF protein measurement and VEGF mRNA showed a 1.4- and 1.6-fold increase only at 0.5 J/cm(2). In vivo subcurative PDT showed an increase in VEGF by both immunohistochemistry and ELISA. In vitro analysis showed no activation of hypoxia-inducible factor-1alpha (HIF-1alpha) or cyclooxygenase-2 (COX-2) following subcurative PDT; furthermore, small interfering RNA inhibition of HIF-1alpha and COX-2 inhibitor treatment had no effect on PDT induction of VEGF. PDT in the presence of phosphatidylinositol 3-kinase/AKT inhibitor or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor still induced VEGF. However, subcurative PDT increased phosphorylated p38 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase. The p38 MAPK inhibitor abolished PDT induction of VEGF. The results establish the importance of VEGF in subcurative BPD-PDT of prostate cancer and suggest possible molecular pathways for its induction. These findings should provide the basis for the development of molecular-based interventions for enhancing PDT and merit further studies.

COMMD5/HCaRG Hooks Endosomes on Cytoskeleton and Coordinates EGFR Trafficking.

COMMD5/HCaRG is involved in tissue repair, and its low expression is associated with tumorigenicity. Cell growth, migration, and differentiation are controlled by COMMD5. We previously reported that COMMD5 inhibited the growth of renal carcinoma cells by regulating expression or phosphorylation of ErbB members. Here, we demonstrate that COMMD5 is crucial for the stability of the cytoskeleton. Its silencing leads to a major re-organization of actin and microtubule networks. The N terminus of COMMD5 binds to the endosomal Rab5, and its C terminus, including the COMMD domain, binds to the cytoskeletal scaffolding. COMMD5 participates in long-range endosome transport, including epidermal growth factor receptor (EGFR) recycling, and provides the strength to deform and assist the scission of vesicles into sorting endosomes. This study establishes the molecular mechanism by which COMMD5 acts as an adaptor protein to coordinate endosomal trafficking and reveals its important role for EGFR transport and activity.

Effect of tumor host microenvironment on photodynamic therapy in a rat prostate tumor model.

PURPOSE: Tumor host microenvironment plays an important role in tumor growth, metastasis, and response to cancer therapy. In this study, the influence of tumor host environment on tumor pathophysiology, photosensitizer distribution, and photodynamic therapy (PDT) treatment effect was examined in the metastatic at lymph node and lung (MatLyLu) rat prostate tumor. EXPERIMENTAL DESIGN: MatLyLu tumors implanted in different host environment [i.e., orthotopically (in the prostate) or s.c.] were compared for difference in vessel density, average vessel size, vascular permeability, tumor vascular endothelial growth factor production, and tumor oxygenation. Uptake of photosensitizer verteporfin in tumors in both sites was determined by fluorescence microscopy. To compare tumor response to PDT, both orthotopic and s.c. MatLyLu tumors were given the same doses of verteporfin and laser light treatment, and PDT-induced tumor necrotic area was measured histologically. RESULTS: Orthotopic MatLyLu tumors were found to grow faster, have higher vessel density and more permeable vasculature, have higher vascular endothelial growth factor protein levels, and have lower tumor hypoxic fraction than the s.c. tumors. Uptake of photosensitizer verteporfin in the orthotopic tumor was higher than in the s.c. tumors at 15 minutes after injection (1 mg/kg, i.v.), and became similar at 3 hours after injection. For the vascular targeting PDT treatment (0.25 mg/kg verteporfin, 50 J/cm(2) at 50 mW/cm(2), 15 minutes drug-light interval), there was no significant difference in PDT-induced tumor necrotic area between the orthotopic and s.c. tumors, with 85% to 90% necrosis in both types of tumors. However, tumor necrosis induced by the cellular targeting PDT (1 mg/kg verteporfin, 50 J/cm(2) at 50 mW/cm(2), 3 hours drug-light interval) was significantly different in the orthotopic (64%) versus the s.c. (29%) tumors. CONCLUSIONS: Tumor host environment can significantly affect photosensitizer verteporfin distribution and PDT treatment effect. Verteporfin-PDT regimen targeting tumor cells is more sensitive to such influence than the vascular targeting PDT. Our study showed the importance of tumor host environment in determining tumor physiologic properties and tumor response to PDT. To obtain clinically relevant information, orthotopic tumor model should be used in the experimental studies.

In vivo flow cytometry: a new method for enumerating circulating cancer cells.

The fate of circulating tumor cells is an important determinant of their ability to form distant metastasis. Here, we demonstrate the use of in vivo flow cytometry as a powerful new method for detecting quantitatively circulating cancer cells. We specifically examine the circulation kinetics of two prostate cancer cell lines with different metastatic potential in mice and rats. We find that the cell line and the host environment affect the circulation kinetics of prostate cancer cells, with the intrinsic cell line properties determining the initial rate of cell depletion from the circulation and the host affecting cell circulation at later time points.

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.

ALK1-Fc inhibits multiple mediators of angiogenesis and suppresses tumor growth.

Activin receptor-like kinase-1 (ALK1) is a type I, endothelial cell-specific member of the transforming growth factor-beta superfamily of receptors known to play an essential role in modulating angiogenesis and vessel maintenance. In the present study, we sought to examine the angiogenic and tumorigenic effects mediated upon the inhibition of ALK1 signaling using a soluble chimeric protein (ALK1-Fc). Of 29 transforming growth factor-beta-related ligands screened by surface plasmon resonance, only bone morphogenetic protein (BMP9) and BMP10 displayed high-affinity binding to ALK1-Fc. In cell-based assays, ALK1-Fc inhibited BMP9-mediated Id-1 expression in human umbilical vein endothelial cells and inhibited cord formation by these cells on a Matrigel substrate. In a chick chorioallantoic membrane assay, ALK1-Fc reduced vascular endothelial growth factor-, fibroblast growth factor-, and BMP10-mediated vessel formation. The growth of B16 melanoma explants was also inhibited significantly by ALK1-Fc in this assay. Finally, ALK1-Fc treatment reduced tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells. These data show the efficacy of chimeric ALK1-Fc proteins in mitigating vessel formation and support the view that ALK1-Fc is a powerful antiangiogenic agent capable of blocking vascularization.