Yumi Yokoyama

Inhibition of the mammary carcinoma angiogenic switch in C3(1)/SV40 transgenic mice by a mutated form of human endostatin

Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth‐inhibitory effect on endothelial cell proliferation than wild‐type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF188 mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase‐1 (Flk‐1) and Fms‐like tyrosine kinase‐1 (Flt‐1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin‐2 mRNA levels are also increased during tumor progression. CD‐31 (platelet‐endothelial cell adhesion molecule [PECAM]) staining revealed that blood vessels developed in tumors larger than 1 mm The administration of P125A human endostatin in C3(1)/Tag females resulted in a significant delay in tumor onset, decreased tumor multiplicity and tumor burden and prolonged survival of the animals. Endostatin treatment did not reduce the number of preinvasive lesions, proliferation rates or apoptotic index, compared with controls. However, mRNA levels of a variety of proangiogenic factors (VEGF, VEGF receptors Flk‐1 and Flt‐1, angiopoietin‐2, Tie‐1, cadherin‐5 and PECAM) were significantly decreased in the endostatin‐treated group compared with controls. These results demonstrate that P125A endostatin inhibits the angiogenic switch during mammary gland adenocarcinoma tumor progression in the C3(1)/Tag transgenic model.

Addition of integrin binding sequence to a mutant human endostatin improves inhibition of tumor growth

Tumor vasculatures express high levels of αVβ3/αVβ5 and α5β1 integrins. Consequently, peptides containing the RGD (Arg‐Gly‐Asp) sequence, which is present in ligands of integrins, is effective in targeting therapeutic reagents to tumor vascular endothelium. In our study, we investigated whether the biologic activity of endostatin can be enhanced by the addition of an integrin targeting sequence. RGD sequence was added to either the amino or carboxyl terminus of endostatin containing a point mutation, P125A‐endostatin. Earlier we have shown that the P125A mutation did not affect the biologic activity of endostatin but in fact had better antiangiogenic activity when compared to the native molecule. Further modification of P125A‐endostatin with the RGD motif showed specific and increased binding to endothelial cells, and the increased binding coincided with improved antiangiogenic properties. Both amino and carboxyl terminal RGD‐modification of P125A‐endostatin resulted in greater inhibition of endothelial cell migration and proliferation. RGD modification increased tumor localization without affecting the circulatory half‐life of P125A‐endostatin, and RGD‐modified P125A‐endostatin was found to be more effective when compared to the P125A‐endostatin in inhibiting ovarian and colon cancer growth in athymic mice. Complete inhibition of ovarian tumor growth was observed when P125A‐endostatin‐RGD was encapsulated into alginate beads. These studies demonstrate that addition of a vascular targeting sequence can enhance the biologic activity of an antiangiogenic molecule.

Addition of an aminopeptidase N-binding sequence to human endostatin improves inhibition of ovarian carcinoma growth.

Blood vessels in tumors express higher level of aminopeptidase N (APN) compared with normal tissues. It has been reported that peptides that contain asparagine‐glycine‐arginine (NGR) sequence home to APN in tumor vasculature. Increased expression of APN in tumor vascular endothelium, therefore, offers an opportunity to target NGR peptide‐linked therapeutic reagents to tumors.

TRAIL and Triptolide: An Effective Combination that Induces Apoptosis in Pancreatic Cancer Cells

IntroductionAn emerging therapy in oncology is the induction of apoptotic cell death through anti-death receptor therapy. However, pancreatic cancer is resistant to apoptosis including anti-death receptor therapy. We have previously described how triptolide decreases resistance to apoptosis in pancreatic cancer cells in vitro and in vivo. We hypothesized that triptolide decreases tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in pancreatic cancer cells. The aim of this study was to evaluate the effects that combined therapy with TRAIL and triptolide have on different parameters of apoptosis.MethodsFour different pancreatic cancer cell lines were exposed to triptolide, TRAIL, or a combination of both drugs. We assessed the effects that combined therapy with TRAIL and triptolide has on cell viability, apoptosis, caspase-3 and caspase-9 activities, and poly(ADP)-ribose polymerase cleavage.ResultsPancreatic cancer cells were resistant to TRAIL therapy; however, combined therapy with triptolide and TRAIL significantly decreased the cell viability in all the cell lines and increased apoptotic cell death as a result of caspase-3 and caspase-9 activation.ConclusionsPancreatic cancer is highly resistant to anti-death receptor therapy, but combined therapy with TRAIL and triptolide is an effective therapy that induces apoptotic cell death in pancreatic cancer cells.

Endostatin binding to ovarian cancer cells inhibits peritoneal attachment and dissemination

Ovarian cancer cells use integrins to attach to the peritoneal wall. Integrin alpha(5)beta(1) is also the target for the angiogenesis inhibitor, endostatin. Therefore, the ability of endostatin to competitively inhibit tumor cell seeding of the peritoneum was investigated. An imaging method was developed to determine early phases of peritoneal dissemination of ovarian cancer cells. Using this method, endostatin was found to bind ovarian cancer cells through integrin alpha(5)beta(1) and inhibit vessel cooption efficiently. Although both angiostatin and endostatin are potent inhibitors of tumor angiogenesis, peritoneal attachment and vessel cooption was blocked only by the endostatin. Knocking down the expression of integrins alpha(5) and beta(1) in ovarian cancer cells interfered with endostatin-mediated inhibition of peritoneal seeding. Furthermore, adenovirus-mediated in situ expression of endostatin either inside the peritoneum or by the ovarian tumor cells inhibited peritoneal seeding and dissemination in vivo. Endostatin treatment also prevented primary ovarian cancer cells from attaching to mouse peritoneal wall. These studies show a paraendothelial mechanism by which endostatin can inhibit peritoneal dissemination of ovarian cancer cells and raises the possibility of intraperitoneal expression of endostatin to reduce recurrence.

Binding of endostatin to human ovarian cancer cells inhibits cell attachment.

Endostatin, a C‐terminal fragment of collagen type XVIII, is one of the well‐characterized endogenous inhibitors of angiogenesis. Endostatin is known to bind integrin α5β1, which is upregulated on tumor endothelium. Most of the ovarian cancer cells express significant amounts of α5β1 integrin, which is important for ovarian cancer cells to attach to the peritoneal wall. Therefore we investigated whether endostatin could directly bind ovarian cancer cells and inhibit tumor cell attachment to extracellular matrix. Binding of endostatin to ovarian cancer cells was characterized by preincubation with function blocking antibodies to integrin subunits. These studies showed that ovarian cancer cell attachment to fibronectin‐coated wells can be inhibited by α5β1 integrin specific antibodies as well as endostatin. Downregulation of integrin α5 and β1 by siRNA abrogated the binding of OVCAR5 and human umbilical vein endothelial cell to endostatin. Although endostatin treatment did not affect ovarian cancer cell migration, treated cells failed to attach mouse peritoneal wall preparations. These studies suggest an extra‐antiangiogenic role for endostatin, which can be used prevent peritoneal attachment and dissemination of ovarian cancer cells.

VEGF—DT385 Toxin Conjugate Inhibits Mammary Adenocarcinoma Development in a Transgenic Mouse Model of Spontaneous Tumorigenesis

Previous experiments have shown that a vascular endothelial growth factor (VEGF)—DT385 toxin conjugate inhibits endothelial cell proliferation, angiogenesis and solid tumor growth in a xenotransplant model system. Here, we report that VEGF—DT385 toxin conjugate effectively inhibits spontaneous tumorigenesis. The C3(1)/SV40 TAg transgenic mouse model of mammary gland carcinogenesis was used to determine the effectiveness of VEGF—DT385 toxin conjugate in delaying the onset of disease and the development of solid tumors. Animals were treated daily with conjugate for a period of 7 days. Therapy was initiated at week 14 of development before any visible adenocarcinomas were evident. Treatment of mice with VEGF—DT385 toxin conjugate significantly delayed the onset of tumorigenesis and inhibited solid tumor growth by more than 92%. Furthermore, conjugate treated animals showed less than twice the number of tumor nodules when compared to control mice. Finally, this vascular targeting agent significantly increased survival time of animals by 5 weeks. VEGF—DT385 toxin conjugate resulted in temporary weight loss and no long-lasting toxicity was seen. More importantly, using this established tumor model, VEGF—DT385 toxin conjugate appeared to be as effective as a similar treatment schedule with recombinant human endostatin. Our results suggest that VEGF—DT385 toxin conjugate is a potent inhibitor of mammary adenocarcinoma growth and might be useful in breast cancer therapy.

Abstract 4191: RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor, demonstrates efficacy in TAM-driven tumors

In recent years, the TAM (TYRO3, AXL, and MER) family of receptor tyrosine kinases (RTKs) has emerged as attractive targets for oncology therapeutics. Under homeostatic conditions, the TAM RTKs are expressed by a number of immune cells where they play key roles in the negative regulation of the immune response. However, their expression on cancer cells has been specifically associated with epithelial-to-mesenchymal transition, an invasive phenotype, and more generally, with a poor prognostic outcome in human cancers. Here, we sought (1) to establish whether expression of the TAM was sufficient to drive cellular transformation, (2) to demonstrate that RXDX-106, a small molecule inhibitor of TAM RTKs, could inhibit tumors harboring activating TAM gene fusions, and furthermore, (3) to decipher how pharmacological inhibition of TAM signaling pathways both on cancer cells and immune cells would be beneficial, given their complex regulation and intimate relationship in the tumor microenvironment. TAM expression on cancer cells has emerged as one of the key mechanisms of resistance to targeted therapies, particularly to EGFR tyrosine kinase inhibitors (TKI). In addition, novel gene rearrangements (fusions) have been identified in the TCGA database for both Mertk (TMEM87B-Mertk) and Axl (Axl-MBIP) that retain the functionality of the tyrosine kinase domain, but have fusion partners that alter the expression profile or dimerization frequency, respectively. Here, we demonstrate both in vitro and in vivo that expression of either wild type receptors or TMEM87B-MER and AXL-MBIP fusion proteins is sufficient to drive oncogenic transformation by both ligand dependent and independent mechanisms. Treatment of these TAM-expressing cellular populations with RXDX-106 completely inhibits TAM phosphorylation and downstream signaling and, consistent with our signaling data, RXDX-106 completely inhibits cellular proliferation and viability at sub-nanomolar concentrations. Furthermore, we demonstrate that, in the co-culture of TAM-expressing cancer cells with immune cells such as macrophages, TAM ligands such as Gas6 are secreted to drive ligand dependent activation of TAM on cancer cells, leading to their survival and proliferation. In summary, our data suggest that TAM RTKs can act as traditional oncodrivers when activated either in a ligand dependent or ligand independent manner. In addition, we demonstrate that TAM fusions are tractable therapeutic targets and that patients with tumors harboring such molecular alterations may derive clinical benefit from RXDX-106. Finally, we show that RXDX-106 not only has the potential to inhibit cellular proliferation and survival on the cancer cell itself, but also affect the TAM-expressing tumor microenvironment to result in a global anti-cancer environment. Citation Format: Erin D. Lew, Elizabeth A. Tindall, Joanne Oh, Colin Walsh, Maria Barrera, Heather Ely, Amy Diliberto, Yumi Yokoyama, Gary Li, Amanda Albert. RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor, demonstrates efficacy in TAM-driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4191. doi:10.1158/1538-7445.AM2017-4191

Abstract LB-080: Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern) activates the direct killing functions of innate immune cells in concert with tumor targeting antibodies

Imprime PGG (Imprime) is a soluble yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern). As a PAMP, Imprime triggers innate immune function, activating the direct killing functions of innate immune cells, facilitating MDSC differentiation and macrophage repolarization as well as enabling dendritic cell maturation and antigen presentation, driving T cell expansion and activation. In the clinic, Imprime is administered intravenously and is well-tolerated. In multiple clinical trials (> 400 subjects), including randomized phase 2 studies in NSCLC, Imprime has consistently shown promising increases in both objective tumor response and patient survival. To date, the clinical experience with Imprime has centered on combinations with tumor targeting monoclonal antibodies (Mabs). For instance, Imprime combined with rituximab and alemtuzumab in CLL patients yielded a 65% complete response rate (vs 37% historical CR rate for alemtuzumab plus rituximab). We sought to better characterize the effect of Imprime in concert with tumor-targeting mAbs. We show that Imprime enhances the effector functions of multiple innate immune cell lineages. We first evaluated the generation of Reactive Oxygen Species (ROS) in neutrophils isolated from human healthy volunteer whole blood. These neutrophils, but not those from vehicle treated whole blood, specifically recognized B cell lymphomas (Raji) only after opsonization with anti-CD20 Mabs (rituximab, ofatumumab, obinatuzumab), generating a substantial ROS burst that coincided with enhanced tumor cell cytotoxicity. Similarly, increased antibody dependent cellular phagocytosis (ADCP) mediated by monocyte-derived macrophages was evident against antibody-opsonized lymphomas (Z138 B cell lymphomas with obinutuzumab) and solid tumor cells (SKBr3 breast cancer cells with trastuzumab) from Imprime-treated whole blood. Likewise, increased Natural Killer (NK) cell-mediated antibody dependent cellular cytotoxicity (ADCC) was evident only after Imprime treatment against antibody-opsonized cancer cells (SKBR3 with trastuzumab, K562 erythroleukemia cells with anti-glycophorin-A). In vivo, we now show that Imprime administered intravenously significantly enhances the anti-tumor efficacy of trastuzumab in a patient derived xenograft model of breast cancer, reducing mean tumor volume to ∼ 50% of that achieved by trastuzumab alone. In the B16 lung experimental metastasis model, the addition of Imprime to the anti-TRP1 tumor targeting antibody TA-99 significantly reduces both the number and size of B16 lung metastases. Together, these data show that Imprime stimulates the innate immune system, augmenting the anti-tumor efficacy of a diverse array of tumor targeting antibodies in multiple tumor types. Citation Format: Steven M. Leonardo, Ross B. Fulton, Keith B. Gorden, Katy Fraser, Ben Harrison, Takashi Kangas, Adria Jonas, Yumi Yokoyama, Nadine Ottoson, Nandita Bose, Jeremy R. Graff. Imprime PGG, a β-glucan PAMP (pathogen-associated molecular pattern) activates the direct killing functions of innate immune cells in concert with tumor targeting antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-080.

Abstract 3767: Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing

Cancer therapy has been reshaped by checkpoint inhibitors (CPIs), making it possible for durable responses against cancers with traditionally low cure rates. Current efforts are focused on combination therapies in the hopes of evading resistance to CPIs and improving overall response. One escape mechanism attributed to acquired resistance to CPIs includes defective antigen presentation, namely a loss in MHC class I expression. This leads to loss of CD8 T cell-mediated tumor killing and disease relapse. This recent revelation has stimulated a need for therapies that activate other cytotoxic effector cells such as NK cells to kill tumors. Imprime PGG (Imprime) is a soluble, systemically delivered yeast 1,3/1,6 β-glucan PAMP (pathogen-associated molecular pattern) capable of triggering innate immune cell function leading to a cascade of immune activation and enhanced tumor killing. Imprime activates the innate immune system via dectin-1, eliciting production of a variety of chemokines and cytokines, including type I IFN, leading to the mobilization and stimulation of innate cell types including dendritic cells and monocytes. Unlike other PAMPs which systemic administration often leads to toxic side effects, Imprime has been administered safely by intravenous infusion to >400 human subjects. Currently, Imprime PGG is being evaluated in combination with αPD1 therapy in multiple clinical trials. Previously we have shown that combination therapy of anti-Trp1 antibody and Imprime leads to a significant reduction in both number and size of lung metastases in the B16F10 metastatic melanoma tumor model over anti-Trp1 antibody alone. This reduction of metastases is highly dependent on NK cells but not CD8 T cells. To explore the impact of Imprime on NK-mediated cytotoxicity, we further evaluated in vivo killing of MHC class I deficient TapKO cells after intravenous administration of Imprime. In these experiments Imprime was able to enhance the NK cytotoxic killing of the targets. All NK cell killing observed was dependent on type I IFN, phagocytic cells and dectin 1. Imprime treatment increased cytokines that drive enhanced NK activation and effector phenotype. Significant increases were observed in the cytokines IL15/IL15rα, IL18, IL12p70 in lymph node lysates as well as increases in the effector molecules CD69, GrB, and CD107a on splenic NK cells. The upregulation of all of these molecules during Imprime treatment was dependent on dectin 1. Additionally, IL15/IL15rα production was also dependent on type I IFN, and phagocytic cells. Interestingly, Ly6c hi monocytes, which are increased after Imprime treatment, also show enhancement of IL15rα expression. Collectively, these data demonstrate that Imprime contributes to enhanced NK functionality and killing which may provide a unique immunotherapeutic approach to complement existing therapies. Citation Format: Kathryn A. Fraser, Takashi Kangas, Ross B. Fulton, Steven M. Leonardo, Ben Harrison, Yumi Yokoyama, Nandita Bose, Jeremy R. Graff, Mark Uhlik, Keith B. Gorden. Imprime PGG, a soluble yeast b-glucan PAMP, enhancement of anti-tumor responses in combination with tumor targeting antibody is highly dependent on NK cell killing [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 3767.