Benjamin Y. Owusu

Erythrocyte storage increases rates of NO and nitrite scavenging: implications for transfusion-related toxicity.

Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2-4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25 days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.

Nitric oxide formation versus scavenging: the red blood cell balancing act

Abstract  Nitric oxide (NO) is a key modulator of vascular homeostasis controlling critical functions related to blood flow, respiration, cell death and proliferation, and protecting the vasculature from pro‐inflammatory and coagulative stresses. Inhibition of NO formation, and/or diversion of NO away from its physiological signalling targets lead to dysregulated NO bioavailability, a hallmark of numerous vascular and pulmonary diseases. Current concepts suggest that the balance between NO formation and NO scavenging is critical in disease development, with the corollary being that redressing the balance offers a target for therapeutic intervention. Evidence presented over the last two decades has seen red blood cells (RBCs) and haemoglobin specifically emerge as prominent effectors in this paradigm. In this symposium review article, we discuss recent insights into the mechanisms by which RBCs may modulate the balance between NO‐formation and inhibition. We discuss how these mechanisms may become dysfunctional to cause disease, highlight key questions that remain, and discuss the potential impact of these insights on therapeutic opportunities.

Inhibition of pro-HGF activation by SRI31215, a novel approach to block oncogenic HGF/MET signaling

The binding of hepatocyte growth factor (HGF) to its receptor MET activates a signaling cascade that promotes cell survival, proliferation, cell scattering, migration and invasion of malignant cells. HGF is secreted by cancer cells or by tumor-associated fibroblasts as pro-HGF, an inactive precursor. A key step in the regulation of HGF/MET signaling is proteolytic processing of pro-HGF to its active form by one of the three serine proteases, matriptase, hepsin or HGF activator (HGFA). We developed SRI 31215, a small molecule that acts as a triplex inhibitor of matriptase, hepsin and HGFA and mimics the activity of HAI-1/2, endogenous inhibitors of HGF activation. We demonstrated that SRI 31215 inhibits fibroblast-induced MET activation, epithelial-mesenchymal transition and migration of cancer cells. SRI 31215 overcomes primary resistance to cetuximab and gefitinib in HGF-producing colon cancer cells and prevents fibroblast-mediated resistance to EGFR inhibitors. Thus, SRI 31215 blocks signaling between cancer cells and fibroblasts and inhibits the tumor-promoting activity of cancer-associated fibroblasts. Aberrant HGF/MET signaling supports cell survival, proliferation, angiogenesis, invasion and metastatic spread of cancer cells, establishing HGF and MET as valid therapeutic targets. Our data demonstrate that inhibitors of HGF activation, such as SRI 31215, merit investigation as potential therapeutics in tumors that are addicted to HGF/MET signaling. The findings reported here also indicate that inhibitors of HGF activation overcome primary and acquired resistance to anti-EGFR therapy, providing a rationale for concurrent inhibition of EGFR and HGF to prevent therapeutic resistance and to improve the outcome of cancer patients.

Effects of Erythrocyte Aging on Nitric Oxide and Nitrite Metabolism

AIMS Recent studies have suggested that in addition to oxygen transport, red blood cells (RBC) are key regulators of vascular function by both inhibiting and promoting nitric oxide (NO)-mediated vasodilation. Most studies assume that RBC are homogenous, but, in fact, they comprise cells of differing morphology and biochemical composition which are dependent on their age, parameters that control NO reactions. We tested the hypothesis that distinct RBC populations will have differential effects on NO signaling. RESULTS Young and old RBC were separated by density gradient centrifugation. Consistent with previous reports, old RBC had decreased levels of surface N-acetyl neuraminic acid and increased oxygen binding affinities. Competition kinetic experiments showed that older RBCs scavenged NO∼2-fold faster compared with younger RBC, which translated to a more potent inhibition of both acetylcholine and NO-donor dependent vasodilation of isolated aortic rings. Moreover, nitrite oxidation kinetics was faster with older RBC compared with younger RBC; whereas no differences in nitrite-reduction kinetics were observed. This translated to increased inhibitory effect of older RBC to nitrite-dependent vasodilation under oxygenated and deoxygenated conditions. Finally, leukodepleted RBC storage also resulted in more dense RBC, which may contribute to the greater NO-inhibitory potential of stored RBC. INNOVATION These results suggest that a key element in vascular NO-homeostasis mechanisms is the distribution of RBC ages across the physiological spectrum (0-120 days) and suggest a novel mechanism for inhibited NO bioavailability in diseases which are characterized by a shift to an older RBC phenotype. CONCLUSION Older RBC inhibit NO bioavailability by increasing NO- and nitrite scavenging.

Targeting the tumor-promoting microenvironment in MET-amplified NSCLC cells with a novel inhibitor of pro-HGF activation

Targeted therapeutic agents, such as inhibitors of epithelial growth factor receptor (EGFR), have transformed the management of non-small cell lung cancer (NSCLC) patients. MET-amplified NSCLC cells display resistance to EGFR-targeting agents, but are addicted to MET signaling for survival and proliferation and are sensitive to MET inhibition. However, responsive cancer cells invariably develop resistance to MET-targeted treatment.The tumor microenvironment plays a major role in resistance to anticancer therapy. We demonstrated that fibroblasts block the response of MET-amplified NSCLC cells to the MET kinase inhibitor, JNJ38877605 in an HGF-dependent manner. Thus, MET-amplified NSCLC cells become addicted to HGF upon pharmacological inhibition of MET. HGF restored phosphorylation of MET, EGFR and RON, and maintained pro-survival AKT and ERK signaling in MET-inhibited cells.We developed a small molecule inhibitor of pro-HGF activation, SRI31215, which acts as a triplex inhibitor of the pro-HGF activating proteases matriptase, hepsin and HGF activator (HGFA). SRI31215 blocked crosstalk between tumor cells and fibroblasts and overcame fibroblast-mediated resistance to MET inhibition by preventing fibroblast-mediated reactivation of AKT and ERK signaling. Structurally unrelated triplex inhibitors of matriptase, hepsin and HGFA that we developed in parallel showed similar biological activity.Our data suggest that simultaneous inhibition of HGF and MET is required to overcome resistance to MET inhibitors in MET-amplified NSCLC cells. This provides a rationale for the development of novel combination therapeutic strategies for the treatment of NSCLC patients with MET amplification.Targeted therapeutic agents, such as inhibitors of epithelial growth factor receptor (EGFR), have transformed the management of non-small cell lung cancer (NSCLC) patients. MET-amplified NSCLC cells display resistance to EGFR-targeting agents, but are addicted to MET signaling for survival and proliferation and are sensitive to MET inhibition. However, responsive cancer cells invariably develop resistance to MET-targeted treatment. The tumor microenvironment plays a major role in resistance to anticancer therapy. We demonstrated that fibroblasts block the response of MET-amplified NSCLC cells to the MET kinase inhibitor, JNJ38877605 in an HGF-dependent manner. Thus, MET-amplified NSCLC cells become addicted to HGF upon pharmacological inhibition of MET. HGF restored phosphorylation of MET, EGFR and RON, and maintained pro-survival AKT and ERK signaling in MET-inhibited cells. We developed a small molecule inhibitor of pro-HGF activation, SRI31215, which acts as a triplex inhibitor of the pro-HGF activating proteases matriptase, hepsin and HGF activator (HGFA). SRI31215 blocked crosstalk between tumor cells and fibroblasts and overcame fibroblast-mediated resistance to MET inhibition by preventing fibroblast-mediated reactivation of AKT and ERK signaling. Structurally unrelated triplex inhibitors of matriptase, hepsin and HGFA that we developed in parallel showed similar biological activity. Our data suggest that simultaneous inhibition of HGF and MET is required to overcome resistance to MET inhibitors in MET-amplified NSCLC cells. This provides a rationale for the development of novel combination therapeutic strategies for the treatment of NSCLC patients with MET amplification.

Hepatocyte growth factor, a key tumor-promoting factor in the tumor microenvironment

The tumor microenvironment plays a key role in tumor development and progression. Stromal cells secrete growth factors, cytokines and extracellular matrix proteins which promote growth, survival and metastatic spread of cancer cells. Fibroblasts are the predominant constituent of the tumor stroma and Hepatocyte Growth Factor (HGF), the specific ligand for the tyrosine kinase receptor c-MET, is a major component of their secretome. Indeed, cancer-associated fibroblasts have been shown to promote growth, survival and migration of cancer cells in an HGF-dependent manner. Fibroblasts also confer resistance to anti-cancer therapy through HGF-induced epithelial mesenchymal transition (EMT) and activation of pro-survival signaling pathways such as ERK and AKT in tumor cells. Constitutive HGF/MET signaling in cancer cells is associated with increased tumor aggressiveness and predicts poor outcome in cancer patients. Due to its role in tumor progression and therapeutic resistance, both HGF and MET have emerged as valid therapeutic targets. Several inhibitors of MET and HGF are currently being tested in clinical trials. Preclinical data provide a strong indication that inhibitors of HGF/MET signaling overcome both primary and acquired resistance to EGFR, HER2, and BRAF targeting agents. These findings support the notion that co-targeting of cancer cells and stromal cells is required to prevent therapeutic resistance and to increase the overall survival rate of cancer patients. HGF dependence has emerged as a hallmark of therapeutic resistance, suggesting that inhibitors of biological activity of HGF should be included into therapeutic regimens of cancer patients.

The role of the endoplasmic reticulum protein calreticulin in mediating TGF-β-stimulated extracellular matrix production in fibrotic disease

Endoplasmic reticulum (ER) stress is a key factor contributing to fibrotic disease. Although ER stress is a short-term adaptive response, with chronic stimulation, it can activate pathways leading to fibrosis. ER stress can induce TGF-β signaling, a central driver of extracellular matrix production in fibrosis. This review will discuss the role of an ER protein, calreticulin (CRT), which has both chaperone and calcium regulatory functions, in fibrosis. CRT expression is upregulated in multiple different fibrotic diseases. The roles of CRT in regulation of fibronectin extracellular matrix assembly, extracellular matrix transcription, and collagen secretion and processing into the extracellular matrix will be discussed. Evidence for the importance of CRT in ER calcium release and NFAT activation downstream of TGF-β signaling will be presented. Finally, we will summarize evidence from animal models in which CRT expression is genetically reduced or experimentally downregulated in targeted tissues of adult animals and discuss how these models define a key role for CRT in fibrotic diseases.

Prognostic and Predictive Significance of Stromal Fibroblasts and Macrophages in Colon Cancer.

Colon cancer development and malignant progression are driven by genetic and epigenetic alterations in tumor cells and by factors from the tumor microenvironment. Cancer cells become reliant on the activity of specific oncogenes and on prosurvival and proliferative signals they receive from the abnormal environment they create and reside in. Accordingly, the response to anticancer therapy is determined by genetic and epigenetic changes that are intrinsic to tumor cells and by the factors present in the tumor microenvironment. Recent advances in the understanding of the involvement of the tumor microenvironment in tumor progression and therapeutic response are optimizing the application of prognostic and predictive factors in colon cancer. Moreover, new targets in the tumor microenvironment that are amenable to therapeutic intervention have been identified. Because stromal cells are with rare exceptions genetically stable, the tumor microenvironment has emerged as a preferred target for therapeutic drugs. In this review, we discuss the role of stromal fibroblasts and macrophages in colon cancer progression and in the response of colon cancer patients to therapy.

Abstract 1583: SRI31215, a novel inhibitor of oncogenic HGF/MET signaling

Constitutive activation of MET signaling, frequently observed in cancer, triggers signaling by AKT, ERK 1/2 and STAT3, and promotes survival, proliferation, migration and invasion of cancer cells. Accordingly, MET amplification or overexpression of its ligand, hepatocyte growth factor (HGF), are associated with tumor aggressiveness, resistance to therapy and poor prognosis in many cancer patients. Cancer cells can secrete HGF which triggers autocrine HGF/MET signaling, however, fibroblasts are the predominant source of HGF and activate MET expressed on tumor cells in a paracrine manner. Cancer cells and fibroblasts secrete HGF as an inactive precursor, pro-HGF. A crucial step in the regulation of HGF/MET signaling is the proteolytic processing of the inactive precursor, pro-HGF, to its active form by one of the three serine proteases, matriptase, hepsin or HGF activator (HGFA). This is the rate-limiting step in the HGF/MET signaling pathway, controlled by the inhibitors of HGF activation, hepatocyte growth factor activator inhibitors 1 and 2 (HAI-1/2). We synthesized a novel small molecule, SRI31215, a triplex inhibitor of matriptase, hepsin and HGFA, mimicking the biological activity of HA1-1/2. We confirmed that SRI31215 inhibits the proteolytic activation of pro-HGF and blocks HGF-induced MET activation and signaling by AKT, ERK 1/2 and STAT3. SRI31215 blocked fibroblast-induced epithelial-to-mesenchymal transition (EMT) and prevented scattering and migration of cancer cells. Finally, we demonstrated that SRI31215 sensitized HGF-producing colon cancer cells to epidermal growth factor receptor inhibitors (EGFRi) and inhibited fibroblast-mediated, HGF-dependent, resistance to EGFRi in colon cancer. Thus, SRI31215 blocks signaling between cancer cells and fibroblasts and inhibits the tumor-promoting activity of cancer associated fibroblasts. Taken together, our data demonstrate that inhibitors of HGF activation, such as SRI31215, represent a novel approach to curb the growth and metastatic spread of tumor cells that are addicted to HGF/MET signaling and to overcome primary and acquired resistance to EGFR-targeted therapy. Citation Format: Benjamin Y. Owusu, Phanindra K. Venukadasula, Robert A. Galemmo, Lidija Klampfer. SRI31215, a novel inhibitor of oncogenic HGF/MET signaling. [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 1583.