Inhibition of αvß5 Integrin Attenuates Vascular Permeability and Protects against Renal Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is a leading cause of AKI. This common clinical complication lacks effective therapies and can lead to the development of CKD. The αvß5 integrin may have an important role in acute injury, including septic shock and acute lung injury. To examine its function in AKI, we utilized a specific function-blocking antibody to inhibit αvß5 in a rat model of renal IRI. Pretreatment with this anti-αvß5 antibody significantly reduced serum creatinine levels, diminished renal damage detected by histopathologic evaluation, and decreased levels of injury biomarkers. Notably, therapeutic treatment with the αvß5 antibody 8 hours after IRI also provided protection from injury. Global gene expression profiling of post-ischemic kidneys showed that αvß5 inhibition affected established injury markers and induced pathway alterations previously shown to be protective. Intravital imaging of post-ischemic kidneys revealed reduced vascular leak with αvß5 antibody treatment. Immunostaining for αvß5 in the kidney detected evident expression in perivascular cells, with negligible expression in the endothelium. Studies in a three-dimensional microfluidics system identified a pericyte-dependent role for αvß5 in modulating vascular leak. Additional studies showed αvß5 functions in the adhesion and migration of kidney pericytes in vitro Initial studies monitoring renal blood flow after IRI did not find significant effects with αvß5 inhibition; however, future studies should explore the contribution of vasomotor effects. These studies identify a role for αvß5 in modulating injury-induced renal vascular leak, possibly through effects on pericyte adhesion and migration, and reveal αvß5 inhibition as a promising therapeutic strategy for AKI.

Treatment with the PARP inhibitor, niraparib, sensitizes colorectal cancer cell lines to irinotecan regardless of MSI/MSS status.

BACKGROUND: Cells with homologous recombination (HR) deficiency, most notably caused by mutations in the BRCA1 or BRCA2 genes, are sensitive to PARP inhibition. Microsatellite instability (MSI) accounts for 10-15% of colorectal cancer (CRC) and is hypothesized to lead to HR defects due to altered expression of Mre11, a protein required for double strand break (DSB) repair. Indeed, others have reported that PARP inhibition is efficacious in MSI CRC. METHODS: Here we examine the response to niraparib, a potent PARP-1/PARP-2 inhibitor currently under clinical evaluation, in MSI versus microsatellite stable (MSS) CRC cell lines in vitro and in vivo. We compiled a large panel of MSI and MSS CRC cell lines and evaluated the anti-proliferative activity of niraparib. In addition to testing single agent cytotoxic activity of niraparib, we also tested irinotecan (or SN-38, the active metabolite of irinotecan) activity alone and in combination with niraparib in vitro and in vivo. RESULTS: In contrast to earlier reports, MSI CRC cell lines were not more sensitive to niraparib than MSS CRC cell lines¸ suggesting that the MSI phenotype does not sensitize CRC cell lines to PARP inhibition. Moreover, even the most sensitive MSI cell lines had niraparib EC50s greater than 10 fold higher than BRCA-deficient cell lines. However, MSI lines were more sensitive to SN-38 than MSS lines, consistent with previous findings. We have also demonstrated that combination of niraparib and irinotecan was more efficacious than either agent alone in both MSI and MSS cell lines both in vitro and in vivo, and that niraparib potentiates the effect of irinotecan regardless of MSI status. CONCLUSIONS: Our results support the clinical evaluation of this combination in all CRC patients, regardless of MSI status.

MCL1 and BCL-xL levels in solid tumors are predictive of dinaciclib-induced apoptosis.

Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.

Overexpression of the alphavbeta6 integrin in endometrial cancer.

The alpha(v)beta(6) integrin (alphavbeta6) has been shown to be up-regulated in adenocarcinoma of the breast, colon, stomach, and ovary, generally reflecting a more aggressive phenotype. Expression in endometrial cancer has not been reported. We analyzed alphavbeta6 expression in the tissue from primary endometrial carcinomas (endometrioid type) using a mouse monoclonal antibody against human alphavbeta6, and correlated the findings with grade, stage, and nodal involvement. Normal cycling endometrium was studied for comparison. alphavbeta6 was only weakly expressed in normal epithelium and infrequently expressed in precancers, but up-regulated in the majority of endometrial carcinomas, especially with high grade. Nodal metastases strongly expressed alphavbeta6, even when the primary tumor showed only focal expression. No correlation was found between expression and depth of invasion or the presence of metastases. Overexpression of alphavbeta6 in endometrial carcinoma is common. Expression is high in metastatic lesions. The level of expression of the primary tumor was not indicative of the presence of nodal metastasis; however, the number of cases with nodal metastases was limited.

Antibody-mediated blockade of integrin alpha v beta 6 inhibits tumor progression in vivo by a transforming growth factor-beta-regulated mechanism.

The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.

Role of alphavbeta6 integrin in acute biliary fibrosis.

UNLABELLED: Acute biliary obstruction leads to periductal myofibroblasts and fibrosis, the origin of which is uncertain. Our study provides new information on this question in mice and humans. We show that bile duct obstruction induces a striking increase in cholangiocyte alphavbeta6 integrin and that expression of this integrin is directly linked to fibrogenesis through activation of transforming growth factor beta (TGF-beta). Administration of blocking antibody to alphavbeta6 significantly reduces the extent of acute fibrosis after bile duct ligation. Moreover, in beta6-null mice subjected to the injury, fibrosis is reduced by 50% relative to that seen in wild-type mice, whereas inflammation occurs to the same extent. The data indicate that alphavbeta6, rather than inflammation, is linked to fibrogenesis. It is known that alphavbeta6 binds latent TGF-beta and that binding results in release of active TGFbeta. Consistent with this, intracellular signaling from the TGFbeta receptor is increased after bile duct ligation in wild-type mice but not in beta6(-/-) mice, and a competitive inhibitor of the TGFbeta receptor type II blocks fibrosis to the same extent as antibody to alphavbeta6. In a survey of human liver disease, expression of alphavbeta6 is increased in acute, but not chronic, biliary injury and is localized to cholangiocyte-like cells. CONCLUSION: Cholangiocytes respond to acute bile duct obstruction with markedly increased expression of alphavbeta6 integrin, which is closely linked to periductal fibrogenesis. The findings provide a rationale for the use of inhibitors of alphavbeta6 integrin or TGFbeta for down-regulating fibrosis in the setting of acute or ongoing biliary injury.

Alphav beta6 integrin regulates renal fibrosis and inflammation in Alport mouse.

The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.

Anti-alpha4 integrin monoclonal antibody inhibits multiple myeloma growth in a murine model.

In a syngeneic murine model of multiple myeloma with many of the characteristics of the human disease, a monoclonal antibody (mAb) to the integrin very late antigen-4 (VLA-4), given after the myeloma has already homed to and begun to establish itself within the bone marrow compartment, produces statistically significant effects on multiple disease variables. These include reductions in circulating levels of IgG2b; percentage of IgG2b-positive myeloma cells circulating in blood; spleen weight; and myeloma cell burden in spleen, bone marrow, and liver. mAb therapy had no effect on nonmalignant hematopoietic cells. An acute 6-day regimen of mAb treatment, initiated very late in disease to avoid mAb elimination in the immunocompetent animals, still significantly reduced spleen and blood myeloma cell burden. The ability of the (VLA-4) mAb to affect multiple variables in this model, even as monotherapy, suggests this pathway plays a central role in disease progression.

Function-blocking integrin alphavbeta6 monoclonal antibodies: distinct ligand-mimetic and nonligand-mimetic classes.

We have generated a panel of potent, selective monoclonal antibodies that bind human and mouse alpha(v)beta(6) integrin with high affinity (up to 15 pm). A subset of these antibodies blocked the binding of alpha(v)beta(6) to the transforming growth factor-beta1 latency-associated peptide with IC(50) values as low as 18 pm, and prevented the subsequent alpha(v)beta(6)-mediated activation of transforming growth factor-beta1. The antibodies also inhibited alpha(v)beta(6) binding to fibronectin. The blocking antibodies form two biochemical classes. One class, exemplified by the ligand-mimetic antibody 6.8G6, bound to the integrin in a divalent cation-dependent manner, contained an RGD motif or a related sequence in CDR3 of the heavy chain, was blocked by RGD-containing peptides, and was internalized by alpha(v)beta(6)-expressing cells. Despite containing an RGD sequence, 6.8G6 was specific for alpha(v)beta(6) and showed no cross-reactivity with the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(8),or alpha(IIb)beta(3). The nonligand-mimetic blocking antibodies, exemplified by 6.3G9, were cation-independent, were not blocked by RGD-containing peptides, were not internalized, and did not contain RGD or related sequences. These two classes of antibody were unable to bind simultaneously to alpha(v)beta(6), suggesting that they may bind overlapping epitopes. The "ligand-mimetic" antibodies are the first to be described for alpha(v)beta(6) and resemble those described for alpha(IIb)beta(3). We also report for the first time the relative abilities of divalent cations to promote alpha(v)beta(6) binding to latency-associated peptide and to the ligand-mimetic antibodies. These antibodies should provide valuable tools to study the ligand-receptor interactions of alpha(v)beta(6) as well as the role of alpha(v)beta(6) in vivo.