Therapeutic efficacy of anti-MMP9 antibody in combination with nab-paclitaxel-based chemotherapy in pre-clinical models of pancreatic cancer

Matrix metalloproteinase 9 (MMP9) is involved in the proteolysis of extracellular proteins and plays a critical role in pancreatic ductal adenocarcinoma (PDAC) progression, invasion and metastasis. The therapeutic potential of an anti-MMP9 antibody (αMMP9) was evaluated in combination with nab-paclitaxel (NPT)-based standard cytotoxic therapy in pre-clinical models of PDAC. Tumour progression and survival studies were performed in NOD/SCID mice. The mechanistic evaluation involved RNA-Seq, Luminex, IHC and Immunoblot analyses of tumour samples. Median animal survival compared to controls was significantly increased after 2-week therapy with NPT (59%), Gem (29%) and NPT+Gem (76%). Addition of αMMP9 antibody exhibited further extension in survival: NPT+αMMP9 (76%), Gem+αMMP9 (47%) and NPT+Gem+αMMP9 (94%). Six-week maintenance therapy revealed that median animal survival was significantly increased after NPT+Gem (186%) and further improved by the addition of αMMP9 antibody (218%). Qualitative assessment of mice exhibited that αMMP9 therapy led to a reduction in jaundice, bloody ascites and metastatic burden. Anti-MMP9 antibody increased the levels of tumour-associated IL-28 (1.5-fold) and decreased stromal markers (collagen I, αSMA) and the EMT marker vimentin. Subcutaneous tumours revealed low but detectable levels of MMP9 in all therapy groups but no difference in MMP9 expression. Anti-MMP9 antibody monotherapy resulted in more gene expression changes in the mouse stroma compared to the human tumour compartment. These findings suggest that anti-MMP9 antibody can exert specific stroma-directed effects that could be exploited in combination with currently used cytotoxics to improve clinical PDAC therapy

Wnt signaling and Loxl2 promote aggressive osteosarcoma

Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 coexpression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS

TGF beta inhibits HGF, FGF7, and FGF10 expression in normal and IPF lung fibroblasts

TGF beta is a multifunctional cytokine that is important in the pathogenesis of pulmonary fibrosis. The ability of TGF beta to stimulate smooth muscle actin and extracellular matrix gene expression in fibroblasts is well established. In this report, we evaluated the effect of TGF beta on the expression of HGF, FGF7 (KGF), and FGF10, important growth and survival factors for the alveolar epithelium. These growth factors are important for maintaining type II cells and for restoration of the epithelium after lung injury. Under conditions of normal serum supplementation or serum withdrawal TGF beta inhibited fibroblast expression of HGF, FGF7, and FGF10. We confirmed these observations with genome wide RNA sequencing of the response of control and IPF fibroblasts to TGF beta. In general, gene expression in IPF fibroblasts was similar to control fibroblasts. Reduced expression of HGF, FGF7, and FGF10 is another means whereby TGF beta impairs epithelial healing and promotes fibrosis after lung injury.TGF beta inhibits the important growth factors HGF, FGF7, and FGF10. This inhibition may be a very important component for the development of pulmonary fibrosis. Control and IPF fibroblasts were inhibited similarly.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145584/1/phy213794_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145584/2/phy213794.pd

Selective targeting of lysyl oxidase-like 2 (LOXL2) suppresses hepatic fibrosis progression and accelerates its reversal

Background/Aims We studied the role of lysyl oxidase-like 2 (LOXL2) in collagen crosslinking and hepatic progenitor cell (HPC) differentiation, and the therapeutic efficacy of a LOXL2-blocking monoclonal antibody on liver fibrosis progression/reversal in mice. Methods: Anti-LOXL2 antibody, control antilysyl oxidase antibody or placebo was administered during thioacetamide (TAA)-induced fibrosis progression or during recovery. Therapeutic efficacy in biliary fibrosis was tested in BALB/c.Mdr2−/− and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice. Collagen crosslinking, fibrosis progression and reversal were assessed histologically and biochemically. HPC differentiation was studied in primary EpCAM(+) liver cells in vitro. Results: LOXL2 was virtually absent from healthy but strongly induced in fibrotic liver, with predominant localisation within fibrotic septa. Delayed anti-LOXL2 treatment of active TAA fibrosis significantly reduced collagen crosslinking and histological signs of bridging fibrosis, with a 53% reduction in morphometric collagen deposition. In established TAA fibrosis, LOXL2 inhibition promoted fibrosis reversal, with enhanced splitting and thinning of fibrotic septa, and a 45% decrease in collagen area at 4 weeks of recovery. In the Mdr2−/− and DDC-induced models of biliary fibrosis, anti-LOXL2 antibody similarly achieved significant antifibrotic efficacy and suppressed the ductular reaction, while hepatocyte replication increased. Blocking LOXL2 had a profound direct effect on primary EpCAM(+) HPC behaviour in vitro, promoting their differentiation towards hepatocytes, while inhibiting ductal cell lineage commitment. Conclusions: LOXL2 mediates collagen crosslinking and fibrotic matrix stabilisation during liver fibrosis, and independently promotes fibrogenic HPC differentiation. By blocking these two convergent profibrotic pathways, therapeutic LOXL2 inhibition attenuates both parenchymal and biliary fibrosis and promotes fibrosis reversal

Wnt signaling and Loxl2 promote aggressive osteosarcoma

Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 coexpression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS

Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer

<div><p>Expression of matrix metalloproteinase 9 (MMP9) is elevated in a variety of inflammatory and oncology indications, including ulcerative colitis and colorectal cancer. MMP9 is a downstream effector and an upstream mediator of pathways involved in growth and inflammation, and has long been viewed as a promising therapeutic target. However, previous efforts to target matrix metalloproteinases (MMPs), including MMP9, have utilized broad-spectrum or semi-selective inhibitors. While some of these drugs showed signs of efficacy in patients, all MMP-targeted inhibitors have been hampered by dose-limiting toxicity or insufficient clinical benefit, likely due to their lack of specificity. Here, we show that selective inhibition of MMP9 did not induce musculoskeletal syndrome (a characteristic toxicity of pan-MMP inhibitors) in a rat model, but did reduce disease severity in a dextran sodium sulfate-induced mouse model of ulcerative colitis. We also found that MMP9 inhibition decreased tumor growth and metastases incidence in a surgical orthotopic xenograft model of colorectal carcinoma, and that inhibition of either tumor- or stroma-derived MMP9 was sufficient to reduce primary tumor growth. Collectively, these data suggest that selective MMP9 inhibition is a promising therapeutic strategy for treatment of inflammatory and oncology indications in which MMP9 is upregulated and is associated with disease pathology, such as ulcerative colitis and colorectal cancer. In addition, we report the development of a potent and highly selective allosteric MMP9 inhibitor, the humanized monoclonal antibody GS-5745, which can be used to evaluate the therapeutic potential of MMP9 inhibition in patients.</p></div

MMP9 expression in human CRC and in an orthotopic xenograft mouse model of CRC.

<p>IHC analysis of HCT116-derived xenograft tumors (A, B) or of human CRC tumors (D, E). MMP9 staining from various cellular sources is highlighted as follows: blue arrows, tumor cells; yellow arrows, inflammatory cells; white arrows, stromal cells such as fibroblasts or smooth muscle cells. (A, B) Immunohistochemical staining for MMP9 in HCT116-derived tumors at 200x (A) or 400x (B) magnification. (D, E) Immunohistochemical staining for MMP9 in a human colorectal carcinoma at 200x (D) or 400x (E) magnification. Panels C (HCT116-derived tumors) and F (human CRC) show tissue sections that were incubated with secondary antibody only and demonstrate the absence of non-specific secondary antibody binding (200x magnification).</p

Efficacy of MMP9-targeting antibody in mouse DSS model of colitis.

<p>Treatment was initiated after establishment of colitis (Day 6): Vehicle control, IgG control (30 mg/kg), and AB0046 (30 mg/kg) were dosed every three days, and entanercept (10 mg/kg) was dosed every two days (A) The area under the curve (AUC) was calculated for daily body weight changes in each animal by the trapezoidal rule method;</p><p></p><p></p><p><mi>A</mi><mi>r</mi><mi>e</mi><mi>a</mi><mo>=</mo></p><p><mo>(</mo></p><p></p><p><mi>t</mi><mn>2</mn></p><mo>−</mo><p><mi>t</mi><mn>1</mn></p><p></p><mo>)</mo><p></p><p><mo>[</mo></p><p></p><p></p><p></p><p></p><p><mo>∫</mo></p><p></p><p><mo>(</mo></p><p></p><p><mi>t</mi><mn>1</mn></p><p></p><mo>)</mo><p></p><mo>+</mo><p></p><p><mo>∫</mo></p><p></p><p><mo>(</mo></p><p></p><p><mi>t</mi><mn>2</mn></p><p></p><mo>)</mo><p></p><p></p><p></p><p></p><p></p><p></p><p></p><p></p><mn>2</mn><p></p><p></p><mo>]</mo><p></p><p></p><p></p><p></p>. (B) The incidence of diarrhea was recorded daily and the AUC calculation was performed as above. (C) Endoscopic evaluation was performed on all groups at study termination. Scoring was based on the single most severe lesion observed in the distal 5 cm of colon. (D) Blinded histopathological analysis was performed on colons excised at study termination. The degree of inflammation (primarily macrophages and neutrophils), edema, and necrosis was scored. (E) Images representative of study groups (40X magnification) were taken by a pathologist and highlight areas of inflammation/mucosal necrosis (black arrows) and edema (blue arrows), which are reduced in AB0046 and etanercept-treated animals. Statistical significance was assessed by one-way ANOVA with Dunnett’s Multiple Comparison post-test. P value designations are as follows: * < 0.05, ** < 0.01, *** <0.001, **** < 0.0001.<p></p

Characterization of the affinity, species specificity, and potency of anti-MMP9 antibodies.

<p>^a. Data is presented in the format of X ± Y, where X is the mean value of and Y is the standard deviation of three independent experiments</p><p>Characterization of the affinity, species specificity, and potency of anti-MMP9 antibodies.</p