VEGF is an important mediator of tumor angiogenesis in malignant lesions in a genetically engineered mouse model of lung adenocarcinoma.

BACKGROUND: VEGF is one of the key drivers of physiological or pathological angiogenesis hence several VEGF inhibitors are in different stages of clinical development. To further dissect the role of VEGF in different stages of tumor progression in lung tumors, we utilized KrasG12D-LSL GEMMs (genetically engineered mouse models). METHODS: Intranasal delivery of adenoviruses expressing cre recombinase in KrasG12D-LSL mice results in the expression of mutant Kras that leads to development of tumor lesions ranging from adenomatous hyperplasia to large adenoma and adenocarcinoma over time in lung. In the current study, we treated KrasG12D-LSL mice at 14 weeks post inhalation with three different angiogenic inhibitors including axitinib and PF-00337210 both of which are selective inhibitors of VEGFR and sunitinib which targets VEGFR, C-SF1-R, PDGFR and KIT. RESULTS: Pathology findings showed no significant difference in percentage of adenomatous hyperplastic lesions between the vehicle vs. any of the treatments suggesting that angiogenesis may not play a major role at early stages of tumorigenesis. However, each inhibitor suppressed percentage of benign adenoma lesions and almost fully inhibited growth of adenocarcinoma lesions in the recipients which was consistent with a reduction in tumor vasculature. Treatment with sunitinib which is a multi-targeted RTKI did not provide any advantage compared to selective VEGFR inhibitor further emphasizing role of VEGF in tumor angiogenesis in this model. CONCLUSION: Overall, our studies indicate significance of VEGF and angiogenesis in a spontaneous model of lung tumorigenesis and provide a proof of mechanism for anti-cancer activity of VEGF inhibitors in this model.

The untranslated exon B of human surfactant protein A2 mRNAs is an enhancer for transcription and translation.

Two human genes, SFTPA1 (SP-A1) and SFTPA2 (SP-A2), encode surfactant protein A, a molecule of innate immunity and surfactant-related functions. Several genetic variants have been identified for both genes. These include nucleotide (nt) polymorphisms, as well as alternative splicing patterns at the 5' untranslated region (5'UTR). Exon B (eB) is included in the 5'UTR of most SP-A2, but not SP-A1 splice variants. We investigated the role of eB in the regulation of gene expression and translation efficiency. A luciferase (Luc) reporter gene was cloned downstream of the entire (AeBD) or eB deletion mutants (del_mut) of the SP-A2 5'UTR, or heterologous 5'UTRs containing the eB sequence, or a random sequence of equal length. The del_mut constructs consisted in consecutive deletions of five nucleotides (n = 8) within eB and the exon-exon junctions in the AeBD 5'UTR. Luc activities and mRNA levels were compared after transfection of NCI-H441 cells. We found that 1) eB increased Luc mRNA levels when placed upstream of heterologous 5'UTR sequences or the promoter region, regardless of its position and orientation; 2) translation efficiency of in vitro-generated mRNAs containing eB was higher than that of mRNAs without eB; and 3) the integrity of eB sequence is crucial for transcription and translation of the reporter gene. Thus eB 1) is a transcription enhancer, because it increases mRNA content regardless of position and orientation, 2) enhances translation when placed in either orientation within its natural 5'UTR sequence and in heterologous 5'UTRs, and 3) contains potential regulatory elements for both transcription and translation. We conclude that eB sequence and length are determinants of transcription and translation efficiency.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia.

BACKGROUND: The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. METHODS: Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. RESULTS: PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine. CONCLUSIONS: We show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

A novel CXCR4 antagonist IgG1 antibody (PF-06747143) for the treatment of hematologic malignancies.

The chemokine receptor CXCR4 is highly expressed and associated with poor prognosis in multiple malignancies. Upon engagement by its ligand, CXCL12, CXCR4 triggers intracellular signaling pathways that control trafficking of cells to tissues where the ligand is expressed, such as the bone marrow (BM). In hematologic cancers, CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism driving disease and therapy resistance. We developed a humanized CXCR4 immunoglobulin G1 (IgG1) antibody (Ab), PF-06747143, which binds to CXCR4 and inhibits CXCL12-mediated signaling pathways, as well as cell migration. In in vivo preclinical studies, PF-06747143 monotherapy rapidly and transiently mobilized cells from the BM into the peripheral blood. In addition, PF-06747143 effectively induced tumor cell death via its Fc constant region-mediated effector function. This Fc-mediated cell killing mechanism not only enhanced antitumor efficacy, but also played a role in reducing the duration of cell mobilization, when compared with an IgG4 version of the Ab, which does not have Fc-effector function. PF-06747143 treatment showed strong antitumor effect in multiple hematologic tumor models including non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and multiple myeloma (MM). Importantly, PF-06747143 synergized with standard-of-care agents in a chemoresistant AML patient-derived xenograft model and in an MM model. These findings suggest that PF-06747143 is a potential best-in-class anti-CXCR4 antagonist for the treatment of hematologic malignancies, including in the resistant setting. PF-06747143 is currently in phase 1 clinical trial evaluation (registered at www.clinicaltrials.gov as #NCT02954653).

Comparison of dynamic contrast-enhanced MR, ultrasound and optical imaging modalities to evaluate the antiangiogenic effect of PF-03084014 and sunitinib.

Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the K(trans) values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.

Contrast agents for quantitative microCT of lung tumors in mice.

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (µCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available µCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. µCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4-ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative µCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced µCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.

HGF/c-Met pathway is one of the mediators of sunitinib-induced tumor cell type-dependent metastasis.

Recent studies in several tumor models indicated that treatment with angiogenic inhibitors may trigger induction of metastasis to other organs. Here we investigated modes of resistance and invasion in several tumor cell lines including 4T1 (breast), H460 (lung) and Colo205 (colorectal) using sunitinib at doses comparable to clinically utilized regimen. In comparison with vehicle-treated tumors, sunitinib increased metastasis to lung in 4T1 tumors and to peritoneal lymph node in Colo205 tumors. However, the same treatment did not induce invasiveness in H460 tumors, further suggesting that accelerating metastasis during treatment with angiogenic inhibitors is tumor cell-type dependent. Interestingly, Crizotinib (a dual inhibitor of c-Met and ALK pathways) as single agent or in combination with sunitinib reduced metastasis in all models tested suggesting a role for c-Met/HGF pathway in intrinsic- or sunitinib-induced-metastasis. Moreover, ELISA data showed that while c-Met is highly enriched in tumor cells, HGF is secreted mainly by the stroma (mouse HGF) suggesting a paracrine fashion for c-Met pathway activation in the tumors. In conclusion, our findings indicate that sunitinib-induced metastasis is tumor cell-type dependent and further supports a rationale for combination of anti-angiogenics and c-Met inhibition in the clinic.

Combination of a MEK inhibitor at sub-MTD with a PI3K/mTOR inhibitor significantly suppresses growth of lung adenocarcinoma tumors in Kras(G12D-LSL) mice.

The role of PI3K and MAPK pathways in tumor initiation and progression is well established; hence, several inhibitors of these pathways are currently in different stages of clinical trials. Recent studies identified a PI3K/mTOR (PF-04691502) and a MEK inhibitor (PD-0325901) with strong potency and efficacy in different cell lines and tumor models. PD-0325901, however, showed adverse effects when administered at or above MTD (maximum tolerated dose) in the clinic. Here, we show in preclinical models that PD-0325901 at doses well below MTD (sub-MTD 1.5 mg/kg SID) is still a potent compound as single agent or in combination with PF-04691502. We first observed that PD-0325901 at 1.5 mg/kg SID and in combination with PF-04691502 (7.5 mg/kg; SID) significantly inhibited growth of H460 (carry Kras and PIK3CA mutations) orthotopic lung tumors. Additionally, we tested efficacy of PD-0325901 in Kras(G12D-LSL) conditional GEMMs (genetically engineered mouse models) which are a valuable tool in translational research to study tumor progression. Intranasal delivery of adenoviruses expressing Cre recombinase (Adeno-Cre) resulted in expression of mutant Kras leading to development of tumor lesions in lungs including adenomatous hyperplasia, large adenoma, and adenocarcinoma. Similar to H460 tumors, PD-0325901 as single agent or in combination with PF-04691502 significantly inhibited growth of tumor lesions in lungs in Kras(G12D-LSL) mice when treatment started at adenocarcinoma stage (at 14 weeks post-Adeno-Cre inhalation). In addition, immunohistochemistry showed inhibition of pS6 (phosphorylated ribosomal S6) in the treated animals particularly in the combination group providing a proof of mechanism for tumor growth inhibition. Finally, m-CT imaging in live Kras(G12D-LSL) mice showed reduction of tumor burdens in PD-0325901-treated animals at sub-MTD dose. In conclusion, our data suggest that PD-0325901 at doses below MTD is still a potent compound capable of tumor growth inhibition where Kras and/or PI3K are drivers of tumor growth and progression.

Osteopontin induces growth of metastatic tumors in a preclinical model of non-small lung cancer.

Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvß3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvß3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvß3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a Kras(G12D-LSL)p53(fl/fl) subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.

Targeting activin receptor-like kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

HGF/c-Met acts as an alternative angiogenic pathway in sunitinib-resistant tumors.

Molecular and cellular mechanisms underlying resistance/low responsiveness to antiangiogenic compounds are under extensive investigations. Both populations of tumor and stroma (nontumor compartment) seem to contribute in inherent/acquired resistance to antiangiogenic therapy. Here, investigating in vivo efficacy of sunitinib in experimental models resulted in the identification of tumors that were resistant/sensitive to the therapy. Analysis of tumor protein lysates indicated a greater concentration of hepatocyte growth factor (HGF) in resistant tumors than in sensitive ones. In addition, using flow cytometry, c-Met expression was found to be significantly higher in endothelial cells than in tumor cells, suggesting that HGF might target the vascular endothelial cells in resistant tumors. Combination of sunitinib and a selective c-Met inhibitor significantly inhibited tumor growth compared with sunitinib or c-Met inhibitor alone in resistant tumors. Histology and in vitro analyses suggested that combination treatment mainly targeted the vasculature in the resistant tumors. Conversely, systemic injection of HGF in the sensitive tumor models conferred resistance to sunitinib through maintenance of tumor angiogenesis. In conclusion, our study indicates a role for HGF/c-Met pathway in development of resistance to antiangiogenic therapy and suggests a potential strategy to circumvent resistance to vascular endothelial growth factor receptor tyrosine kinase inhibitor in the clinic.

Pyridine-3-propanoic acids: Discovery of dual PPARalpha/gamma agonists as antidiabetic agents.

A series of novel pyridine-3-propanoic acids was synthesized. A structure-activity relationship study of these compounds led to the identification of potent dual PPARalpha/gamma agonists with varied isoform selectivity. Based on the results of efficacy studies in diabetic (db/db) mice, and the desired pharmacokinetic parameters, compounds (S)-14 and (S)-19 were selected for further profiling.