Brett H. Simmons

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.

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 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.

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 a nti-O PN m onoclonal 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 αvβ3 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 (αvβ3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvβ3 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 KrasG12D-LSLp53fl/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.

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 Ktrans 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.

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

BackgroundThe 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.MethodsPatient-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.ResultsPF-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.ConclusionsWe 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).

Abstract 2649: Fc-effector function activity of the CXCR4 IgG1 antibody PF-06747143: a novel clinical candidate for the treatment of hematologic malignancies

The chemokine receptor CXCR4 triggers signaling pathways that control cell migration to tissues where its ligand, CXCL12, is highly expressed, including the bone marrow (BM). In hematologic cancers, CXCR4 expression is associated with poor prognosis. CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism of chemotherapy resistance. PF-06747143 is a novel humanized IgG1 therapeutic antibody that binds to CXCR4 and inhibits CXCL12-driven pathways. Human IgG1 antibodies can induce strong cytotoxicity mediated by the antibody Fc-region, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-driven cytotoxicity, while human IgG4 antibodies show minimal or no Fc-driven cytotoxicity. Here we generated an IgG4 version of PF-06747143 (m15-IgG4), which has similar binding to CXCR4 as the IgG1 antibody. We then characterized the role of Fc-driven cytotoxic function, comparing both antibodies in efficacy and safety studies. In an ADCC assay, PF-06747143 showed strong cytotoxicity of non-Hodgkin’s lymphoma (NHL) and acute myeloid leukemia (AML), while m15-IgG4 had no significant cytotoxicity. In a NHL mouse tumor model, the IgG1 CXCR4 antibody resulted in superior tumor growth inhibition, with 50% of mice exhibiting complete tumor regressions, compared to the m15-IgG4 antibody, which had limited activity, with no tumor regressions (p 4 days. Since both antibodies had comparable exposures, the different mobilization duration is likely due to the ability of the IgG1 CXCR4 antibody to reduce the number of mobilized cells via Fc-driven cytotoxic function. Finally, CXCR4 has been shown to play a key role in chemotherapy resistance. In a chemo-resistant PDX AML mouse model, in which the standard of care agents daunorubicin and cytarabine had limited activity, resulting in 30% of tumor cells remaining in the BM post-treatment, we show that combination of PF-06747143 with these chemo agents led to synergistic activity, with tumor burden reduced to 0.3% tumor cells in the BM. In conclusion, PF-06747143 attributes offer potential efficacy- and safety-related advantages over other CXCR4 antagonists currently in development, which do not have Fc-driven cytotoxic activity. PF-06747143 is now being evaluated in a Phase 1 clinical trial in relapsed and refractory AML (NCTID 02954653). Citation Format: Flavia Pernasetti, Shu-Hui Liu, Gu Yin, Bernadette Pascual, Zhengming Yan, Max Hallin, Rolla Yafawi, Cathy Zhang, Connie Fang, Wenlian Wang, Justine Lam, Mary E. Spilker, Eileen Blasi, Brett Simmons, Nanni Huser, Wei-Hsien Ho, Kevin Lindquist, Thomas-Toan Tran, Jyothirmayee Kudaravalli, Jing-Tyan Ma, Gretchen Jimenez, Ishita Barman, Colleen Brown, Sherman-Michael Chin, Maria Costa, David Shelton, Tod Smeal, Valeria R. Fantin. Fc-effector function activity of the CXCR4 IgG1 antibody PF-06747143: a novel clinical candidate for the treatment of hematologic malignancies [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 2649. doi:10.1158/1538-7445.AM2017-2649

Abstract 322: Dual targeting of the PI3K/mTOR pathways by PF-04691502 or PF-05212384 exhibits therapeutic effects against the metastatic disease progression in the Ovcar 3 disseminated model.

Ovarian cancer has the highest mortality rate of all gynecological cancers primarily due to the high incidence of metastatic spread prior to diagnosis. Understanding the biology of this malignancy and developing models that reflect the patient disease phenotype will create better pre-clinical opportunities for testing targeted therapy. We used the Ovcar 3 cell line to establish a metastasis model through serial in vivo passage. The dual PI3K/mTOR inhibitors PF-04691502 and PF-05212384 were then investigated in this model for their therapeutic effect against metastatic disease progression. Notably, the stromal environment and tumor initiating subpopulation play critical roles on the Ovcar 3MET disease invasiveness and progression. Based on the molecular profile, these invasive Ovcar 3MET cells exhibited a more EMT phenotype when compared to parental cells. Impairing PI3K/mTOR pathway with the dual PI3K/mTOR inhibitors resulted in significant anti-metastatic activity and increased survival in the Ovcar 3MET model. Serum CA125 level, which is a validated clinical marker, correlated with anatomic measurement of tumor burdens via BLI and mouse survival time in these studies, providing means for longitudinal monitoring of disease progression and response to therapy. In addition, using ex vivo tumorsphere analysis under serum-free condition, both PF-04691502 and PF-05212384 demonstrated robust activity against the self-renewal ability of Ovcar 3 tumor cells. In summary, this translational tumor model may serve as a valuable tool for testing targeted therapies such as PI3K/mTOR inhibitors for their ability to impact progression of metastatic ovarian disease. Citation Format: Zhengming Yan, Douglas Fang, Brett Simmons, Jing Yuan, Julie Kan, Cathy C. Zhang. Dual targeting of the PI3K/mTOR pathways by PF-04691502 or PF-05212384 exhibits therapeutic effects against the metastatic disease progression in the Ovcar 3 disseminated model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2013-322

Abstract 1512: Sunitinib-induced metastasis is tumor dependent and is mediated by c-Met/HGF pathway

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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 tumors 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 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 dependent and further supports a rationale for combination of anti-angiogenesis and c-Met inhibition in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1512. doi:1538-7445.AM2012-1512