Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective smallmolecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G2/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.cancer ͉ mitosis ͉ apoptosis

Dysregulated fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of human cancers. Aberrant activation of FGF receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification, has been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand-binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We surveyed a broad panel of human cancer cell lines for the dysregulation of FGFR2 signaling and discovered that breast and gastric cancer cell lines harboring FGFR2 amplification predominantly express the IIIb isoform of the receptor. Therefore, we used an FGFR2-IIIb-specific antibody, GP369, to investigate the importance of FGFR2 signaling in vitro and in vivo. GP369 specifically and potently suppressed ligand-induced phosphorylation of FGFR2-IIIb and downstream signaling, as well as FGFR2-driven proliferation in vitro. The administration of GP369 in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support the hypothesis that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies.

Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients.Experimental Design: To investigate TG02, an agent with known penetration of the blood-brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays.Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue.Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial.

TG02 is a novel cyclin‐dependent kinase (CDK) inhibitor and thought to act mainly via CDK‐9 inhibition‐dependent depletion of short‐lived oncoproteins such as MCL‐1 or c‐MYC. We studied the activity of TG02 in 9 human long‐term glioma cell lines (LTC) and 5 glioma‐initiating cell lines (GIC) using various cell death assays in vitro and in the LN‐229 LTC and ZH‐161 GIC models in vivo. TG02 exhibits strong anti‐tumor cell activity with EC50 concentrations in the nanomolar range. Median survival in the LN‐229 and ZH‐161 models was moderately prolonged by TG02. Neither constitutive CDK levels nor those of MCL‐1 or c‐MYC correlated with sensitivity to TG02. Cdk‐9 or cdk‐5 gene silencing alone did not fully reproduce the effects of TG02. C‐myc gene silencing inhibited cell growth, but did not modulate TG02 activity. Electron microscopy revealed cell death to be essentially apoptotic. High concentrations of TG02 induced annexin V binding and minor caspase 3 cleavage, but the pan‐caspase inhibitor, zVAD‐fmk, or BCL‐2 or MCL‐1 gene transfer only moderately attenuated TG02‐induced cell death, and caspase inhibition did not prevent loss of MCL‐1 or c‐MYC. TG02 activity was independent of O6‐methylguanine DNA methyltransferase expression. Repetitive exposure to TG02 did not generate an acquired TG02 resistance phenotype, but accumulation of MCL‐1, loss of c‐MYC, or senescence. TG02 is a highly potent apoptosis‐inducing agent in glioma cells in vitro. Caspase inhibition does not rescue TG02‐treated cells and repetitive exposure fails to confer acquired resistance, supporting the clinical evaluation of TG02 in glioblastoma.

Purpose: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/k ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic.Experimental Design: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models.Results: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models.Conclusions: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.

Hepatocyte growth factor (HGF) is the soluble ligand for the c-Met receptor tyrosine kinase. Signaling through the HGF/c-Met pathway mediates a plethora of cellular activities that are involved in cancer cell dysregulation, tumorigenesis, and metastasis including cell proliferation and survival, angiogenesis, migration, invasion and drug resistance. HGF/c-Met autocrine and paracrine regulatory loops have been reported in a number of non-small cell lung cancer studies. Furthermore, studies have shown that HGF/c-Met pathway upregulation via either c-Met amplification or HGF secretion can result in intrinsic or acquired resistance to EGFR TKIs in lung adenocarcinoma. SCH 900105, formerly known as AV-299, is a humanized IgG1 antibody with high affinity to HGF that neutralizes all its biological functions tested with sub-nM potency. It is currently in phase 1 clinical trials that demonstrated good safety and tolerability. Anti-tumor activity of SCH 900105 was observed in HGF autocrine and paracrine in vivo tumor models, such as GMB, pancreatic cancer and multiple myeloma. Anti-tumor efficacy of SCH 900105 was evaluated in paracrine models of the HGF-dependent NCI-H596 NSCLC cell line xenografted in SCID mice engineered to produce human HGF. In these models, SCH 900105 treatment resulted in dose-dependent decrease in tumor growth with concurrent increases in serum and tumor concentration of SCH900105 and increases in serum SCH 900105/HGF complex. Treatment also led to significant reduction in phospho-c-Met and phospho-Akt levels in tumor lysates. Concurrently, increases in cleaved caspase-3 and decreases in Ki67 and CD31 staining were also observed. Anti-tumor activity of SCH 900105 was also explored in combination with EGFR inhibitors, erlotinib and Cetuximab that resulted in increased efficacy. SCH900105 treatment resulted in decreased phospho-c-Met levels with concurrent increases in phospho-EGFR levels. Conversely, erlotinib treatment decreased phospho-EGFR with concurrent increases in phospho-Met levels. The combination of SCH900105 with Cetuximab resulted in complete response in all animals treated without tumor re-growth 50 days after treatment withdrawal. Potent anti-tumor activity of SCH 900105 in combination with EGFR inhibitors observed in these preclinical models suggests testing the combination in NSCLC is warranted in the clinic. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C173.

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs.MethodsWe developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo.ResultsCLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts.ConclusionsCLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Hepatocyte growth factor (HGF) is a pluripotent growth factor produced predominantly by mesenchymal or stromal cells, and binds to the well-characterized tyrosine kinase receptor, c-Met. The HGF/c-Met pathway is frequently deregulated in different types of human cancers and is thought to play an important role in regulating tumor growth, invasion, metastasis and drug resistance. HGF/c-Met autocrine and paracrine loops have been reported in a number of human cancers including breast, lung, bladder, gastric, head and neck, glioma, multiple myeloma, leukemias, and certain sarcomas. SCH 900105, formerly known as AV-299, is a potent, humanized anti-HGF antibody. It is currently in phase 1 clinical trials that demonstrated good safety and tolerability. It has been shown to neutralize HGF binding to c-Met and inhibits its biological function in vitro, such as cell signaling, growth, motility, invasion and drug resistance. SCH 900105 was also shown to have potent anti-tumor activity in autocrine and paracrine GBM, NSCLC, pancreatic and multiple myeloma xenograft models both as monotherapy and in combinations with chemotherapeutics or targeted agents. In vivo efficacy of systemically administered SCH 900105 was evaluated in an intracranial autocrine U87MG model. In these studies, SCH 900105 treatment resulted in significant survival benefit over an IgG treated control group. Immunohistochemistry staining for SCH 900105 in the intracranial tumor tissue suggested adequate tumor penetration of the antibody. Treatment also led to significantly decreased tumor phospho-c-Met, increased cleaved caspase-3, as well as decreased Ki67 and CD31 staining. Greater survival benefit was also seen when SCH 900105 was combined with temozolomide in U87 intracranial model. These findings suggest evaluating SCH 900105 in GBM is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C181.