Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.

Localized angiopoietin-2 (Ang2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis, making it an attractive candidate for antiangiogenic therapy. A fully human monoclonal antibody (3.19.3) was developed, which may have significant pharmaceutical advantages over synthetic peptide-based approaches in terms of reduced immunogenicity and increased half-life to block Ang2 function. The 3.19.3 antibody potently binds Ang2 with an equilibrium dissociation constant of 86 pmol/L, leading to inhibition of Tie2 receptor phosphorylation in cell-based assays. In preclinical models, 3.19.3 treatment blocked blood vessel formation in Matrigel plug assays and in human tumor xenografts.

MLN2704 is an antibody-chemotherapeutic conjugate designed to target prostate-specific membrane antigen (PSMA). PSMA is a transmembrane receptor whose expression is largely restricted to prostatic epithelium and prostate cancer cells with its expression level increasing during the progression of malignancy. MLN2704 consists of a de-immunized, monoclonal antibody that is specific for PSMA conjugated to drug maytansinoid 1 (DM1), a microtubule-depolymerizing compound. After antibody binding to PSMA and the subsequent cellular internalization of this complex, DM1 is released leading to cell death. MLN2704 has an approximate half-life of 39 hours in scid mice bearing CWR22 tumor tissue, and the antibody effectively penetrates xenograft tumor tissue. Optimization of dosage and schedule of MLN2704 administration defined interdependency between these conditions that maximized efficacy with no apparent toxicity. Tumor growth delays of ϳ100 days could be achieved on the optimized schedule of one dose of 60 mg/kg MLN2704 every 14 days for five doses (q14d؋5). The unconjugated antibody (MLN591) demonstrated essentially no antitumor activity and DM1 alone or a non-PSMA targeted antibody-DM1 conjugate was only weakly active. Furthermore, we show that MLN2704 is active in a novel model of osteoblastic prostate cancer metastasis.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.

◥Purpose: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-x L , has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-x L inhibitor that broadens the therapeutic activity while minimizing Bcl-x L -mediated thrombocytopenia.Experimental Design: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-x L and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time.Results: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-x L , and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2-and Bcl-x L -dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-x L -dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models.Conclusions: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-x L inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

GalNAc␤1-4(NeuAc␣2-3)Gal␤1-4Glc␤1-Cer (GM2)/ GalNAc␤1-4(NeuAc␣2-8NeuAc␣2-3)Gal␤1-4Glc␤1-1Cer (GD2) synthetase [␤-1,4-N-acetyl-galactosaminyl transferase (GalNAc-T)] mRNA, which encodes a key glycosyltransferase for ganglioside GD2 synthesis , was assessed as a molecular marker for detecting metastatic neuroblastoma cells in bone marrow (BM). GalNAc-T mRNA expression by neuroblastoma cell lines (n ‫؍‬ 15), primary untreated neuroblastoma tumors (n ‫؍‬ 29), morphologically normal BM (n ‫؍‬ 22), peripheral blood stem cells (n ‫؍‬ 10) from patients with cancers other than neuroblastoma, and blood mononuclear cells from normal donors (n ‫؍‬ 17) was assessed by using reverse transcriptase-polymerase chain reaction (RT-PCR) and electrochemiluminescence detection assay (RT-PCR/ECL). BM harvested from 15 neuroblastoma patients was tested before and after ex vivo immunomagnetic bead purging, and results were compared to immunocytological analysis of the same specimens. Neuroblastoma, the most common extracranial cancer in children, is derived from the neural crest. Approximately 45% of patients have high-risk, metastatic disease (stage 4, International Neuroblastoma Staging System) at diagnosis, and 86% of these have bone marrow (BM) involvement when assessed by immunocytology.1 High-dose, myeloablative chemo-radiotherapy followed by BM-or blood-derived hematopoietic stem cell rescue (autologous hematopoietic stem cell transplant, AHSCT) is increasingly used to treat these patients and has been shown to improve outcome in a randomized study, especially if followed by 13-cis-retinoic acid therapy.

Despite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin's lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcgammaRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.