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Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

Chemoresistance represents a considerable barrier to improving outcomes for patients with acute myeloid leukemia (AML) and therapeutic approaches using multiple lines of therapy have been unsuccessful as cancer cells acquire resistance to the chemotherapeutic agents to which they are exposed. This vulnerable patient group needs individualizing therapy through careful selection of appropriate agents based on specific signaling pathways. The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow microenvironment, is highly expressed in 25-30% of patients with AML and its expression is correlated with poor prognosis and drug resistance. The purpose of this study was to investigate a new humanized monoclonal IgG1 antibody to CXCR4 (PF-06747143) and its effects as a monotherapy in AML primary patient samples and in chemotherapy resistant patient-derived xenotransplantation (PDX) models. This antibody was previously shown to be able to induce cell death through its effector function (CDC and ADCC) and to be efficacious in cell-based xenograft models of AML, NHL, CLL and MM. Here we have shown that PF-06747143 binds strongly and specifically to AML cell lines and to AML primary cells, by flow cytometry. Of 16 samples evaluated, 7 displayed low CXCR4 expression (CXCR4neg/low) whereas 9 displayed high expression (CXCR4high). A good correlation was observed between 12G5, a commercially available CXCR4 Ab, and PF-06747143 staining, indicating that PF-06747143 can be used to stratify AML patients. Chemotaxis in response to CXCL12 was significantly inhibited in all AML patient primary samples analyzed. Administration of PF-06747143 to mice engrafted with AML patient cells (PDX models) induced rapid malignant cell mobilization into the peripheral blood at 4 hrs after a single antibody dose, with mobilized cell levels going back down to baseline at 24 hrs post-dose. This is in line with the ability of the antibody to block malignant cell homing to the bone marrow, inducing cell mobilization, as well as induction of cell death through effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different models: 1) P15 model characterized by high CXCR4 expression, inducing aggressive disease, with rapid progression of leukemia and widespread dissemination and chemoresistance; 2) P17 model characterized by a low CXCR4 expression, a less aggressive disease and limited dissemination. Weekly administration of PF-06747143 to leukemic mice previously engrafted with P17 or P15 malignant cells induced a sharp reduction of leukemia cells in the bone marrow, spleen and blood leading to increased survival of leukemic mice in both models. Activity of the antibody as monotherapy was superior to daunorubicin in the P15 chemoresistant model. Secondary transplantation of bone marrow cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143 treatment, with slower tumor growth in mice transplanted with PF-06747143-treated cells. In summary, PF-06747143, a CXCR4 IgG1 antibody, is significantly efficacious as a monotherapy and superior to daunorubicin in AML chemoresistant PDX models. These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy. Disclosures No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in the Western world. This leukemia is not curable and resistance to therapy is promoted by factors present in the tumor microenvironment including the chemokine CXCL12 (SDF-1), which interacts with its receptor CXCR4 and is thought to promote cancer cell survival. Here we explored the therapeutic potential of blocking CXCL12-CXCR4 interactions using PF-06747143, a humanized IgG1 antibody specific for CXCR4, which is expressed at high levels by CLL cells. Using primary leukemia cells from CLL patients, we found that PF-06747143 inhibited CXCL12-induced cell migration and blocked cytoskeletal changes via F-actin polymerization similar to AMD-3100 (Mozobil, a small molecule inhibitor of CXCR4). In addition, PF-06747143 induced apoptosis on CLL cells cultured alone or in the presence of human bone marrow-derived stromal cells (stroma-NK-tert). The pro-apoptotic activity of PF-06747143 was independent of high-risk prognostic factors including IGHV mutation status, ZAP-70 expression or TP53 mutation / 17p-deletion. Interestingly, AMD-3100, which binds and inhibits signaling through CXCR4, did not induce cell death in CLL or any of the cell lines tested. PF-06747143 did not induce apoptosis on normal B and T cells, and the ability of this anti-CXCR4 antibody to induce cell death on CLL cells appeared to be dependent on the crosslinking of CXCR4. This was supported by the fact that a Fab only fragment derived from PF-06747143 did not induce apoptosis despite of its high binding affinity for CXCR4. We observed that PF-06747143 induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in CLL cells. However, this antibody did not induce caspase activation but rather its cell death activity appeared to be dependent on the production of reactive oxygen species (ROS) in leukemia cells. This effect was similar to that observed with other ROS dependent antibodies such as obinutuzumab (Gazyva). ROS induction was observed with PF-06747143, but not its Fab derived fragment and preceded apoptosis suggesting that this is critical component of its mechanism of action. We evaluated synergism of PF-06747143 with other CLL therapeutic agents and observed that this antibody synergized with fludarabine, bendamustine, ibrutinib and rituximab in the majority of CLL patient samples tested. In summary, our studies showed that PF-06747143, a CXCR4 IgG1 antibody is a potent inhibitor of the CXCR4-CXCL12 pathway and induces cell death primarily in CLL cells but not in normal lymphocytes. The cytotoxic effect of PF-06747143 was similar in CLL cells cultured alone or with stromal cells, suggesting that this antibody has the potential to overcome the protective effect of the tumor microenvironment. We also showed that PF-06747143 induced programmed cell death on CLL cells was dependent on ROS production and that this antibody synergized with agents currently used for the treatment of CLL patients. Overall, these findings highlight the biological relevance of the CXCR4-CXCL12 pathway in CLL, and provide rationale for clinical evaluation of PF-06747143 in CLL and other cancers. Disclosures Choi: Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a histone deacetylase inhibitor active clinically in cutaneous T-cell lymphoma and preclinically in leukemia. A phase 1 study was conducted to evaluate the safety and activity of oral vorinostat 100 to 300 mg twice or thrice daily for 14 days followed by 1-week rest. Patients with relapsed or refractory leukemias or myelodysplastic syndromes (MDS) and untreated patients who were not candidates for chemotherapy were eligible. Of 41 patients, 31 had acute myeloid leukemia (AML), 4 chronic lymphocytic leukemia, 3 MDS, 2 acute lymphoblastic leukemia, and 1 chronic myelocytic leukemia. The maximum tolerated dose (MTD) was 200 mg twice daily or 250 mg thrice daily. Dose-limiting toxicities were fatigue, nausea, vomiting, and diarrhea. Common drug-related adverse experiences were diarrhea, nausea, fatigue, and anorexia and were mild/moderate in severity. Grade 3/4 drug–related adverse experiences included fatigue (27%), thrombocytopenia (12%), and diarrhea (10%). There were no drug-related deaths; 7 patients had hematologic improvement response, including 2 complete responses and 2 complete responses with incomplete blood count recovery (all with AML treated at/below MTD). Increased histone acetylation was observed at all doses. Antioxidant gene expression may confer vorinostat resistance. Further evaluation of vorinostat in AML/MDS is warranted.

CXCR4 is a chemokine receptor that belongs to the G-coupled protein receptor (GPCR) family. It is over-expressed in various cancers, including solid tumors and hematological malignancies, and correlates with poor prognosis. CXCR4 expressing cells actively respond to CXCL12 (SDF-1), a chemokine constitutively secreted by stromal cells in bone marrow. Activation of CXCR4 induces cell trafficking and homing to the marrow microenvironment, where CXCL12 retains these cells in close contact with marrow stromal cells that provide growth signals, promote self-renewal, and contribute to drug resistance, leading to poor prognosis and relapse. Here we describe the generation of a highly potent and selective anti-CXCR4 humanized IgG1 antagonist Ab (PF-06747143) that binds to human CXCR4 with high affinity and blocks SDF-1-induced Calcium flux and cAMP signaling. We have also characterized the ability of PF-06747143 to induce cell death through three different mechanisms: a) mobilization of cells from CXCL12-rich niches, making them more sensitive to chemotherapy b) direct cell-death through a mechanism dependent on the antibody’s bivalency; c) ADCC- and CDC-dependent cell death through the Fc-region in IgG1 backbone, when in the presence of effector cells or serum proteins. Weekly administration of PF-06747143 at 10 mg/kg, as a monotherapy, significantly improved survival, induced sustained regression and reduced bone marrow tumor burden in various patient population relevant murine disseminated tumor models of Acute Myeloid Leukemia (MV4-11, PDXs), Non Hodgkin Lymphoma (Raji and Ramos), Chronic Lymphocytic Leukemia (JVM-13) and Multiple Myeloma (OPM-2). The CXCR4 IgG1 antibody was also shown to be similar or more efficacious than approved standards of care agents currently employed for treatment of hematological malignancies. The safety and PK/PD profile of PF-06747143 were evaluated in a Non-Human Primate (NHP) exploratory toxicology study. Results from this study indicate that the CXCR4 IgG1 Ab was well tolerated in a two-week exploratory study at pharmacologically relevant doses. Upon treatment with PF-06747143, egression of white blood cells (WBC) from bone marrow (leukocytosis) was noted, which is consistent with target (CXCR4) modulation. Following the peak of leukocytosis between 1-6 hours post antibody administration, the number of circulating WBCs rapidly decreased back to baseline levels at 24 hrs. These results are likely explained by the direct cell killing through the effector function of this IgG1 CXCR4 antibody. Altogether, the promising preclinical efficacy and safety data support clinical evaluation of PF-06747143 in hematological malignacies. Disclosures Pernasetti: Pfizer: Employment. Liu:Pfizer: Employment. Hallin:Pfizer: Employment. Gu:Pfizer: Employment. Ho:Pfizer: Employment. Zhang:Pfizer: Employment. Pascual:Pfizer: Employment. Simmons:Pfizer: Employment. Yan:Pfizer: Employment. Huser:Pfizer: Employment. Wang:Pfizer: Employment. Lam:Pfizer: Employment. Spilker:Pfizer: Employment. Blasi:Pfizer: Employment. Tran:Pfizer: Employment. Kudaravalli:Pfizer: Employment. Ma:Pfizer: Employment. Chin:Pfizer: Employment. Shelton:Pfizer: Employment. Smeal:Pfizer: Employment. Fantin:Pfizer: Employment.

Abstract 2509 Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes have recently been shown to be recurrent, frequent and prognostic in acute myeloid leukemia (AML) patients. IDH1 and IDH2 encode NADP+ dependent enzymes responsible for oxidative decarboxylation of isocitrate to α-ketoglutarate (AKG). Heterozygous point mutations in IDH1 and IDH2 result in the loss of this activity, concomitant with a neomorphic ability to convert AKG to 2-hydroxyglutarate (2HG), resulting in significant elevations in this oncometabolite. The ten-eleven translocation 2 (TET2) gene converts 5-methylcytosine (5MC) to 5-hydroxymethylcytosine (5HMC), leading to DNA demethylation. TET2 encodes a dioxygenase that is dependent on AKG, and therefore, increased levels of 2HG may disrupt TET2 function. We explored the potential of 2HG to serve as a clinical biomarker of response to therapy in AML patients with IDH mutations, and correlated fluctuations of 2HG with changes in 5MC and 5HMC levels in a clinical setting. In a Phase I/II clinical trial of 44 elderly, previously untreated AML patients who received sequential azacitidine and lenalidomide, we identified 12 patients with IDH1/2 mutations (27%). Five had IDH1 mutations (all R132), and seven had IDH2 mutations (R140 N=5, R172 N=2). 2HG levels were measured by mass spectrometry from plasma obtained from bone marrow aspirations. Six patients had pre- and post-treatment samples available for comparison. Clinical responses were determined per LeukemiaNet criteria. Two of the six patients achieved a complete remission (CR), one with an IDH1 R132 and the other with an IDH2 R140. A comparison of pre- and post-treatment samples from these patients revealed decreases in 2HG levels of −16.9 and −17.9 fold after three cycles of therapy. Four patients did not achieve a CR, although some did experience decreases in the percentage of blasts. These patients had substantially smaller decreases, or increases, in 2HG levels when measured after one cycle (Figure 1). A non-parametric comparison (Mann-Whitney U test) in this small sample set showed a trend toward a significant change in pre- vs. post-treatment 2HG levels between patients who experienced a CR and those who did not (p=0.067), suggesting 2HG levels may correlate with leukemic blast clearance. Both patients who achieved a CR continued to have detectable 2HG levels. Furthermore, patient 8 ultimately relapsed, and at the time of this relapse, which was unexpected based on clinical indicators or routine peripheral blood laboratory findings, there was a 1.8-fold increase in 2HG levels. Therefore, for AML patients with IDH1 and IDH2 mutations, 2HG may provide a useful, non-invasive marker of minimal residual disease. We hypothesized that decreases in 2HG would allow functional TET2 activity, resulting in increased 5HMC and decreased 5MC. Therefore, we correlated changes in 2HG levels with changes in global 5MC and 5HMC levels, as measured by mass spectrometry, in the patients who achieved a CR. Patient 8 was wild type for TET2. As predicted, after one cycle of therapy, 2HG decreased 6.1 fold, 5HMC increased 7.8 fold and 5MC decreased 1.4 fold. At relapse, when 2HG rose 1.8 fold, 5HMC decreased 5.4 fold; the expected increase in 5MC was not observed (Figure 2). Although concomitant mutations in IDH and TET2 are exceedingly rare, patient 22 did have this genotype, which allowed for an instructive negative control. After one cycle of therapy, 2HG decreased 2.6 fold. However, in contrast to patient 8, in which it appeared TET2 became functional as shown by increased 5HMC after 2HG decreased, patient 22's mutant TET2 resulted in marginal changes in 5HMC and 5MC levels (1.3-fold increase and 1.1-fold decrease, respectively) (Figure 3). These observations provide in vivo evidence for the interaction between IDH and TET2, and suggest that these interactions are dynamic and can change with therapy. Disclosures: Off Label Use: Lenalidomide in AML. Schenkein:Agios: Employment. Fantin:Agios: Employment. Straley:Agios: Employment. Medeiros:Celgene: Honoraria, Research Funding.

Vorinostat is a histone deacetylase inhibitor currently under evaluation in numerous oncology clinical trials. In a Phase IIb trial, oral vorinostat resulted in a 29.7% overall objective response rate in patients (pts) with advanced cutaneous T-cell lymphoma (CTCL) and had an acceptable safety profile. These results prompted efforts to identify gene expression patterns that could elucidate the molecular mechanism of action (MOA), assess exposure to vorinostat and enrich for pts who are likely to respond. In the Phase IIb trial, gene expression profiles were obtained from 24 predose and 30 postdose (2 hr postdose on Day 15) PBMC samples. The gene expression associated with Sezary burden was easily identified in predose samples and consistent with published results. Although the power of this dataset was limited for development of a predose predictor of response, we identified three biologically-relevant pathways that correlated with response and deserve further validation. First, we found a coherent cluster of proliferation/cell cycle genes to be associated with resistance to therapy. This may imply that tumor aggressiveness is an important factor for clinical response. Second, a set of antioxidant genes was upregulated in non-responders. The generation of reactive oxygen species (ROS) is a component of the vorinostat MOA and increased ROS scavenging ability may confer resistance. Finally, cytotoxic cell markers were upregulated in responders and may represent another factor associated with contribution of T and NK cells to response. Each of these 3 patterns, if confirmed, would allow for 20–50% responder enrichment. We observed robust postdose gene expression changes in which ~942 genes exhibited significant regulation (fold-change>2, P

The hydroxamic acid vorinostat (SAHA) is an HDAC inhibitor that induces differentiation, growth arrest and/or apoptosis of malignant cells both in vitro and in vivo, and is currently being tested in the clinic for a variety of indications. In particular, vorinostat has demonstrated an overall response rate of approximately 30% in advanced cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers that predict response to vorinostat in CTCL using preclinical model systems based upon gene expression profiling and pathogenesis of lymphoma. Herein we report the results of our evaluation of the STAT (signal transducer and activator of transcription) signaling pathway. The relative level of mRNA and protein expression as well as activation status of several members of this family have been evaluated in lymphoid cell lines with diverse vorinostat sensitivity (proliferation/viability IC50 range: 0.3–14 mM). Among them, the more responsive CTCL cells lines HH and HUT102 exhibited an IC50 ≃ 0.8 mM. These cells underwent apoptosis in response to vorinostat as assessed by TUNEL assay, in contrast to Hut78 and MJ cells (IC50 > 2 mM) that did not show signs of DNA fragmentation upon incubation in vorinostat-containing media for 48 h. Our results indicate that in lymphoma cell lines elevated protein levels and persistent activation of STAT1, 3 and 5 correlate with resistance to vorinostat. Immunofluorescence microscopy studies revealed that STAT proteins preferentially localize to the nuclear compartment in cells with impaired vorinostat response, consistent with the expected distribution for this group of transcription factors in the active state. Simultaneous treatment with a pan-JAK inhibitor (JAK inhibitor I; Calbiochem) resulted in an additive antiproliferative effect consistent with a survival, antiapoptotic role for STAT proteins in the response to vorinostat treatment. Immunohistochemical analysis of STAT1 in skin biopsies isolated from CTCL patients (N = 49) enrolled in the vorinostat Phase IIb clinical trial showed that malignant T cells were positively stained in approximately half of the samples (21 positively stained; 25 negatively stained; 3 poor quality specimens), and in those cases a relationship between nuclear accumulation of STAT1 and lack of response to treatment exists (p=0.007

Vorinostat is a histone deacetylase inhibitor with potent in vitro and in vivo anti-leukemia activity. Several gene expression signatures were developed from preclinical models as biomarkers for clinical response to vorinostat. The signatures were evaluated in peripheral blood mononuclear cell (PBMC) samples from a Phase I study of oral vorinostat in patients with advanced leukemias or myelodysplastic syndromes (MDS). Of 41 registered patients, 31 had acute myeloid leukemia (AML), 4 had chronic lymphocytic leukemia, 3 had MDS, 2 had acute lymphoblastic leukemia, and 1 had chronic myeloid leukemia. Seven of 41 patients showed improvement with treatment, 2 complete responses, 2 complete responses that were not maintained for 28 days, and 3 with >50% decreases in blast counts without count recovery. All responses occurred in patients with AML at or below the MTD. The following three clinical gene expression signatures were shown to be predictive of clinical response in PBMC samples of AML patients. First, a positive prediction trend was observed when a preclinical 40-gene resistance prediction signature was evaluated in baseline (pre-dose and first day of dosing) AML PBMC samples (8 responder and 21 non-responder samples) or all PBMC samples (14 responder and 56 non-responder samples, including non-AML patients). If this trend is maintained in pretreatment samples of a larger patient population, it may allow for exclusion of up to 50% of non-responders from future clinical trials. Second, a similar prediction of resistance was obtained based on expression levels of known reactive oxygen species scavengers that were upregulated in non-responders relative to responders, indicating that cellular ability to withstand oxidative stress may be protective with respect to vorinostat exposure. This supports the hypotheses that oxidative stress pathways play an important role in the response to vorinostat. Finally, overexpression of p21 and several p53-responsive genes was also associated with resistance, consistent with preclinical observations. These results are being validated at the protein level in a tissue array platform. Post-dose downregulation of genes associated with cellular proliferation was also observed in responders but not in non-responder PBMC samples. This suggests that proliferation may be used as a post-dose efficacy biomarker. Further evaluation of vorinostat in patients with leukemias and MDS, including analysis of a potentially predictive gene expression signature for response to vorinostat, is warranted.