Christina R. Lee

Therapy-Induced Acute Recruitment of Circulating Endothelial Progenitor Cells to Tumors

The contribution of bone marrow–derived circulating endothelial progenitor cells (CEPs) to tumor angiogenesis has been controversial, primarily because of their low numbers in blood vessels of untreated tumors. We show that treatment of tumor-bearing mice with vascular disrupting agents (VDAs) leads to an acute mobilization of CEPs, which home to the viable tumor rim that characteristically remains after such therapy. Disruption of this CEP spike by antiangiogenic drugs or by genetic manipulation resulted in marked reductions in tumor rim size and blood flow as well as enhanced VDA antitumor activity. These findings also provide a mechanistic rationale for the enhanced efficacy of VDAs when combined with antiangiogenic drugs.

Multiple circulating proangiogenic factors induced by sunitinib malate are tumor-independent and correlate with antitumor efficacy

Cancer patients treated with antiangiogenic multitargeted receptor tyrosine kinase (RTK) inhibitors show increased levels of plasma VEGF and placental growth factor and decreased levels of soluble VEGF receptor-2, thus implicating these overall changes as a possible class effect of such drugs and raising the possibility of their exploitation as surrogate biomarkers for pharmacodynamic drug activity/exposure and patient benefit. A postulated mechanism for these changes is that they are tumor-dependent, resulting from drug-induced decreases in vascular function, increases in tumor hypoxia, and changes in hypoxia-regulated genes. However, here we report that an identical pattern of change is observed in normal nontumor-bearing mice treated with SU11248/sunitinib, a small-molecule inhibitor of VEGF and PDGF RTKs. The changes were dose-dependent, plateaued after 4 days of consecutive treatment, reversed after discontinuation of therapy, and correlated with antitumor activity. Altered protein expression was found in a broad variety of tissues, and dose-dependent elevations were observed of several plasma proteins previously unassociated with this class of inhibitor, including G-CSF, SDF-1α, SCF, and osteopontin. Our results suggest that observed sunitinib-induced molecular plasma changes, including those both directly and indirectly targeted by drug, represent a systemic tumor-independent response to therapy and may correlate with the most efficacious antitumor doses, potentially having utility for defining the optimal biologic dose range for this drug class but not as predictive markers of tumor response or clinical benefit. They may also be relevant to drug-associated toxicities, drug resistance, and observed rapid tumor (re)growth seen after cessation of therapy.

Glioma tumor stem-like cells promote tumor angiogenesis and vasculogenesis via vascular endothelial growth factor and stromal-derived factor 1.

Cancer stem cells (CSC) are predicted to be critical drivers of tumor progression due to their self-renewal capacity and limitless proliferative potential. An emerging area of research suggests that CSC may also support tumor progression by promoting tumor angiogenesis. To investigate how CSC contribute to tumor vascular development, we used an approach comparing tumor xenografts of the C6 glioma cell line containing either a low or a high fraction of CSC. Compared with CSC-low tumors, CSC-high tumors exhibited increased microvessel density and blood perfusion and induced increased mobilization and tumor recruitment of bone marrow-derived endothelial progenitor cells (EPC). CSC-high C6 cell cultures also induced higher levels of endothelial cell proliferation and tubule organization in vitro compared with CSC-low cultures. CSC-high cultures and tumors expressed increased levels of the proangiogenic factors vascular endothelial growth factor and stromal-derived factor 1, and when signaling by either factor was blocked, all aspects of angiogenesis observed in CSC-high cultures and tumors, including microvessel density, perfusion, EPC mobilization/recruitment, and stimulation of endothelial cell activity, were reduced to levels comparable with those observed in CSC-low cultures/tumors. These results suggest that CSC contribute to tumor angiogenesis by promoting both local endothelial cell activity and systemic angiogenic processes involving bone marrow-derived EPC in a vascular endothelial growth factor-dependent and stromal-derived factor 1-dependent manner.

Tumor and Host-Mediated Pathways of Resistance and Disease Progression in Response to Antiangiogenic Therapy

Despite early benefits seen in cancer patients treated with antivascular endothelial growth factor (VEGF) pathway-targeted drugs, the clinical benefits obtained in terms of progression-free or overall survival have been more modest than expected. This outcome is, at least in part, due to antiangiogenic drug resistance mechanisms that involve pathways mediated largely by the tumor, whether intrinsic or acquired in response to therapy, or by the host, which is either responding directly to therapy or indirectly to tumoral cues. The focus of this review is to distinguish, where possible, between such host and tumor-mediated pathways of resistance and discuss key challenges facing the preclinical and clinical development of antiangiogenic agents, including potential differences in drug efficacies when treating primary tumors or various stages of metastatic disease. (Clin Cancer Res 2009;15(16):5020–5)

Autologous and Allogeneic Stem-Cell Transplantation for Transformed Follicular Lymphoma: A Report of the Canadian Blood and Marrow Transplant Group

PURPOSE To determine whether autologous (auto) or allogeneic (allo) stem-cell transplantation (SCT) improves outcome in patients with transformed follicular lymphoma compared with rituximab-containing chemotherapy alone. PATIENTS AND METHODS This was a multicenter cohort study of patients with follicular lymphoma and subsequent biopsy-proven aggressive histology transformation. Patient, treatment, and outcome data were collected from each transplantation center and combined for analysis. A separate control group was composed of patients with transformation treated with rituximab-containing chemotherapy but not SCT. The primary end point was overall survival (OS) after transformation. RESULTS One hundred seventy-two patients were identified: 22 (13%) treated with alloSCT, 97 (56%) with autoSCT, and 53 (31%) with rituximab-containing chemotherapy. Five-year OS after transformation was 46% for patients treated with alloSCT, 65% with autoSCT, and 61% with rituximab-containing chemotherapy (P = .24). Five-year progression-free survival (PFS) after transformation was 46% for those treated with alloSCT, 55% with autoSCT, and 40% with rituximab-containing chemotherapy (P = .12). In multivariate analysis, patients treated with autoSCT had improved OS compared with those who received rituximab-containing chemotherapy (hazard ratio [HR], 0.13; 95% CI, 0.05 to 0.34; P < .001). On the other hand, there was no OS difference between those treated with alloSCT and rituximab-containing chemotherapy (HR, 0.44; 95% CI, 0.16 to 1.24; P = .12). OS and PFS after SCT were similar between those treated with autoSCT and alloSCT. Five-year transplantation-related mortality was 23% for those treated with alloSCT and 5% for autoSCT. CONCLUSION Patients undergoing autoSCT had better outcomes than those treated with rituximab-containing chemotherapy alone. AlloSCT did not improve outcome compared with rituximab-containing chemotherapy and was associated with clinically significant toxicity.

Influence of formulation vehicle on metronomic taxane chemotherapy: albumin-bound versus cremophor EL-based paclitaxel.

Purpose: Low-dose metronomic chemotherapy treatments, especially when combined with ‘dedicated’ antiangiogenic agents, can induce significant antitumor activity without serious toxicity in various preclinical models. It remains unclear, however, whether some cytotoxic drugs are better suited for metronomic regimens than others. Paclitaxel appears to be a strong candidate for metronomic chemotherapy given its ability to inhibit endothelial cell functions relevant to angiogenesis in vitro at extraordinarily low concentrations and broad-spectrum antitumor activity. Clinically relevant concentrations of the formulation vehicle cremophor EL in Taxol, however, were previously reported to nullify the antiangiogenic effect of paclitaxel, the result of which would hamper its usefulness in metronomic regimens. We hypothesized that ABI-007, a cremophor EL–free, albumin-bound, 130-nm form of paclitaxel, could potentially alleviate this problem. Experimental Design: The antiangiogenic activity of ABI-007 was assessed by multiple in vitro assays. The in vivo optimal dose of ABI-007 for metronomic chemotherapy was determined by measuring circulating endothelial progenitors in peripheral blood. The antitumor effects of metronomic and maximum tolerated dose ABI-007 and Taxol were then evaluated and compared in severe combined immunodeficient mice bearing human MDA-MD-231 breast cancer and PC3 prostate cancer xenografts. Results: ABI-007 significantly inhibited rat aortic microvessel outgrowth, human endothelial cell proliferation, and tube formation. The optimal metronomic dose of ABI-007 was determined to be between 3 and 10 mg/kg. Metronomic ABI-007 but not Taxol, significantly suppressed tumor growth in both xenograft models. Furthermore, the antitumor effect of minimally toxic metronomic ABI-007 approximated that of the maximum tolerated dose of Taxol. Conclusions: Our results underscore the influence of formulation vehicles on the selection of cytotoxic drugs for metronomic chemotherapy.

Discovery and Optimization of Tetramethylpiperidinyl Benzamides as Inhibitors of EZH2

The identification and development of a novel series of small molecule Enhancer of Zeste Homologue 2 (EZH2) inhibitors is described. A concise and modular synthesis enabled the rapid development of structure-activity relationships, which led to the identification of 44 as a potent, SAM-competitive inhibitor of EZH2 that dose-dependently decreased global H3K27me3 in KARPAS-422 lymphoma cells.

Co-option of Liver Vessels and Not Sprouting Angiogenesis Drives Acquired Sorafenib Resistance in Hepatocellular Carcinoma

Background: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is ‘vessel co-option,’ ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents. Methods: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology. Results: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic ‘rebound’ effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option. Conclusions: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling.

Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent and Selective Inhibitor of Histone Methyltransferase EZH2, Suitable for Phase I Clinical Trials for B-Cell Lymphomas.

Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 μM, cellular EC50 = 0.032 μM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.

Differential Post-Surgical Metastasis and Survival in SCID, NOD-SCID and NOD-SCID-IL-2Rγnull Mice with Parental and Subline Variants of Human Breast Cancer: Implications for Host Defense Mechanisms Regulating Metastasis

We compare for the first time, the metastatic aggressiveness of the parental MDA-MB-231 breast cancer cell line and two luciferase-tagged in vivo-derived and selected pro-metastatic variants (LM2-4/luc+ and 164/8-1B/luc+) in SCID, NOD-SCID and NOD-SCID-IL-2Rγnull (NSG) mice following orthotopic implantation and primary tumour resection. The variants are known to be more aggressively metastatic in SCID mice, compared to the parental line which has limited spontaneous metastatic competence in these mice. When 2×106 cells were injected into the mammary fat pad, the growth of the resultant primary tumours was identical for the various cell lines in the three strains of mice. However, metastatic spread of all three cell lines, including the MDA-MB-231 parental cell line, was strikingly more aggressive in the highly immunocompromised NSG mice compared to both NOD-SCID and SCID mice, resulting in extensive multi-organ metastases and a significant reduction in overall survival. While these studies were facilitated by monitoring post-surgical spontaneous metastases using whole body bioluminescence imaging, we observed that the luciferase-tagged parental line showed altered growth and diminished metastatic properties compared to its untagged counterpart. Our results are the first to show that host immunity can have a profound impact on the spread of spontaneous visceral metastases and survival following resection of a primary tumour in circumstances where the growth of primary tumours is not similarly affected; as such they highlight the importance of immunity in the metastatic process, and by extension, suggest certain therapeutic strategies that may have a significant impact on reducing metastasis.