Lee J. Helman

Tumor Regression in Patients With Metastatic Synovial Cell Sarcoma and Melanoma Using Genetically Engineered Lymphocytes Reactive With NY-ESO-1

PURPOSE Adoptive immunotherapy using tumor-infiltrating lymphocytes represents an effective cancer treatment for patients with metastatic melanoma. The NY-ESO-1 cancer/testis antigen, which is expressed in 80% of patients with synovial cell sarcoma and approximately 25% of patients with melanoma and common epithelial tumors, represents an attractive target for immune-based therapies. The current trial was carried out to evaluate the ability of adoptively transferred autologous T cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic melanoma and synovial cell sarcoma. PATIENTS AND METHODS A clinical trial was performed in patients with metastatic melanoma or metastatic synovial cell sarcoma refractory to all standard treatments. Patients with NY-ESO-1-positive tumors were treated with autologous TCR-transduced T cells plus 720,000 iU/kg of interleukin-2 to tolerance after preparative chemotherapy. Objective clinical responses were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS Objective clinical responses were observed in four of six patients with synovial cell sarcoma and five of 11 patients with melanoma bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted after 1 year. A partial response lasting 18 months was observed in one patient with synovial cell sarcoma. CONCLUSION These observations indicate that TCR-based gene therapies directed against NY-ESO-1 represent a new and effective therapeutic approach for patients with melanoma and synovial cell sarcoma. To our knowledge, this represents the first demonstration of the successful treatment of a nonmelanoma tumor using TCR-transduced T cells.

Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism

Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)-II-treated C2C12 mouse myoblasts and IGF-II-overexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycin-induced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.

The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.

Metastatic cancers, once established, are the primary cause of mortality associated with cancer. Previously, we used a genomic approach to identify metastasis-associated genes in cancer. From this genomic data, we selected ezrin for further study based on its role in physically and functionally connecting the actin cytoskeleton to the cell membrane. In a mouse model of osteosarcoma, a highly metastatic pediatric cancer, we found ezrin to be necessary for metastasis. By imaging metastatic cells in the lungs of mice, we showed that ezrin expression provided an early survival advantage for cancer cells that reached the lung. AKT and MAPK phosphorylation and activity were reduced when ezrin protein was suppressed. Ezrin-mediated early metastatic survival was partially dependent on activation of MAPK, but not AKT. To define the relevance of ezrin in the biology of metastasis, beyond the founding mouse model, we examined ezrin expression in dogs that naturally developed osteosarcoma. High ezrin expression in dog tumors was associated with early development of metastases. Consistent with this data, we found a significant association between high ezrin expression and poor outcome in pediatric osteosarcoma patients.

Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators

Patients presenting with metastatic rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children, have a very poor clinical prognosis. This is due, in large part, to our rudimentary knowledge of the molecular events that dictate metastatic potential. We used cDNA microarray analysis of RMS cell lines, derived from Ink4a/Arf-deficient mice transgenic for hepatocyte growth factor/scatter factor (HGF/SF), to identify a set of genes whose expression was significantly different between highly and poorly metastatic cells. Subsequent in vivo functional studies revealed that the actin filament–plasma membrane linker ezrin (encoded by Vil2) and the homeodomain-containing transcription factor Six-1 (sine oculis–related homeobox-1 homolog) had essential roles in determining the metastatic fate of RMS cells. VIL2 and SIX1 expression was enhanced in human RMS tissue, significantly correlating with clinical stage. The identification of ezrin and Six-1 as critical regulators of metastasis in RMS provides new mechanistic and therapeutic insights into this pediatric cancer.

Defects in succinate dehydrogenase in gastrointestinal stromal tumors lacking KIT and PDGFRA mutations

Carney-Stratakis syndrome, an inherited condition predisposing affected individuals to gastrointestinal stromal tumor (GIST) and paraganglioma, is caused by germline mutations in succinate dehydrogenase (SDH) subunits B, C, or D, leading to dysfunction of complex II of the electron transport chain. We evaluated the role of defective cellular respiration in sporadic GIST lacking mutations in KIT or PDGFRA (WT). Thirty-four patients with WT GIST without a personal or family history of paraganglioma were tested for SDH germline mutations. WT GISTs lacking demonstrable SDH genetic inactivation were evaluated for SDHB expression by immunohistochemistry and Western blotting and for complex II activity. For comparison, SDHB expression was also determined in KIT mutant and neurofibromatosis-1–associated GIST, and complex II activity was also measured in SDH-deficient paraganglioma and KIT mutant GIST; 4 of 34 patients (12%) with WT GIST without a personal or family history of paraganglioma had germline mutations in SDHB or SDHC. WT GISTs lacking somatic mutations or deletions in SDH subunits had either complete loss of or substantial reduction in SDHB protein expression, whereas most KIT mutant GISTs had strong SDHB expression. Complex II activity was substantially decreased in WT GISTs. WT GISTs, particularly those in younger patients, have defects in SDH mitochondrial complex II, and in a subset of these patients, GIST seems to arise from germline-inactivating SDH mutations. Testing for germline mutations in SDH is recommended in patients with WT GIST. These findings highlight a potential central role of SDH dysregulation in WT GIST oncogenesis.

Mechanisms of sarcoma development.

Sarcomas are a rare and diverse group of tumours that are derived from connective tissues, including bone, muscle and cartilage. Although there are instances of hereditary predisposition to sarcomas, the overwhelming majority of such tumours are sporadic. In the past decade, we have gained much insight into the genetic abnormalities that seem to underlie the pathogenesis of these tumours. This information has already led to new classification of many sarcomas, as well as to successful therapies that are targeted at specific genetic abnormalities. It is likely that this approach will lead to continued refinements in classification and treatment of these tumours.

Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T (Treg) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking Treg cells develop severe autoimmune disease, and depletion of Treg cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of Treg cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2–responsive Treg cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain Treg cells, the question arises as to the effects of IL-2 therapy on them. We monitored Treg cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4+CD25hi cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the Treg cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent Treg cells in normal hosts, and IL-2–induced Treg cell expansion was further augmented by lymphopenia. On a per-cell basis, Treg cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to Treg cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4+CD25+ Treg cell homeostasis.

Insulin-like Growth Factors and Cancer

Dr. Derek LeRoith (Molecular and Cellular Physiology Section, National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK], National Institutes of Health [NIH], Bethesda, Maryland): The insulin-like growth factor (IGF) system comprises a collection of ligands, receptors, and binding proteins Table 1 [1]. Insulin, IGF-I, and IGF-II are polypeptides that affect many tissues and result in diverse biological actions. The major role of insulin is controlling metabolic homeostasis. In contrast, IGF-I and IGF-II are vital for normal growth and development during fetal, neonatal, and pubertal stages [2]. In addition, IGFs have specialized functions in differentiated tissues, including the reproductive, cardiovascular, and neurologic systems. The biological functions of the IGFs are initiated by their interactions with cell-surface receptors, in particular the IGF-I receptor. When activated, this receptor initiates a cascade of events that begins with the activation of tyrosine kinase and results in divergent effects depending on specific cell types [3]. Table 1. The Insulin-like Growth Factor System* Circulating IGFs are synthesized primarily in the liver and serve an endocrine function, whereas locally produced IGFs act in an autocrine-paracrine mode. Both forms are bound by a family of binding proteins, six of which have been well characterized Table 1 [4]. Insulin-like growth factor-binding proteins are responsible for protecting IGFs in the circulation, prolonging their half-lives, and delivering them to their specific target tissues. At the local level, IGF-binding proteins may regulate the interaction of IGFs with their receptors by either inhibiting or augmenting the interaction. In addition, IGF-binding proteins may have some actions that are independent of interactions between IGF and IGF receptors. As could be predicted from the importance of IGFs, their binding proteins, and their receptors in normal cellular growth and development, it has become apparent over the past few years that IGFs are important mitogens in many types of malignancies [5]. Although these conclusions were initially derived from in vitro studies, IGFs may enhance in vivo tumor cell formation, growth, and even metastasis. Insulin-like growth factors may reach tumors either from the circulation (endocrine) or as a result of local production by the tumor itself (autocrine) or by adjacent stromal tissue (paracrine). Tumors also express many of the IGF-binding proteins, which modulate IGF action, and IGF receptors, which mediate the effects of IGFs on tumors. We highlight important aspects of IGFs in normal cell growth and their role in certain malignancies. The syndrome of hypoglycemia with non-islet cell tumors, although not covered in this review, deserves special mention because it was one of the earliest links of IGFs to tumors and was derived from studies done in the mid-1970s at the Diabetes Branch of the NIDDK. The clinical syndrome was described shortly after insulinomas were first described in the late 1920s. The advent of the radioimmunoassay for insulin in the early 1960s showed that insulinomas release insulin but that the nonislet tumors that produce hypoglycemia usually lack insulin, a finding that triggered intense speculation about the mechanism of hypoglycemia. In the mid-1970s, the NIH group devised a novel radioreceptor assay for IGF-II. Using this assay, they showed that in patients with this syndrome, IGF-II-like material is often present in elevated amounts in the circulation and in the tumors [6]. Of further interest is that the major clinical culprit may be a higher-molecular-weight precursor form of IGF-II; this IGF-II-like material probably binds to insulin receptors, activates them, and thereby produces hypoglycemia [7, 8]. The Role of the Insulin-like Growth Factor I Receptor in Cell Growth and Transformation Dr. Renato Baserga (Jefferson Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania): Mammalian cell growth in vitro and in vivo is regulated by factors that interact with specific cell-surface receptors. Most normal cells require at least two factors for optimal growth. Insulin-like growth factor I is often one of them and is required for the growth of such cells as fibroblasts, epithelial cells, bone marrow stem cells, and osteoblasts [9]. The other required growth factor varies depending on the cell type, but in fibroblasts, platelet-derived growth factor and epidermal growth factor act with IGF-I to stimulate cell proliferation. In culture, neither of these factors alone can sustain cell growth. The recent finding that mice in which the IGF-I and IGF-I receptor genes had been inactivated grow to only 30% of the size of normal littermates underscores the role of the IGF-I receptor in murine development [10, 11]. Further support for the role of the IGF-I receptor in growth and tumorigenesis has come from studies showing the transforming potential of transfected cells overexpressing the IGF-I receptor [12] and abrogation of this effect by specific mutations of the receptor [13]. On the basis of research using fibroblast cell lines derived from IGF-I receptor-deficient mice [R-cells], it has been possible to show that 1) IGF-I receptors are essential for the growth of cells in serum-free media supplemented with factors that support the growth of normal mouse cells [W cells that are fibroblasts derived from normal mice or 3T3 cells]; 2) IGF-I receptors are not necessary for growth in media containing 10% serum but are required for optimal growth; 3) IGF-I receptors are also required for platelet-derived growth factor-stimulated or epidermal growth factor-stimulated growth and transformation; and 4) IGF-I receptors stimulate both ras-dependent and ras-independent signaling pathways [14]. It has been shown that SV40 T antigen increases IGF-I expression, leading to transformation of BALB/c 3T3 cells [15]. The obligate role of the IGF-I receptor in T-antigen-mediated transformation was confirmed by the inability of T antigen to transform the R-cells described above. Further studies have shown that R- cells are also refractory to transformation by v-src and by bovine papilloma virus, both of which efficiently transform cells expressing IGF-I receptors; thus, some oncogenic viruses require IGF-I receptors to transform mouse embryo fibroblasts. To ascertain the role of IGF-I receptors in the growth and transformation of other cell types, C6 rat glioblastoma cells were rendered IGF-I receptor-deficient by expressing an antisense RNA that prevented efficient expression of the endogenous IGF-I receptor gene. These cells were then evaluated for their ability to form tumors when transplanted into syngeneic rats in comparison with wild-type C6 cells. In all 50 rats injected with wild-type C6 cells, the C6 cells grew well and formed large tumors. Insulin-like growth factor I receptor-deficient C6 cells did not grow in the 27 animals into which they were injected, and no tumors were formed [16]. These results suggest that IGF-I receptors are extremely important in establishing and maintaining the transformed phenotype and that they may represent a suitable target for the inhibition of cell proliferation in vivo. It is currently accepted that a major signal transduction pathway triggered by the IGF-I receptor and by other receptor tyrosine kinases such as the receptors for insulin, epidermal growth factor, and platelet-derived growth factor involves activation of ras, the protein kinase Raf-1, and the mitogen-activated protein kinase cascade [17]. It is interesting that overexpression of an activated ras or Raf-1 could not confer in IGF-I receptor-deficient R- cells the ability to grow in soft agar or in a serum-free medium supplemented with purified growth factors [14, 15, 18]. Thus, the IGF-I receptor must also use a ras (and Raf-1)-independent pathway to stimulate cell proliferation and transformation. Insulin-like Growth Factors and Breast Cancer Derek LeRoith (Section on Molecular and Cellular Physiology, Diabetes Branch, NIDDK, NIH): Breast cancer is a common malignancy that affects almost 1 in every 7 women and is the leading cause of death from cancer in women in North America. During normal development, estrogen is primarily involved in promoting the development of breast ducts, whereas progesterone promotes lobuloalveolar development. Many cancers, especially those developing in the postmenopausal period, express estrogen and progesterone receptors. The presence of these receptors and the likelihood that these cancers will respond to endocrine therapy are strongly correlated. Initial therapy for breast cancer is primarily surgical, but once metastatic disease has developed, endocrine therapy is appropriate. In premenopausal patients, lowering hormone levels by removing the ovaries is useful, whereas in postmenopausal patients, antiestrogens such as tamoxifen have proved useful [19]. In addition to classic hormones, several growth factors, including transforming growth factors, epidermal growth factors, and IGFs, have been shown to be involved in breast cancer. The cellular proto-oncogene products such as c-myc, c-fos, and c-jun are also involved Table 2 [20]. Table 2. Growth Factors and Oncogenes Involved in Breast Cancer Breast cancer cells in vivo express low levels of IGF-II [21], whereas the adjacent stromal tissue expresses IGF-I [22]. In addition, most breast cancer cells express insulin and IGF receptors [23]. Different cancers express different combinations of the IGF-binding proteins: In vitro, estrogen receptor-positive cancer cells synthesize IGF-binding proteins 2, 4, and 5, and estrogen receptor-negative cancer cells synthesize IGF-binding proteins 1, 3, 4, and 5 [24]. Examination of biopsy specimens of breast cancer cells has confirmed this specific pattern of IGF-binding protein expression [25]. Because it has been shown that the proliferation of breast cancer cell lines i

Phase 0 Clinical Trial of the Poly (ADP-Ribose) Polymerase Inhibitor ABT-888 in Patients With Advanced Malignancies

PURPOSE We conducted the first phase 0 clinical trial in oncology of a therapeutic agent under the Exploratory Investigational New Drug Guidance of the US Food and Drug Administration. It was a first-in-human study of the poly (ADP-ribose) polymerase (PARP) inhibitor ABT-888 in patients with advanced malignancies. PATIENTS AND METHODS ABT-888 was administered as a single oral dose of 10, 25, or 50 mg to determine the dose range and time course over which ABT-888 inhibits PARP activity in tumor samples and peripheral blood mononuclear cells, and to evaluate ABT-888 pharmacokinetics. Blood samples and tumor biopsies were obtained pre- and postdrug administration for evaluation of PARP activity and pharmacokinetics. A novel statistical approach was developed and utilized to study pharmacodynamic modulation as the primary end point for trials of limited sample size. RESULTS Thirteen patients with advanced malignancies received the study drug; nine patients underwent paired tumor biopsies. ABT-888 demonstrated good oral bioavailability and was well tolerated. Statistically significant inhibition of poly (ADP-ribose) levels was observed in tumor biopsies and peripheral blood mononuclear cells at the 25-mg and 50-mg dose levels. CONCLUSION Within 5 months of study activation, we obtained pivotal biochemical and pharmacokinetic data that have guided the design of subsequent phase I trials of ABT-888 in combination with DNA-damaging agents. In addition to accelerating the development of ABT-888, the rapid conclusion of this trial demonstrates the feasibility of conducting proof-of-principle phase 0 trials as part of an alternative paradigm for early drug development in oncology.

Rhabdomyosarcoma – working out the pathways

Rhabdomyosarcomas constitute a collection of childhood malignancies thought to arise as a consequence of regulatory disruption of skeletal muscle progenitor cell growth and differentiation. Our understanding of the pathogenesis of this neoplasm has recently benefited from the study of normal and malignant myogenic cells in vitro, facilitating the identification of diagnostic cytogenetic markers and the elucidation of mechanisms by which myogenesis is regulated. It is now appreciated that the delicate balance between proliferation and differentiation, mutually exclusive yet intimately associated processes, is normally controlled in large part through the action of a multitude of growth factors, whose signals are interpreted by members of the MyoD family of helix – loop – helix proteins, and key regulatory cell cycle factors. The latter have proven to be frequent targets of mutational events that subvert myogenesis and promote the development of rhabdomyosarcoma. Although significant progress has been made in the treatment of rhabdomyosarcoma, patients presenting with metastatic disease or certain high risk features are still faced with a dismal prognosis. Only now are genetically engineered mouse models becoming available that are certain to provide fresh insights into the molecular/genetic pathways by which rhabdomyosarcomas arise and progress, and to suggest novel avenues of therapeutic opportunity.