Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl.

The Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50 <30 nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants. Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107 prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolonged survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and Ph+ ALL.

Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins.

Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM.

Expression-based screening identifies the combination of histone deacetylase inhibitors and retinoids for neuroblastoma differentiation.

The discovery of new small molecules and their testing in rational combination poses an ongoing problem for rare diseases, in particular, for pediatric cancers such as neuroblastoma. Despite maximal cytotoxic therapy with double autologous stem cell transplantation, outcome remains poor for children with high-stage disease. Because differentiation is aberrant in this malignancy, compounds that modulate transcription, such as histone deacetylase (HDAC) inhibitors, are of particular interest. However, as single agents, HDAC inhibitors have had limited efficacy. In the present study, we use an HDAC inhibitor as an enhancer to screen a small-molecule library for compounds inducing neuroblastoma maturation. To quantify differentiation, we use an enabling gene expression-based screening strategy. The top hit identified in the screen was all-trans-retinoic acid. Secondary assays confirmed greater neuroblastoma differentiation with the combination of an HDAC inhibitor and a retinoid versus either alone. Furthermore, effects of combination therapy were synergistic with respect to inhibition of cellular viability and induction of apoptosis. In a xenograft model of neuroblastoma, animals treated with combination therapy had the longest survival. This work suggests that testing of an HDAC inhibitor and retinoid in combination is warranted for children with neuroblastoma and demonstrates the success of a signature-based screening approach to prioritize compound combinations for testing in rare diseases.

Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells.

An attractive target for therapeutic intervention is constitutively activated, mutant FLT3, which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing, and which is currently in late-stage clinical trials. However, the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel, structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here, we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487, which selectively targets mutant FLT3 protein kinase activity, is also shown to override PKC412 resistance in vitro, and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally, the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus, we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target, and which could potentially be used to override drug resistance in AML.

Stromal-mediated protection of tyrosine kinase inhibitor-treated BCR-ABL-expressing leukemia cells.

Clinical studies of patients with chronic myeloid leukemia revealed that a common pattern of response is a dramatic fall in the circulating population of blast cells, with a minimal or delayed decrease in marrow blasts, suggesting a protective environment. These observations suggest that a greater understanding of the interaction of stromal cells with leukemic cells is essential. Here, we present an in vivo system for monitoring relative tumor accumulation in leukemic mice and residual disease in leukemic mice treated with a tyrosine kinase inhibitor and an in vitro system for identifying integral factors involved in stromal-mediated cytoprotection. Using the in vivo model, we observed high tumor burden/residual disease in tissues characterized as significant sources of hematopoiesis-promoting stroma, with bone marrow stroma most frequently showing the highest accumulation of leukemia in untreated and nilotinib-treated mice as well as partial protection of leukemic cells from the inhibitory effects of nilotinib. These studies, which showed a pattern of leukemia distribution consistent with what is observed in imatinib- and nilotinib-treated chronic myeloid leukemia patients, were followed by a more in-depth analysis of stroma-leukemia cell interactions that lead to protection of leukemia cells from nilotinib-induced cytotoxicity. For the latter, we used the human BCR-ABL-positive cell line, KU812F, and the human bone marrow stroma cell line, HS-5, to more closely approximate the bone marrow-associated cytoprotection observed in drug-treated leukemia patients. This in vitro system helped to elucidate stromal-secreted viability factors that may play a role in stromal-mediated cytoprotection of tyrosine kinase inhibitor-treated leukemia cells.

Potentiation of antileukemic therapies by Smac mimetic, LBW242: effects on mutant FLT3-expressing cells.

Members of the inhibitor of apoptosis protein (IAP) family play a role in mediating apoptosis. Studies suggest that these proteins may be a viable target in leukemia because they have been found to be variably expressed in acute leukemias and are associated with chemosensitivity, chemoresistance, disease progression, remission, and patient survival. Another promising therapeutic target, FLT3, is mutated in about one third of acute myelogenous leukemia (AML) patients; promising results have recently been achieved in clinical trials investigating the effects of the protein tyrosine kinase inhibitor PKC412 on AML patients harboring mutations in the FLT3 protein. Of growing concern, however, is the development of drug resistance resulting from the emergence of point mutations in targeted tyrosine kinases used for treatment of acute leukemia patients. One approach to overriding resistance is to combine structurally unrelated inhibitors and/or inhibitors of different signaling pathways. The proapoptotic IAP inhibitor, LBW242, was shown in proliferation studies done in vitro to enhance the killing of PKC412-sensitive and PKC412-resistant cell lines expressing mutant FLT3 when combined with either PKC412 or standard cytotoxic agents (doxorubicin and Ara-c). In addition, in an in vivo imaging assay using bioluminescence as a measure of tumor burden, a total of 12 male NCr-nude mice were treated for 10 days with p.o. administration of vehicle, LBW242 (50 mg/kg/day), PKC412 (40 mg/kg/day), or a combination of LBW242 and PKC412; the lowest tumor burden was observed in the drug combination group. Finally, the combination of LBW242 and PKC412 was sufficient to override stromal-mediated viability signaling conferring resistance to PKC412.

Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma.

BACKGROUND: The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression-based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application. METHODS AND FINDINGS: A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted. CONCLUSIONS: We demonstrate that a gene expression-based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers.

Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis.

The disease state of cancer appears late in tumor development. Before being diagnosed, a tumor can remain for prolonged periods of time in a dormant state. Dormant human cancer is commonly defined as a microscopic tumor that does not expand in size and remains asymptomatic. Dormant tumors represent an early stage in tumor development and may therefore be a potential target for nontoxic, antiangiogenic therapy that could prevent tumor recurrence. Here, we characterize an experimental model that recapitulates the clinical dormancy of human tumors in mice. We demonstrate that these microscopic dormant cancers switch to the angiogenic phenotype at a predictable time. We further show that while angiogenic liposarcomas expand rapidly after inoculation of tumor cells in mice, nonangiogenic dormant liposarcomas remain microscopic up to one-third of the normal severe combined immune deficiency (SCID) mouse life span, although they contain proliferating tumor cells. Nonangiogenic dormant tumors follow a similar growth pattern in subcutaneous (s.c.) and orthotopic environments. Throughout the dormancy period, development of intratumoral vessels is impaired. In nonangogenic dormant tumors, small clusters of endothelial cells without lumens are observed early after tumor cell inoculation, but the nonangiogenic tumor cannot sustain these vessels, and they disappear within weeks. There is a concomitant decrease in microvessel density, and the nonangiogenic dormant tumor remains harmless to the host. In contrast, microvessel density in tumors increases rapidly after the angiogenic switch and correlates with rapid expansion of tumor mass. Both tumor types cultured in vitro contain fully transformed cells, but only cells from the nonangiogenic human liposarcoma secrete relatively high levels of the angiogenesis inhibitors thrombospondin-1 and TIMP-1. This model suggests that as improved blood or urine molecular biomarkers are developed, the microscopic, nonangiogenic, dormant phase of human cancer may be vulnerable to antiangiogenic therapy years before symptoms, or before anatomical location of a tumor can be detected, by conventional methods.

Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL- and mutant FLT3-expressing cells.

Mediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhibitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL-, and induce apoptosis of BCR-ABL-expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL- and mutant FLT3-expressing cells both in vitro and in vivo.

Beneficial effects of combining nilotinib and imatinib in preclinical models of BCR-ABL+ leukemias.

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor, nilotinib (AMN107), is significantly more potent against BCR-ABL than imatinib, and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib, indicating that sequential therapy with these 2 agents has clinical value. However, simultaneous, rather than sequential, administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons, including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here, we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity, including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further, using a highly quantifiable bioluminescent in vivo model, drug combinations were at least additive in antileukemic activity, compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase, the combination of imatinib and nilotinib is highly efficacious in these models, indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted.

A novel noninvasive model of endometriosis for monitoring the efficacy of antiangiogenic therapy.

Endometriosis, the presence of ectopic endometrial tissue, is a common disease associated with high morbidity and socioeconomic problems. Angiogenesis, the formation of new blood vessels, plays an important role in the formation and growth of endometriotic lesions. We have created a novel, noninvasive model to monitor the growth of these lesions and the associated angiogenesis in vivo. First, we generated luciferase-expressing transgenic mice by inserting the human ubiquitin C promoter coupled to the firefly luciferase reporter. Injection of luciferin in these mice causes full-body bioluminescence, which can be detected using a low-light CCD camera. Endometrial tissue from these transgenic mice was surgically implanted into nonluminescent recipients. Bioluminescence of lesions was noninvasively imaged after intravenous or intraperitoneal injection of luciferin. Transabdominal luminescence compared well with the location of the transgenic endometriotic lesions, and lesion size correlated with the intensity of luminescence. Systemic treatment with the angiogenesis inhibitors caplostatin and endostatin peptide mP-1 delayed and suppressed the onset and intensity of the luminescent signal. Caplostatin suppressed the growth of endometriotic lesions by 59% compared with controls. This novel, noninvasive model of endometriosis provides a means to study early angiogenesis in vivo and to monitor endometriotic growth and the efficacy of systemic antiangiogenic therapy.

Effects of PKC412, nilotinib, and imatinib against GIST-associated PDGFRA mutants with differential imatinib sensitivity.

BACKGROUND & AIMS: Activating mutations in platelet-derived growth factor receptor alpha (PDGFRA) have been reported in a subset of gastrointestinal stromal tumor (GIST) patients who do not express the mutant stem cell factor receptor c-kit. The responsiveness of mutant PDGFRA-positive GIST to imatinib depends on the location of the PDGFRA mutation; for example, the V561D juxtamembrane domain mutation is more sensitive to imatinib than the D842V kinase domain mutation. In this study, we compare the effects of 3 tyrosine kinase inhibitors, PKC412 and nilotinib, and imatinib, on 2 GIST-related PDGFRA mutants, V561D and D842V, which possess differential sensitivity to imatinib. METHODS: The effects of PKC412, nilotinib, and imatinib, alone and in combination, were evaluated via in vitro proliferation studies performed with V561D- or D842V-PDGFRA mutants. The effects of nilotinib and PKC412, alone and combined, were investigated in vivo. RESULTS: PKC412 potently inhibited the V561D-PDGFRA mutant in vitro and the D842V-PDGFRA mutant in vitro and in vivo. Both imatinib and nilotinib displayed potent activity in vitro against the V561D-PDGFRA mutant but were significantly less efficacious against D842V-PDGFRA. However, when combined with either imatinib or PKC412, nilotinib showed no evidence for antagonism and acted in a cooperative fashion against D842V-PDGFRA. CONCLUSIONS: Our findings support the clinical testing of PKC412 for treatment of mutant PDGFRA-GIST. The data also support the use of nilotinib as a treatment option for V561D-PDGFRA-associated GIST, although the reduced sensitivity of D842V-PDGFRA probably limits the potential of nilotinib monotherapy for D842V-PDGFRA-associated GIST.

Tuberculous epididymitis with extensive retroperitoneal and mediastinal involvement.

The incidence of tuberculosis in the United States is on the rise, in part, because of its association with acquired immunodeficiency syndrome. Genitourinary tuberculosis remains one of the most common forms of secondary or extrapulmonary disease. We present an unusual case of tuberculous epididymitis with extensive retroperitoneal and mediastinal spread. The possible routes of dissemination, as well as the efficacy of antimycobacterial therapy in the management of tuberculous epididymitis, are discussed and the relevant literature is reviewed.

Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix.

BCL-2 family proteins constitute a critical control point for the regulation of apoptosis. Protein interaction between BCL-2 members is a prominent mechanism of control and is mediated through the amphipathic alpha-helical BH3 segment, an essential death domain. We used a chemical strategy, termed hydrocarbon stapling, to generate BH3 peptides with improved pharmacologic properties. The stapled peptides, called "stabilized alpha-helix of BCL-2 domains" (SAHBs), proved to be helical, protease-resistant, and cell-permeable molecules that bound with increased affinity to multidomain BCL-2 member pockets. A SAHB of the BH3 domain from the BID protein specifically activated the apoptotic pathway to kill leukemia cells. In addition, SAHB effectively inhibited the growth of human leukemia xenografts in vivo. Hydrocarbon stapling of native peptides may provide a useful strategy for experimental and therapeutic modulation of protein-protein interactions in many signaling pathways.