ABSTRACT
Targeting critical epigenetic regulators reverses aberrant transcription in cancer, thereby restoring normal tissue function1-3. The interaction of menin with lysine methyltransferase 2A (KMT2A), an epigenetic regulator, is a dependence in acute leukaemia caused by either rearrangement of KMT2A or mutation of the nucleophosmin 1 gene (NPM1)4-6. KMT2A rearrangements occur in up to 10% of acute leukaemias and have an adverse prognosis, whereas NPM1 mutations occur in up to 30%, forming the most common genetic alteration in acute myeloid leukaemia7,8. Here, we describe the results of the first-in-human phase 1 clinical trial investigating revumenib (SNDX-5613), a potent and selective oral inhibitor of the menin-KMT2A interaction, in patients with relapsed or refractory acute leukaemia (ClinicalTrials.gov, NCT04065399). We show that therapy with revumenib was associated with a low frequency of grade 3 or higher treatment-related adverse events and a 30% rate of complete remission or complete remission with partial haematologic recovery (CR/CRh) in the efficacy analysis population. Asymptomatic prolongation of the QT interval on electrocardiography was identified as the only dose-limiting toxicity. Remissions occurred in leukaemias refractory to multiple previous lines of therapy. We demonstrate clearance of residual disease using sensitive clinical assays and identify hallmarks of differentiation into normal haematopoietic cells, including differentiation syndrome. These data establish menin inhibition as a therapeutic strategy for susceptible acute leukaemia subtypes.
Subject(s)
Antineoplastic Agents , Histone-Lysine N-Methyltransferase , Leukemia, Myeloid, Acute , Nucleophosmin , Proto-Oncogene Proteins , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Neoplasm, Residual/drug therapy , Nucleophosmin/genetics , Prognosis , Protein Binding/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Remission InductionABSTRACT
BACKGROUND: Elacestrant is an oral selective estrogen receptor (ER) degrader. This phase 1b open-label, non-randomized study (RAD1901-106) was initiated to determine the effect of elacestrant on the availability of ER in lesions from postmenopausal women with ER+ advanced breast cancer (ABC) using 16α-18F-fluoro-17ß-estradiol positron emission tomography with low-dose computed tomography (FES-PET/CT). METHODS: Eligible patients were postmenopausal women with ER+, HER2- ABC; tumor progression after ≥ 6 months of 1-3 lines of endocrine treatment for ABC; and measurable or evaluable disease. Two 8-patient cohorts were enrolled: one treated with 400 mg elacestrant once daily (QD) and one treated with 200 mg elacestrant QD with dose escalation to 400 mg QD after 14 days. Elacestrant was dosed continuously until progressive disease, toxicity, or withdrawal. FES-PET/CT was performed pre-dose at baseline and 4 h post-dose on day 14. The primary endpoint was the percentage difference in FES uptake in tumor lesions (maximum 20) after 14 days of treatment compared to baseline. Overall response was investigator-assessed by Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1. RESULTS: Patients (n = 16; median age, 53.5 years) had ABC with a median 2.5 prior lines of endocrine therapy. Median reduction in tumor FES uptake from baseline to day 14 was 89.1% (Q1, Q3: 75.1%, 94.1%) and was similar in both cohorts (89.1% [Q1, Q3: 67.4%, 94.2%], 200/400 mg and 88.7% [Q1, Q3: 79.5%, 94.1%], 400 mg). Residual ER availability (> 25% persistence in FES uptake) on day 14 was observed in 3 patients receiving 200/400 mg (3/78, 37.5%) and 1 patient receiving 400 mg (1/8, 12.5%). The overall response rate (ORR) was 11.1% (1 partial response), and clinical benefit rate (CBR) was 30.8%. Median percentage change in FES uptake did not correlate with ORR or CBR. Adverse events occurring in > 20% of the patients were nausea (68.8%), fatigue (50.0%), dyspepsia (43.8%), vomiting (37.5%), and decreased appetite, dysphagia, and hot flush (31.3% each). Most events were grade 2 in severity. CONCLUSION: Elacestrant 200 mg and 400 mg QD greatly reduced ER availability measured by FES-PET/CT. In a heavily pretreated population, elacestrant was associated with antitumor activity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02650817 . Registered on 08 January 2016.
Subject(s)
Breast Neoplasms/pathology , Estradiol/metabolism , Positron Emission Tomography Computed Tomography/methods , Receptors, Estrogen/metabolism , Tetrahydronaphthalenes/therapeutic use , Adult , Aged , Aged, 80 and over , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Female , Fluorine Radioisotopes/metabolism , Humans , Middle Aged , Neoplasm Metastasis , Patient Safety , Radiopharmaceuticals/metabolism , Selective Estrogen Receptor Modulators/pharmacokinetics , Selective Estrogen Receptor Modulators/therapeutic use , Tetrahydronaphthalenes/pharmacokinetics , Tissue Distribution , Treatment OutcomeABSTRACT
BACKGROUND: Glembatumumab vedotin is an antibody-drug conjugate that produced preliminary clinical activity against advanced melanoma in a phase 1 dose-escalation trial. The objective of the current study was to investigate further the antitumor activity of glembatumumab vedotin at the recommended phase 2 dose in heavily pretreated patients with melanoma. METHODS: This single-arm, phase 2 study enrolled patients with stage IV melanoma who were refractory to checkpoint inhibition and to B-raf proto-oncogene, serine/threonine kinase (BRAF)/mitogen-activated protein kinase kinase (MEK) inhibition (in the presence of a BRAF valine mutation at codon 600). Patients received 1.9 mg/kg glembatumumab vedotin intravenously every 3 weeks until they developed disease progression or intolerance. The primary endpoint was objective response rate (ORR), which was determined according to Response Evaluation Criteria in Solid Tumors, version 1.1. Secondary endpoints included progression-free survival (PFS), duration of response, overall survival (OS), safety, and clinical efficacy versus tumor glycoprotein NMB (gpNMB) expression. Tumor expression of gpNMB was assessed using immunohistochemistry. RESULTS: In total, 62 patients received treatment. The ORR was 11% and the median response duration was 6.0 months (95% confidence interval [CI], 4.1 months to not reached). The median PFS was 4.4 months (95% CI, 2.6-5.5 months), and the median OS was 9.0 months (95% CI, 6.1-11.7 months). For patients who developed rash during the first cycle versus those who did not, the ORR was 21% versus 7%, respectively, and there was an overall improvement in PFS (hazard ratio, 0.43; P = .013) and OS (hazard ratio, 0.43; P = .017). The most frequent adverse events were alopecia, neuropathy, rash, fatigue, and neutropenia. With one exception, all evaluable tumors were positive for gpNMB, and 46 of 59 tumors (76%) had 100% gpNMB-positive epithelial cells. CONCLUSIONS: Glembatumumab vedotin had modest activity and an acceptable safety profile in patients with advanced melanoma who were refractory to checkpoint inhibitors and MEK/BRAF inhibition. Treatment-related rash may be associated with response.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Immunoconjugates/therapeutic use , Melanoma/drug therapy , Skin Neoplasms/drug therapy , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Melanoma/metabolism , Melanoma/pathology , Membrane Glycoproteins/metabolism , Middle Aged , Neoplasm Staging , Progression-Free Survival , Proportional Hazards Models , Proto-Oncogene Mas , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Treatment OutcomeABSTRACT
JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post-PV myelofibrosis (PPMF) and rapid post-essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2(V617F) mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2-independent. Interestingly, we originally found a decreased in the JAK2(V617F) allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.
Subject(s)
Janus Kinase 2/antagonists & inhibitors , Polycythemia Vera/drug therapy , Primary Myelofibrosis/drug therapy , Protein Kinase Inhibitors/administration & dosage , Pyrrolidines/administration & dosage , Sulfonamides/administration & dosage , Thrombocythemia, Essential/drug therapy , Animals , Disease Progression , Mice , Platelet Count , Polycythemia Vera/blood , Polycythemia Vera/physiopathology , Primary Myelofibrosis/blood , Primary Myelofibrosis/physiopathology , Splenomegaly/drug therapy , Thrombocythemia, Essential/blood , Thrombocythemia, Essential/physiopathologyABSTRACT
PURPOSE: Revumenib, an oral, small molecule inhibitor of the menin-lysine methyltransferase 2A (KMT2A) interaction, showed promising efficacy and safety in a phase I study of heavily pretreated patients with KMT2A-rearranged (KMT2Ar) acute leukemia. Here, we evaluated the activity of revumenib in individuals with relapsed/refractory (R/R) KMT2Ar acute leukemia. METHODS: AUGMENT-101 is a phase I/II, open-label, dose-escalation and expansion study of revumenib conducted across 22 clinical sites in five countries (ClinicalTrials.gov identifier: NCT04065399). We report results from the phase II, registration-enabling portion. Individuals age ≥30 days with R/R KMT2Ar acute leukemia or with AML and nucleophosmin 1 (NPM1) mutation were enrolled. Revumenib was administered once every 12 hours, at 163 mg (95 mg/m2 if weight <40 kg) with a strong cytochrome P450 inhibitor, in 28-day cycles. The primary end points were the rate of complete remission (CR) or CR with partial hematologic recovery (CR + CRh) and safety. At a prespecified interim analysis, safety was assessed in all KMT2Ar treated patients; efficacy was assessed in those with centrally confirmed KMT2Ar. The separate NPM1 cohort of the trial is ongoing. RESULTS: From October 1, 2021, to July 24, 2023, N = 94 patients (median [range] age, 37 [1.3-75] years) were treated. Grade ≥3 adverse events included febrile neutropenia (37.2%), differentiation syndrome (16.0%), and QTc prolongation (13.8%). In the efficacy-evaluable patients (n = 57), the CR + CRh rate was 22.8% (95% CI, 12.7 to 35.8), exceeding the null hypothesis of 10% (P = .0036). Overall response rate was 63.2% (95% CI, 49.3 to 75.6), with 15 of 22 patients (68.2%) having no detectable residual disease. CONCLUSION: Revumenib led to high remission rates with a predictable safety profile in R/R KMT2Ar acute leukemia. To our knowledge, this trial represents the largest evaluation of a targeted therapy for these patients.
ABSTRACT
INTRODUCTION/BACKGROUND: This first-in-human, phase 1 study aimed to characterize the safety, tolerability, maximum tolerated dose (MTD), pharmacokinetic (PK) profile, and antitumor activity of RAD140, an oral selective androgen receptor (AR) modulator (SARM). PATIENTS AND METHODS: This dose-escalation study with a 3 + 3 design and PK expansion cohort enrolled postmenopausal women with ER+/HER2- metastatic breast cancer (mBC). Serum sex hormone-binding globulin (SHBG) and prostate-specific antigen (PSA) were used as surrogate markers of AR engagement. RESULTS: Twenty-two (21 AR+) heavily pretreated mBC patients were enrolled. Dose levels included 50 mg (n = 6), 100 mg (n = 13), and 150 mg (n = 3) once daily (QD). Most frequent (> 10%) treatment-emergent adverse events (TEAEs) were elevated AST (59.1%), ALT (45.5%), and total blood bilirubin (27.3%), and vomiting, dehydration, and decreased appetite and weight (27.3% each). Grade 3/4 TEAEs occurred in 16 (72.7%) patients and included elevations in AST/ALT and hypophosphatemia (22.7% each). Treatment-related TEAEs occurred in 17 per 22 patients (77.3%); 7 (31.8%) were Grade 3; none were Grade 4. The half-life (t1/2) of 44.7 hours supported QD dosing. At the MTD of 100 mg/day, 1 patient with an ESR1 mutation at baseline had a partial response. Overall, clinical benefit rate at 24 weeks was 18.2%, and median progression-free survival was 2.3 months. SHBG decreased in 18 per 18 patients, and PSA increased in 16 per 20 patients. Paired baseline and on-treatment tumor biopsies demonstrated AR engagement. CONCLUSION: RAD140 is a novel oral AR-targeted agent for the treatment of AR+/ER+/HER2- mBC with an acceptable safety profile and preliminary evidence of target engagement and antitumor activity.
Subject(s)
Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Nitriles/therapeutic use , Oxadiazoles/therapeutic use , Administration, Oral , Aged , Breast Neoplasms/pathology , Disease-Free Survival , Dose-Response Relationship, Drug , ErbB Receptors , Female , Humans , Maximum Tolerated Dose , Middle Aged , Neoplasm MetastasisABSTRACT
Antiangiogenesis has been validated as a therapeutic strategy to treat cancer, however, a need remains to identify new targets and therapies for specific diseases and to improve clinical benefit from antiangiogenic agents. Tumor endothelial marker 7 (TEM-7) was investigated as a possible target for therapeutic antiangiogenic intervention in cancer. TEM-7 expression was assessed by in situ hybridization or by immunohistochemistry (IHC) in 130 formalin-fixed paraffin-embedded (FFPE) and 410 frozen human clinical specimens of cancer plus 301 normal tissue samples. In vitro TEM-7 expression was evaluated in 4 human endothelial cell models and in 32 human cancer cell lines by RT-PCR and flow cytometry. An anti-TEM-7 antibody was tested in vitro on human SKOV3 ovarian and MDA-MB-231 breast carcinoma cells that expressed TEM-7 in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. In frozen tumor tissues, TEM-7 mRNA and protein was detected in all but one of the cancer types tested and was infrequently expressed in normal frozen tissues. In FFPE tumor tissues, TEM-7 protein was detected by IHC in colon, breast, lung, bladder, ovarian and endometrial cancers and in sarcomas. TEM-7 protein was not detected in head and neck, prostate or liver cancers. TEM-7 expression was restricted to the vasculature and was absent from tumor cells. In vitro, TEM-7 was not detected in human microvascular endothelial cells (HMVEC) or human umbilical vein endothelial cells (HUVEC) but was induced in endothelial precursor/progenitor cells (EPC) in the presence of the mitogen phorbol ester PMA. An anti-TEM-7 antibody mediated ADCC and phagocytosis in SKOV3 and MDA-MB-231 cell lines infected with an adenovirus expressing TEM-7. These data demonstrate that TEM-7 is a vascular protein associated with angiogenic states. TEM-7 is a novel and attractive target for antiangiogenic therapy.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies/pharmacology , Endothelial Cells/drug effects , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Neovascularization, Pathologic/prevention & control , Receptors, Cell Surface/antagonists & inhibitors , Animals , Antibody-Dependent Cell Cytotoxicity , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Flow Cytometry , Formaldehyde , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , In Situ Hybridization , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/blood supply , Neoplasms/genetics , Neoplasms/metabolism , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Paraffin Embedding , Phagocytosis , Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Tissue Fixation , TransfectionABSTRACT
PURPOSE: This phase I study (RAD1901-005; NCT02338349) evaluated elacestrant, an investigational oral selective estrogen receptor degrader (SERD), in heavily pretreated women with estrogen receptor-positive, human epidermal growth factor receptor 2-negative metastatic breast cancer, including those with estrogen receptor gene alpha (ESR1) mutation. The primary objective was to determine the maximum tolerated dose and/or recommended phase II dose (RP2D). METHODS: The study consisted of a 3 + 3 design (elacestrant capsules) followed by expansion at RP2D (400-mg capsules, then 400-mg tablets) for the evaluation of safety and antitumor activity. Elacestrant was taken once daily until progression or intolerability. RESULTS: Of 57 postmenopausal women enrolled, 50 received RP2D (400 mg once daily): median age, 63 years; median three prior anticancer therapies, including cyclin-dependent kinase 4,6 inhibitors (CDK4/6i; 52%), SERD (52%), and ESR1 mutation (circulating tumor DNA; 50%). No dose-limiting toxicities occurred; the most common adverse events at RP2D (400-mg tablet; n = 24) were nausea (33.3%) and increased blood triglycerides and decreased blood phosphorus (25.0% each). Most adverse events were grade 1-2 in severity. The objective response rate was 19.4% (n = 31 evaluable patients receiving RP2D), 15.0% in patients with prior SERD, 16.7% in patients with prior CDK4/6i, and 33.3% in patients with ESR1 mutation (n = 5/15). The clinical benefit rate (24-week) was 42.6% overall (n = 47 patients receiving RP2D), 56.5% (n = 23, ESR1 mutation), and 30.4% (n = 23, prior CDK4/6i). Elacestrant clinical benefit was associated with decline in ESR1 mutant allele fraction. CONCLUSION: Elacestrant 400 mg orally once daily has an acceptable safety profile and demonstrated single-agent activity with confirmed partial responses in heavily pretreated patients with estrogen receptor-positive metastatic breast cancer. Notably, responses were observed in patients with ESR1 mutation as well as those with prior CDK4/6i and prior SERD. A phase III trial investigating elacestrant versus standard endocrine therapy is ongoing.
Subject(s)
Breast Neoplasms/drug therapy , Receptor, ErbB-2/analysis , Receptors, Estrogen/analysis , Tetrahydronaphthalenes/therapeutic use , Adult , Aged , Aged, 80 and over , Breast Neoplasms/chemistry , Breast Neoplasms/genetics , Estrogen Receptor alpha/genetics , Female , Humans , Middle Aged , Mutation , Tetrahydronaphthalenes/adverse effects , Tetrahydronaphthalenes/pharmacokineticsABSTRACT
The METRIC study (NCT#0199733) explored a novel antibody-drug conjugate, glembatumumab vedotin (GV), targeting gpNMB that is overexpressed in ~40% of patients with triple-negative breast cancer (TNBC) and associated with poor prognosis. The study was a randomized, open-label, phase 2b study that evaluated progression-free survival (PFS) of GV compared with capecitabine in gpNMB-overexpressing TNBC. Patients who had previously received anthracycline and taxane-based therapy were randomized 2:1 to receive, GV (1.88 mg/kg IV q21 days) or capecitabine (2500 mg/m2 PO daily d1-14 q21 days). The primary endpoint was RECIST 1.1 PFS per independent, blinded central review. In all, 327 patients were randomized to GV (213 treated) or capecitabine (92 treated). Median PFS was 2.9 months for GV vs. 2.8 months for capecitabine. The most common grade ≥3 toxicities for GV were neutropenia, rash, and leukopenia, and for capecitabine were fatigue, diarrhea, and palmar-plantar erythrodysesthesia. The study did not meet the primary endpoint of improved PFS over capecitabine or demonstrate a relative risk/benefit improvement over capecitabine.
ABSTRACT
Tumor development is a complex and dynamic process that involves malignant, vascular, and stromal cells. Endosialin is a tumor endothelial marker (TEM) present in the microvasculature and stroma of human tumors. Cancer-associated fibroblasts (CAF) have been implicated in promoting tumor development and have been associated with mesenchymal stem cells (MSC). Since stem/progenitor cells recruited either from bone marrow or residing in nearby tissues can contribute to pathological processes we investigated endosialin in MSC using a novel monoclonal antibody. Endosialin is highly expressed by CAF and human bone marrow-derived MSC. MSC can form networks in a tube formation assay that is inhibited by an anti-endosialin antibody. Immunohistochemistry for human endosialin in xenograft tumors following co-injection of MSC and cancer cells identified MSC in tumor stroma. MSC are a potential target for anticancer therapeutic intervention and endosialin expression offers a new tool for the identification of MSC. Endosialin expression by both CAF and MSC further implies the potential contribution of MSC to tumor stroma via differentiation into tumor stromal fibroblasts.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Neoplasm/biosynthesis , Biomarkers, Tumor/biosynthesis , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Endothelium, Vascular/metabolism , Neoplasms/metabolism , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Differentiation/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Mice , Mice, SCID , Neoplasms/blood supply , Neoplasms/genetics , Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/cytology , Stromal Cells/metabolism , Stromal Cells/pathologyABSTRACT
Nucleoside analogs are rationally designed anticancer agents that disrupt DNA and RNA synthesis. Fludarabine and cladribine have important roles in the treatment of hematologic malignancies. Clofarabine is a next generation nucleoside analog which is under clinical investigation. The bone marrow toxicity, tumor cell cytotoxicity and human tumor xenograft activity of fludarabine, cladribine and clofarabine were compared. Mouse and human bone marrow were subjected to colony forming (CFU-GM) assays over a 5-log concentration range in culture. NCI-60 cell line screening data were compared. In vivo, a range of clofarabine doses was compared with fludarabine for efficacy in several human tumor xenografts. The IC90 concentrations for fludarabine and cladribine for mouse CFU-GM were >30 and 0.93 microM, and for human CFU-GM were 8 and 0.11 microM, giving mouse to human differentials of >3.8- and 8.5-fold. Clofarabine produced IC90s of 1.7 microM in mouse and 0.51 microM in human CFU-GM, thus a 3.3-fold differential between species. In the NCI-60 cell line screen, fludarabine and cladribine showed selective cytotoxicity toward leukemia cell lines while for clofarabine there was no apparent selectivity based upon origin of the tumor cells. In vivo, clofarabine produced a dose-dependent increase in tumor growth delay in the RL lymphoma, the RPMI-8226 multiple myeloma, and HT-29 colon carcinoma models. The PC3 prostate carcinoma was equally responsive to clofarabine and fludarabine. Bringing together bone marrow toxicity data, tumor cell line cytotoxicity data, and human tumor xenograft efficacy provides valuable information for the translation of preclinical findings to the clinic.
Subject(s)
Adenine Nucleotides/therapeutic use , Arabinonucleosides/therapeutic use , Cladribine/therapeutic use , Granulocyte-Macrophage Progenitor Cells/drug effects , Neoplasms/drug therapy , Neoplasms/pathology , Vidarabine Phosphate/analogs & derivatives , Adenine Nucleotides/antagonists & inhibitors , Adenine Nucleotides/pharmacology , Animals , Antimetabolites, Antineoplastic/pharmacology , Antimetabolites, Antineoplastic/therapeutic use , Arabinonucleosides/pharmacology , Bone Marrow Cells/drug effects , Bone Marrow Cells/physiology , Cells, Cultured , Cladribine/pharmacology , Clofarabine , Granulocyte-Macrophage Progenitor Cells/physiology , HT29 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Models, Biological , Treatment Outcome , Vidarabine Phosphate/pharmacology , Vidarabine Phosphate/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Endosialin/CD248/tumor endothelial marker 1 is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. EXPERIMENTAL DESIGN: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. RESULTS: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1 neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. CONCLUSIONS: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Neoplasm/biosynthesis , Carcinoma/metabolism , Immunotoxins/pharmacology , Neoplasms/metabolism , Sarcoma/metabolism , Animals , Antigens, CD/genetics , Antigens, Neoplasm/genetics , Carcinoma/genetics , Cell Line, Tumor , Flow Cytometry , Humans , Immunoglobulin G/pharmacology , Immunohistochemistry , Neoplasms/genetics , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Ribosome Inactivating Proteins, Type 1/toxicity , Saporins , Sarcoma/geneticsABSTRACT
Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.
Subject(s)
Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Endothelium, Vascular/metabolism , Antigens, CD/immunology , Antigens, Neoplasm/immunology , Base Sequence , Cells, Cultured , DNA Primers , Endothelium, Vascular/cytology , Flow Cytometry , Humans , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC(90) values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC(90) values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity.
Subject(s)
Bone Marrow/pathology , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Neoplastic Stem Cells/pathology , Topoisomerase I Inhibitors , Topotecan/pharmacology , Xenograft Model Antitumor Assays , Animals , Antineoplastic Agents/pharmacology , Body Weight/drug effects , Bone Marrow/drug effects , Camptothecin/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Confidence Intervals , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Inhibitory Concentration 50 , Irinotecan , Male , Mice , Mice, Inbred BALB C , Naphthyridines/chemistry , Naphthyridines/pharmacology , Neoplastic Stem Cells/drug effects , Topotecan/chemistry , Weight Loss/drug effectsABSTRACT
The formation of functional, mature blood vessels depends on the interaction between endothelial cells and pericytes. Commonality exists in the processes involved in vasculature development between tissues whether healthy or diseased. Endosialin/TEM 1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumorigenesis but not in normal mature vessels. Antibodies developed to human endosialin were used to investigate endosialin expression and function in human prenatal brain pericytes and pericytes residing in tumors. Anti-endosialin was capable of preventing pericyte tube formation in culture and inhibited migration. Brain pericytes in culture had higher levels of endosialin/TEM 1 than TEMs-2, -3, -4, -5, -7, and -8. Immunocytochemistry revealed that endosialin was present in the cytoplasmic body and in the elongated extensions essential to pericyte function. Transgenic mice engineered to express human endosialin bred on an immunocompromised background allowed the growth of human tumor xenografts. In human colon carcinoma Colo205 and HT29 xenografts grown in human endosialin-transgenic mice, endosialin expression was largely confined to NG2-expressing perivascular cells and not CD31-positive endothelial cells. Similar methods applied to human ovarian and colon tumors confirmed endosialin expression by pericytes. The data indicate that endosialin is strongly expressed by pericytes during periods of active angiogenesis during embryonic and tumor development. Anti-endosialin antibodies may have value in identifying vasculature in malignant tissues. With the appropriate agent, targeting endosialin may interfere with blood vessel growth during tumor development.
Subject(s)
Antigens, CD/metabolism , Antigens, Neoplasm/metabolism , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/metabolism , Neovascularization, Pathologic , Neovascularization, Physiologic , Pericytes/cytology , Pericytes/metabolism , Animals , Antigens, CD/genetics , Antigens, Neoplasm/genetics , Base Sequence , Cell Line, Tumor , Cells, Cultured , DNA Primers/genetics , Embryonic Development , Female , Gene Expression , Humans , Male , Mice , Mice, Inbred C57BL , Mice, SCID , Mice, Transgenic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transplantation, HeterologousABSTRACT
Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including platelet-derived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function-associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cell-based models for tumor vascular development and selection of agents that may provide therapeutic benefit.
Subject(s)
Breast Neoplasms/pathology , Endothelial Cells/cytology , Pericytes/cytology , Stem Cells/cytology , Animals , Breast Neoplasms/blood supply , Cell Communication , Cell Line, Tumor , Coculture Techniques , Endothelial Cells/metabolism , Female , Growth Substances/biosynthesis , Humans , Mice , Mice, SCID , Neoplasm Transplantation , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Pericytes/metabolism , Stem Cells/metabolism , Transplantation, HeterologousABSTRACT
Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor-stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy.
Subject(s)
Gene Expression Regulation, Neoplastic , Genes, Neoplasm/genetics , Neoplasms/enzymology , Salivary Proteins and Peptides/genetics , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Gene Expression/drug effects , Humans , Neoplasms/genetics , Oligonucleotide Array Sequence Analysis , RNA, Messenger/analysis , RNA, Messenger/metabolism , Salivary Proteins and Peptides/analysis , Tetradecanoylphorbol Acetate/pharmacology , Up-RegulationABSTRACT
The diabetogenic major histocompatibility complex (MHC) (H2(g7)) of NOD mice comprises contributions from several class II loci collectively designated as Idd1. Introduction of the H2(gx) haplotype from the related but diabetes-resistant cataract Shionogi (CTS) strain demonstrated an additional MHC-linked locus designated Idd16. The NOD-related alloxan resistant (ALR)/Lt strain is also characterized by the H2(gx) haplotype, which does not differ from H2(g7) from the class I H2-K(d) gene distally through the class II and into the class III region. Polymorphisms distal to the heat shock protein 70 locus (Hspa1b) include a rare H2-D(dx) rather than the H2(g7) encoded D(b) allele. Two differential-length NOD.ALR-H2(gx) congenic stocks (D.R1 and D.R2), both containing H2-D(dx), significantly suppressed diabetogenesis. This protection was lost when ALR alleles between the class III region and H2-D were removed in a shorter interval congenic (D.R3). Because no differences were observed in the ALR-derived interval extending 0.41 mB proximal to H2-K in any of these congenic stocks, a component of what was originally designated "Idd16" was sited to an interval shorter than 7.33 mB, distinguishing D.R2 from D.R3. Evidence supporting the candidacy of the ALR/CTS-shared H2-D(dx) MHC class I variant present in both diabetes-resistant stocks, but not the susceptible stock, is discussed.
Subject(s)
Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Genetic Predisposition to Disease , Major Histocompatibility Complex , Aging , Animals , Biomarkers , Diabetes Mellitus, Type 1/epidemiology , Female , Incidence , Male , Mice , Mice, Inbred NOD , Sex CharacteristicsABSTRACT
Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) have been the standards for cell-based assays in the field of angiogenesis research and in antiangiogenic drug discovery. These normal mature endothelial cells may not be most representative of human tumor endothelial cells. Human AC133+/CD34+ bone marrow progenitor cells were established in cell culture media containing vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and heparin to drive differentiation toward the endothelial phenotype. The resulting cells designated endothelial precursor cells (EPC) have many of the same functional properties as mature endothelial cells represented by HUVEC and HMVEC. By SAGE analysis, the genes expressed by EPC are more similar to the genes expressed by endothelial cells isolated from fresh surgical specimens of human tumors than are the genes expressed by HUVEC and HMVEC. Analysis of several cell surface markers by flow cytometry showed that EPC, HUVEC, and HMVEC have similar expression of P1H12, vascular endothelial growth factor 2, and endoglin but that EPC have much lower expression of ICAM1, ICAM2, VCAM1, and thrombomodulin than do HUVEC and HMVEC. The EPC generated can form tubes/networks on Matrigel, migrate through porous membranes, and invade through thin layers of Matrigel similarly to HUVEC and HMVEC. However, in a coculture assay using human SKOV3 ovarian cancer cell clusters in collagen as a stimulus for invasion through Matrigel, EPC were able to invade into the malignant cell cluster, whereas HMVEC were not able to invade the malignant cell cluster. In vivo, a Matrigel plug assay where human EPC were suspended in the Matrigel allowed tube/network formation by human EPC to be carried out in a murine host. EPC may be a better model of human tumor endothelial cells than HUVEC and HMVEC and, thus, may provide an improved cell-based model for second generation antineoplastic antiangiogenic drug discovery.
Subject(s)
Endothelium, Vascular/cytology , Neoplasms/blood supply , Neovascularization, Pathologic/pathology , Stem Cells/cytology , Antigens, CD34/biosynthesis , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Endothelium, Vascular/metabolism , Humans , Neoplasms/pathology , Neovascularization, Pathologic/metabolism , Stem Cells/metabolism , Vascular Endothelial Growth Factor Receptor-2/biosynthesisABSTRACT
Outbred CD-1 mice carry a spectrum of genetic susceptibilities for obesity and type 2 diabetes. ALS is an inbred strain with low antioxidant defenses produced by inbreeding CD-1 mice, with selection for susceptibility to alloxan, a generator of highly reactive oxygen free radicals and a potent beta-cell toxin. The objective of this study was to determine if the low ability to diffuse free radical stress would contribute to spontaneous type 2 diabetes development in alloxan-untreated males. Indeed, both hyperinsulinemia and impaired glucose tolerance developed spontaneously between 6 and 8 weeks of age in alloxan-untreated males. Further aging was accompanied by increases in body mass, progressively more severe hyperinsulinemia, and development of overt hyperglycemia. Transition from impaired glucose tolerance to overt hyperglycemia correlated with a decreased ratio of reduced to oxidized glutathione. Evidence that the increased oxidative burden elicited the type 2 diabetes syndrome was obtained by the systemic elevation of the antioxidative capacity through daily administration of R-lipoic acid. R-lipoic acid (30 mg/kg) prevented hyperglycemia, reduced insulin levels, and increased free radical scavenging potential. This mouse model with reduced ability to diffuse free radical stress is of obvious interest because free radical-mediated damage is implicated in the pathogenesis and complications of both type 1 and type 2 diabetes.