ABSTRACT
The membrane protein carbonic anhydrase IX (CAIX) is highly expressed in many hypoxic or von Hippel-Lindau tumor suppressor-mutated tumor types. Its restricted expression in healthy tissues makes CAIX an attractive diagnostic and therapeutic target. DPI-4452 is a CAIX-targeting cyclic peptide with a DOTA cage, allowing radionuclide chelation for theranostic purposes. Here, we report CAIX expression in multiple tumor types and provide in vitro and in vivo evaluations of 68Ga-labeled DPI-4452 ([68Ga]Ga-DPI-4452) and 177Lu-labeled DPI-4452 ([177Lu]Lu-DPI-4452). Methods: CAIX expression was assessed by immunohistochemistry with a panel of tumor and healthy tissues. The molecular interactions of complexed and uncomplexed DPI-4452 with CAIX were assessed by surface plasmon resonance and cell-binding assays. In vivo characterization of radiolabeled and nonradiolabeled DPI-4452 was performed in HT-29 colorectal cancer (CRC) and SK-RC-52 clear cell renal cell carcinoma (ccRCC) human xenograft mouse models and in healthy beagle dogs. Results: Overexpression of CAIX was shown in several tumor types, including ccRCC, CRC, and pancreatic ductal adenocarcinoma. DPI-4452 specifically and selectively bound CAIX with subnanomolar affinity. In cell-binding assays, DPI-4452 displayed comparably high affinities for human and canine CAIX but a much lower affinity for murine CAIX, demonstrating that the dog is a relevant species for biodistribution studies. DPI-4452 was rapidly eliminated from the systemic circulation of beagle dogs. The highest uptake of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 was observed in the small intestine and stomach, 2 organs known to express CAIX. Uptake in other organs (e.g., kidneys) was remarkably low. In HT-29 and SK-RC-52 xenograft mouse models, both [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 showed tumor-selective uptake; in addition, [177Lu]Lu-DPI-4452 significantly reduced tumor growth. These results demonstrated the theranostic potential of DPI-4452. Conclusion: DPI-4452 selectively targets CAIX. [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 localized to tumors and were well tolerated in mice. [177Lu]Lu-DPI-4452 demonstrated strong tumor growth inhibition in 2 xenograft mouse models. Thus, the 2 agents potentially provide a theranostic approach for selecting and treating patients with CAIX-expressing tumors such as ccRCC, CRC, and pancreatic ductal adenocarcinoma.
Subject(s)
Carbonic Anhydrase IX , Gallium Radioisotopes , Lutetium , Radioisotopes , Carbonic Anhydrase IX/metabolism , Humans , Animals , Mice , Radioisotopes/therapeutic use , Cell Line, Tumor , Tissue Distribution , Ligands , Antigens, Neoplasm/metabolism , Theranostic Nanomedicine , Precision Medicine , Female , DogsABSTRACT
Acute myelogenous leukemia (AML) is initiated and maintained by leukemia stem cells (LSC). LSCs are therapy-resistant, cause relapse, and represent a major obstacle for the cure of AML. Resistance to therapy is often mediated by aberrant tyrosine kinase (TK) activation. These TKs primarily activate downstream signaling via STAT3/STAT5. In this study, we analyzed the potential to therapeutically target aberrant TK signaling and to eliminate LSCs via the multi-TK inhibitor Debio 0617B. Debio 0617B has a unique profile targeting key kinases upstream of STAT3/STAT5 signaling such as JAK, SRC, ABL, and class III/V receptor TKs. We demonstrate that expression of phospho-STAT3 (pSTAT3) in AML blasts is an independent prognostic factor for overall survival. Furthermore, phospho-STAT5 (pSTAT5) signaling is increased in primary CD34+ AML stem/progenitors. STAT3/STAT5 activation depends on tyrosine phosphorylation, mediated by several upstream TKs. Inhibition of single upstream TKs did not eliminate LSCs. In contrast, the multi-TK inhibitor Debio 0617B reduced maintenance and self-renewal of primary human AML CD34+ stem/progenitor cells in vitro and in xenotransplantation experiments resulting in long-term elimination of human LSCs and leukemia. Therefore, inhibition of multiple TKs upstream of STAT3/5 may result in sustained therapeutic efficacy of targeted therapy in AML and prevent relapses. Mol Cancer Ther; 16(8); 1497-510. ©2017 AACR.
Subject(s)
Antigens, CD34/metabolism , Cell Self Renewal/drug effects , Isoxazoles/pharmacology , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Picolinic Acids/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Animals , Apoptosis/drug effects , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Progression , Female , Humans , Mice, Inbred NOD , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Phosphorylation/drug effects , Prognosis , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , Survival Analysis , Tumor Stem Cell Assay , Xenograft Model Antitumor AssaysABSTRACT
Tumor survival, metastases, chemoresistance, and escape from immune responses have been associated with inappropriate activation of STAT3 and/or STAT5 in various cancers, including solid tumors. Debio 0617B has been developed as a first-in-class kinase inhibitor with a unique profile targeting phospho-STAT3 (pSTAT3) and/or pSTAT5 in tumors through combined inhibition of JAK, SRC, ABL, and class III/V receptor tyrosine kinases (RTK). Debio 0617B showed dose-dependent inhibition of pSTAT3 in STAT3-activated carcinoma cell lines; Debio 0617B also showed potent antiproliferative activity in a panel of cancer cell lines and in patient-derived tumor xenografts tested in an in vitro clonogenic assay. Debio 0617B showed in vivo efficacy by inhibiting tumor growth in several mouse xenograft models. To increase in vivo efficacy and STAT3 inhibition, Debio 0617B was tested in combination with the EGFR inhibitor erlotinib in a non-small cell lung cancer xenograft model. To evaluate the impact of in vivo STAT3 blockade on metastases, Debio 0617B was tested in an orthotopic tumor model. Measurement of primary tumor weight and metastatic counts in lung tissue demonstrated therapeutic efficacy of Debio 0617B in this model. These data show potent activity of Debio 0617B on a broad spectrum of STAT3-driven solid tumors and synergistic activity in combination with EGFR inhibition. Mol Cancer Ther; 15(10); 2334-43. ©2016 AACR.
Subject(s)
Antineoplastic Agents/pharmacology , Janus Kinases/antagonists & inhibitors , Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , STAT3 Transcription Factor/metabolism , src-Family Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Line, Tumor , Disease Models, Animal , Drug Design , Humans , Janus Kinases/chemistry , Mice , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Neoplasm Metastasis , Neoplasms/drug therapy , Neoplasms/pathology , Protein Kinase Inhibitors/chemistry , Receptor Protein-Tyrosine Kinases/chemistry , Signal Transduction/drug effects , Tumor Burden/drug effects , Xenograft Model Antitumor Assays , src-Family Kinases/chemistryABSTRACT
TCRαß thymocytes differentiate into either CD8αß(+) cytotoxic T lymphocytes or CD4(+) helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4(+) T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4(+) T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4(+) cytotoxic T lymphocytes.
Subject(s)
T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Helper-Inducer/immunology , Transcription Factors/genetics , Transcription Factors/metabolism , Animals , Cell Differentiation , Cell Lineage , Citrobacter rodentium/immunology , Histocompatibility Antigens Class II/immunology , Homeodomain Proteins/genetics , Interleukin-7/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/metabolism , Thymocytes/metabolismABSTRACT
APO866 inhibits nicotinamide phosphoribosyltransferase (NMPRTase), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Intracellular NAD is essential for cell survival, and NAD depletion resulting from APO866 treatment elicits tumor cell death. Here, we determine the in vitro and in vivo sensitivities of hematologic cancer cells to APO866 using a panel of cell lines (n = 45) and primary cells (n = 32). Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma), but not normal hematopoietic progenitor cells, were sensitive to low concentrations of APO866 as measured in cytotoxicity and clonogenic assays. Treatment with APO866 decreased intracellular NAD and adenosine triphosphate (ATP) at 24 hours and 48 to72 hours, respectively. The NAD depletion led to cell death. At 96 hours, APO866-mediated cell death occurred in a caspase-independent mode, and was associated with mitochondrial dysfunction and autophagy. Further, in vivo administration of APO866 as a single agent prevented and abrogated tumor growth in animal models of human AML, lymphoblastic lymphoma, and leukemia without significant toxicity to the animals. The results support the potential of APO866 for treating hematologic malignancies.
Subject(s)
Acrylamides/therapeutic use , Antineoplastic Agents/therapeutic use , Cytokines/antagonists & inhibitors , Hematologic Neoplasms/drug therapy , NAD/biosynthesis , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Piperidines/therapeutic use , Acrylamides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Dose-Response Relationship, Drug , HL-60 Cells , Hematologic Neoplasms/metabolism , Humans , Jurkat Cells , K562 Cells , Mice , Mice, Inbred BALB C , Mice, Nude , NAD/drug effects , Piperidines/pharmacology , Tumor Cells, Cultured , U937 Cells , Xenograft Model Antitumor AssaysABSTRACT
The immune system preserves and makes use of autoreactive lymphocytes with specialized functions. Here we showed that one of these populations, CD8alphaalpha(+)TCRalphabeta(+) intestinal intraepithelial lymphocytes (IELs), arose from a unique subset of double-positive thymocytes. This subset of cells was precommitted to preferentially give rise to CD8alphaalpha(+)TCRalphabeta(+) IELs, but they required exposure to self-agonist peptides. The agonist-selected TCRalphabeta(+) thymocytes are CD4 and CD8 double-negative, and their final maturation, including the induction of CD8alphaalpha expression, appeared to occur only after thymus export in the IL-15-rich environment of the gut. These developmental steps, including precommitment of immature thymocytes, TCR-mediated agonist selection, and postthymic differentiation promoted by cytokines, define a unique pathway for the generation of CD8alphaalpha(+)TCRalphabeta(+) IEL.
Subject(s)
Lymphocyte Activation , Receptors, Antigen, T-Cell, alpha-beta/analysis , T-Lymphocyte Subsets/immunology , Thymus Gland/immunology , Animals , CD4 Antigens/analysis , CD8 Antigens/analysis , Cell Differentiation , Cytokines/metabolism , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Mice , Mice, Inbred Strains , Phenotype , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/drug effects , Thymus Gland/cytologyABSTRACT
We developed and tested a potent hexameric Fas agonist, termed MegaFasL, for its cytotoxic effects on a panel of human haematopoietic malignant cells and healthy human haematopoietic progenitor cells (CD34+CD38low). Results demonstrated that MegaFasL induced apoptosis in cell lines and primary cells representing multiple myeloma (MM), acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) and Burkitt's lymphoma. Cells from a chronic myeloid leukaemia (CML) line and from patients with chronic lymphocytic leukaemia (CLL) were resistant. Furthermore, CD34+CD38low progenitor cells were also resistant to MegaFasL. The data indicate that MegaFasL could be a highly efficient therapeutic agent ex vivo or potentially in vivo.
Subject(s)
Apoptosis , Hematologic Neoplasms/pathology , fas Receptor/drug effects , Caspases/metabolism , Cell Line, Tumor , Enzyme Activation , Hematologic Neoplasms/enzymology , HumansABSTRACT
The mouse thymic leukemia (TL) Ag is a nonclassical MHC class I molecule that binds with higher affinity to CD8alphaalpha than CD8alphabeta. The interaction of CD8alphaalpha with TL is important for lymphocyte regulation in the intestine. Therefore, we studied the molecular basis for TL Ag binding to CD8alphaalpha. The stronger affinity of the TL Ag for CD8alphaalpha is largely mediated by three amino acids on exposed loops of the conserved alpha3 domain. Mutant classical class I molecules substituted with TL Ag amino acids at these positions mimic the ability to interact with CD8alphaalpha and modulate lymphocyte function. These data indicate that small changes in the alpha3 domain of class I molecules potentially can have profound physiologic consequences.
Subject(s)
CD8 Antigens/metabolism , Membrane Glycoproteins/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Base Sequence , Binding Sites/genetics , CD8 Antigens/chemistry , CD8 Antigens/genetics , Conserved Sequence , DNA/genetics , Humans , Hybridomas , In Vitro Techniques , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , T-Lymphocytes/immunology , TransfectionABSTRACT
In humans suffering from dialysis-related amyloidosis, the protein beta2-microglobulin (beta2M) is deposited as an amyloid; however, an amyloid of beta2M is unknown in mice. beta2M sequences from human and mouse are 70% identical, but there is a seven-residue peptide in which six residues differ. This peptide from human beta2M forms amyloid in vitro, whereas the mouse peptide does not. Substitution of the human peptide for its counterpart in the mouse sequence results in the formation of amyloid in vitro. These results show that a seven-residue segment of human beta2M is sufficient to convert beta2M to the amyloid state, and that specific residue interactions are crucial to the conversion. These observations are consistent with a proposed Zipper-spine model for beta2M amyloid, in which the spine of the fibril consists of an anhydrous beta-sheet.
Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Models, Molecular , Protein Conformation , beta 2-Microglobulin/chemistry , Amino Acid Sequence , Amyloid/genetics , Animals , Humans , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , X-Ray Diffraction , beta 2-Microglobulin/genetics , beta 2-Microglobulin/metabolismABSTRACT
Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric alpha chains of the CD8 molecule (CD8alphaalpha) are transiently induced on a selected subset of CD8alphabeta+ T cells upon antigenic stimulation. These CD8alphaalpha molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8alphaalpha.
Subject(s)
CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory , Lymphocyte Activation , Animals , Antigen-Presenting Cells/immunology , Arenaviridae Infections/immunology , Cell Differentiation , Cell Survival , Interferon-gamma/biosynthesis , Lymphocytic choriomeningitis virus/immunology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Receptors, Interleukin-7/immunology , Receptors, Interleukin-7/metabolism , T-Lymphocyte Subsets/immunologyABSTRACT
The nonclassical class I molecule, thymic leukemia (TL), has been shown to be expressed on intestinal epithelial cells and to interact with CD8(+) intraepithelial T lymphocytes. We generated recombinant soluble TL (T18(d)) H chains in bacteria as inclusion bodies and refolded them with beta(2)-microglobulin in the presence or absence of a random peptide library. Using a mAb, HD168, that recognizes a conformational epitope on native TL molecules, we observed that protein folds efficiently in the absence of peptide. Circular dichroism analysis demonstrated that TL molecules have structural features similar to classical class I molecules. Moreover, thermal denaturation experiments indicated that the melting temperature for peptide-free TL is similar to values reported previously for conventional class I-peptide complexes. Our results also show that CD8alphaalpha binding is not dependent on either TL-associated peptide or TL glycosylation.
Subject(s)
Antigens, Neoplasm/metabolism , CD8 Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Membrane Glycoproteins/metabolism , Peptides/metabolism , Protein Folding , Animals , Antigens, Neoplasm/chemistry , Biosensing Techniques , CD8 Antigens/chemistry , Circular Dichroism , HLA-A2 Antigen/metabolism , Histocompatibility Antigens Class I/chemistry , Humans , Membrane Glycoproteins/chemistry , Mice , Mice, Inbred C57BL , Peptide Library , Peptides/chemistry , Protein Binding/immunology , T-Lymphocytes/chemistry , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thermodynamics , beta 2-Microglobulin/metabolismABSTRACT
The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.