ABSTRACT
Around 10% of acute leukemias harbor a rearrangement of the MLL/KMT2A gene, and the presence of this translocation results in a highly aggressive, therapy-resistant leukemia subtype with survival rates below 50%. There is a high unmet need to identify safer and more potent therapies for MLL-rearranged (MLL-r) leukemia that can be combined with established chemotherapeutics to decrease treatment-related toxicities. The curaxin, CBL0137, has demonstrated nongenotoxic anticancer and chemopotentiating effects in a number of preclinical cancer models and is currently in adult Phase I clinical trials for solid tumors and hematological malignancies. The aim of our study was to investigate whether CBL0137 has potential as a therapeutic and chemopotentiating compound in MLL-r leukemia through a comprehensive analysis of its efficacy in preclinical models of the disease. CBL0137 decreased the viability of a panel of MLL-r leukemia cell lines (n = 12) and xenograft cells derived from patients with MLL-r acute lymphoblastic leukemia (ALL, n = 3) in vitro with submicromolar IC50s. The small molecule drug was well-tolerated in vivo and significantly reduced leukemia burden in a subcutaneous MV4;11 MLL-r acute myeloid leukemia model and in patient-derived xenograft models of MLL-r ALL (n = 5). The in vivo efficacy of standard of care drugs used in remission induction for pediatric ALL was also potentiated by CBL0137. CBL0137 exerted its anticancer effect by trapping Facilitator of Chromatin Transcription (FACT) into chromatin, activating the p53 pathway and inducing an Interferon response. Our findings support further preclinical evaluation of CBL0137 as a new approach for the treatment of MLL-r leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Carbazoles/pharmacology , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/genetics , Carbazoles/therapeutic use , Cell Line, Tumor , DNA-Binding Proteins/genetics , Disease Models, Animal , Gene Expression Profiling , High Mobility Group Proteins/genetics , Humans , Kaplan-Meier Estimate , Leukemia, Biphenotypic, Acute/diagnosis , Leukemia, Biphenotypic, Acute/drug therapy , Leukemia, Biphenotypic, Acute/genetics , Leukemia, Biphenotypic, Acute/mortality , Mice , Signal Transduction/drug effects , Transcriptional Elongation Factors/genetics , Transcriptome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.
Subject(s)
Crk-Associated Substrate Protein/chemistry , Crk-Associated Substrate Protein/metabolism , Focal Adhesions/metabolism , Mechanotransduction, Cellular , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Cell Line, Tumor , Cell Movement , Extracellular Matrix/metabolism , Focal Adhesions/drug effects , Gene Knockdown Techniques , Humans , Mechanotransduction, Cellular/drug effects , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Phosphorylation , Protein Domains , Protein Transport/drug effects , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Structure-Activity Relationship , Tetracycline/pharmacologyABSTRACT
Chimeric Antigen Receptor (CAR) T-cell therapy, as an approved treatment option for patients with B cell malignancies, demonstrates that genetic modification of autologous immune cells is an effective anti-cancer regimen. Erythropoietin-producing Hepatocellular receptor tyrosine kinase class A2 (EphA2) is a tumour associated antigen expressed on a range of sarcomas, including paediatric osteosarcoma (OS) and Ewing sarcoma (ES). We tested human EphA2 directed CAR T cells for their capacity to target and kill human OS and ES tumour cells using in vitro and in vivo assays, demonstrating that EphA2 CAR T cells have potent anti-tumour efficacy in vitro and can eliminate established OS and ES tumours in vivo in a dose and delivery route dependent manner. Next, in an aggressive metastatic OS model we demonstrated that systemically infused EphA2 CAR T cells can traffic to and eradicate tumour deposits in murine livers and lungs. These results support further pre-clinical evaluation of EphA2 CAR T cells to inform the design of early phase clinical trial protocols to test the feasibility and safety of this immune cell therapy in paediatric bone sarcoma patients.
Subject(s)
Bone Neoplasms/therapy , Immunotherapy, Adoptive/methods , Receptor, EphA2/immunology , T-Lymphocytes/immunology , Animals , Bone Neoplasms/genetics , Bone Neoplasms/immunology , Female , Humans , Mice , Mice, Inbred NOD , Molecular Targeted TherapyABSTRACT
Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt's lymphoma in vitro and in mouse xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further expanded the compound's therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species. These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Benzamides/chemistry , Benzamides/pharmacology , Cytokines/antagonists & inhibitors , Hematologic Neoplasms/drug therapy , NAD/metabolism , Niacin/pharmacology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Animals , Apoptosis , Cell Proliferation , Female , Hematologic Neoplasms/metabolism , Hematologic Neoplasms/pathology , High-Throughput Screening Assays , Humans , Male , Mice , Mice, Inbred C57BL , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The prognosis for children diagnosed with high-risk acute lymphoblastic leukemia (ALL) remains suboptimal, and more potent and less toxic treatments are urgently needed. We investigated the efficacy of a novel nicotinamide phosphoribosyltransferase inhibitor, OT-82, against a panel of patient-derived xenografts (PDXs) established from high-risk and poor outcome pediatric ALL cases. OT-82 was well-tolerated and demonstrated impressive single agent in vivo efficacy, achieving significant leukemia growth delay in 95% (20/21) and disease regression in 86% (18/21) of PDXs. In addition, OT-82 enhanced the efficacy of the established drugs cytarabine and dasatinib and, as a single agent, showed similar efficacy as an induction-type regimen combining three drugs used to treat pediatric ALL. OT-82 exerted its antileukemic action by depleting NAD+ and ATP, inhibiting the NAD+-requiring DNA damage repair enzyme PARP-1, increasing mitochondrial ROS levels and inducing DNA damage, culminating in apoptosis induction. OT-82 sensitivity was associated with the occurrence of mutations in major DNA damage response genes, while OT-82 resistance was characterized by high expression levels of CD38. In conclusion, our study provides evidence that OT-82, as a single agent, and in combination with established drugs, is a promising new therapeutic strategy for a broad spectrum of high-risk pediatric ALL for which improved therapies are urgently needed.
Subject(s)
Antineoplastic Agents/pharmacology , Cytokines/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Animals , Apoptosis/drug effects , Cell Survival/drug effects , Enzyme Inhibitors/pharmacology , Humans , Mice , Xenograft Model Antitumor AssaysABSTRACT
Survival rates for pediatric patients suffering from mixed lineage leukemia (MLL)-rearranged leukemia remain below 50% and more targeted, less toxic therapies are urgently needed. A screening method optimized to discover cytotoxic compounds selective for MLL-rearranged leukemia identified CCI-006 as a novel inhibitor of MLL-rearranged and CALM-AF10 translocated leukemias that share common leukemogenic pathways. CCI-006 inhibited mitochondrial respiration and induced mitochondrial membrane depolarization and apoptosis in a subset (7/11, 64%) of MLL-rearranged leukemia cell lines within a few hours of treatment. The unresponsive MLL-rearranged leukemia cells did not undergo mitochondrial membrane depolarization or apoptosis despite a similar attenuation of mitochondrial respiration by the compound. In comparison to the sensitive cells, the unresponsive MLL-rearranged leukemia cells were characterized by a more glycolytic metabolic phenotype, exemplified by a more pronounced sensitivity to glycolysis inhibitors and elevated HIF1α expression. Silencing of HIF1α expression sensitized an intrinsically unresponsive MLL-rearranged leukemia cell to CCI-006, indicating that this pathway plays a role in determining sensitivity to the compound. In addition, unresponsive MLL-rearranged leukemia cells expressed increased levels of MEIS1, an important leukemogenic MLL target gene that plays a role in regulating metabolic phenotype through HIF1α. MEIS1 expression was also variable in a pediatric MLL-rearranged ALL patient dataset, highlighting the existence of a previously undescribed metabolic variability in MLL-rearranged leukemia that may contribute to the heterogeneity of the disease. This study thus identified a novel small molecule that rapidly kills MLL-rearranged leukemia cells by targeting a metabolic vulnerability in a subset of low HIF1α/low MEIS1-expressing MLL-rearranged leukemia cells.
Subject(s)
Acrylates/pharmacology , Antineoplastic Agents/pharmacology , Furans/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Mitochondria/drug effects , Nitriles/pharmacology , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Leukemic/drug effects , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice, Inbred Strains , Mitochondria/physiology , Myeloid Ecotropic Viral Integration Site 1 Protein/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Unfolded Protein Response/drug effectsABSTRACT
Gene transfer targeting hematopoietic stem cells (HSC) in children has shown sustained therapeutic benefit in the treatment of genetic diseases affecting the immune system, most notably in severe combined immunodeficiencies affecting T-cell function. The HSC compartment has also been successfully targeted using gene transfer in children with genetic diseases affecting the central nervous system, such as metachromatic leukodystrophy and adrenoleukodystrophy. HSCs are also a target for genetic modification in strategies aiming to confer drug resistance to chemotherapy agents so as to reduce off-target toxicity, and to allow for chemotherapy dose escalation with the possibility of enhanced therapeutic benefit. In a trial of this strategy in adult glioma patients, significant engraftment of gene-modified HSCs expressing a mutant of the DNA repair protein O6-methyl-guanine-methyl-transferase (MGMT(P140K)) showed potential in conferring drug resistance against the combined effect of O6-benzylguanine (O6BG)/temozolomide (TMZ) chemotherapy. The aim was to test the safety and feasibility of this approach in children with poor prognosis brain tumors. In this Phase I trial, seven patients received gene-modified HSC following myelo-suppressive conditioning, but with only transient low-level engraftment of MGMT(P140K) gene-modified cells detectable in four patients. All patients received O6BG/TMZ chemotherapy following infusion of gene-modified cells, with five patients eligible for chemotherapy dose escalation, though in the absence of demonstrable transgene-mediated chemoprotection. Since all gene-modified cell products met the criteria for release and assays for engraftment potential met expected outcome measures, inadequate cell dose, conditioning chemotherapy, and/or underlying bone-marrow function may have contributed to the lack of sustained engraftment of gene-modified cells. We were able to demonstrate safe conduct of a technically complex Phase I study encompassing manufacture of the gene therapy vector, genetically modified cells, and a drug product specifically for the trial in compliance with both local and national regulatory requirements.
Subject(s)
Brain Neoplasms/drug therapy , DNA Modification Methylases/therapeutic use , DNA Repair Enzymes/therapeutic use , Genetic Therapy , Hematopoietic Stem Cell Transplantation , Tumor Suppressor Proteins/therapeutic use , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Child , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Drug Resistance, Neoplasm/genetics , Female , Guanine/administration & dosage , Guanine/analogs & derivatives , Hematopoietic Stem Cells/metabolism , Humans , Male , Temozolomide/administration & dosage , Tumor Suppressor Proteins/geneticsABSTRACT
There is an urgent need for the development of less toxic, more selective and targeted therapies for infants with leukemia characterized by translocation of the mixed lineage leukemia (MLL) gene. In this study, we performed a cell-based small molecule library screen on an infant MLL-rearranged (MLL-r) cell line, PER-485, in order to identify selective inhibitors for MLL-r leukemia. After screening initial hits for a cytotoxic effect against a panel of 30 cell lines including MLL-r and MLL wild-type (MLL-wt) leukemia, solid tumours and control cells, small molecule CCI-007 was identified as a compound that selectively and significantly decreased the viability of a subset of MLL-r and related leukemia cell lines with CALM-AF10 and SET-NUP214 translocation. CCI-007 induced a rapid caspase-dependent apoptosis with mitochondrial depolarization within twenty-four hours of treatment. CCI-007 altered the characteristic MLL-r gene expression signature in sensitive cells with downregulation of the expression of HOXA9, MEIS1, CMYC and BCL2, important drivers in MLL-r leukemia, within a few hours of treatment. MLL-r leukemia cells that were resistant to the compound were characterised by significantly higher baseline gene expression levels of MEIS1 and BCL2 in comparison to CCI-007 sensitive MLL-r leukemia cells.In conclusion, we have identified CCI-007 as a novel small molecule that displays rapid toxicity towards a subset of MLL-r, CALM-AF10 and SET-NUP214 leukemia cell lines. Our findings suggest an important new avenue in the development of targeted therapies for these deadly diseases and indicate that different therapeutic strategies might be needed for different subtypes of MLL-r leukemia.