ABSTRACT
Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.
Subject(s)
Drug Resistance, Neoplasm/genetics , Leukemia, Myeloid, Acute/drug therapy , Morpholines/administration & dosage , Niacinamide/analogs & derivatives , Phenylurea Compounds/administration & dosage , Triazines/administration & dosage , fms-Like Tyrosine Kinase 3/genetics , Animals , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mutation , Niacinamide/administration & dosage , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/administration & dosage , Signal Transduction/drug effects , Sorafenib , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Xenograft Model Antitumor AssaysABSTRACT
Spleen tyrosine kinase (SYK) was recently identified as a new target in acute myeloid leukemia (AML); however, its mechanistic role in this disease is poorly understood. Based on the known interaction between SYK and mammalian target of rapamycin (mTOR) signaling in lymphoma, we hypothesized that SYK may regulate mTOR signaling in AML. Both small-molecule inhibition of SYK and SYK-directed shRNA suppressed mTOR and its downstream signaling effectors, as well as its upstream activator, AKT. Moreover, the inhibition of multiple nodes of the phosphatidylinositol 3'-kinase (PI3K) signaling pathway enhanced the effects of SYK suppression on AML cell viability and differentiation. Evaluation of the collateral mitogen-activated protein kinase (MAPK) pathway revealed a heterogeneous response to SYK inhibition in AML with downregulation of MEK and extracellular signal-regulated kinase (ERK) phosphorylation in some AML cell lines but a paradoxical increase in MEK/ERK phosphorylation in RAS-mutated AML. These studies reveal SYK as a regulator of mTOR and MAPK signaling in AML and demonstrate that inhibition of PI3K pathway activity enhances the effects of SYK inhibition on AML cell viability and differentiation.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Protein-Tyrosine Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Apoptosis/drug effects , Blotting, Western , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Leukemia, Myeloid, Acute/genetics , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Syk Kinase , TOR Serine-Threonine Kinases/genetics , Tumor Cells, CulturedABSTRACT
While target-based small-molecule discovery has taken centre-stage in the pharmaceutical industry, there are many cancer-promoting proteins not easily addressed with a traditional target-based screening approach. In order to address this problem, as well as to identify modulators of biological states in the absence of knowing the protein target of the state switch, alternative phenotypic screening approaches, such as gene expression-based and high-content imaging, have been developed. With this renewed interest in phenotypic screening, however, comes the challenge of identifying the binding protein target(s) of small-molecule hits. Emerging technologies have the potential to improve the process of target identification. In this review, we discuss the application of genomic (gene expression-based), genetic (short hairpin RNA and open reading frame screening), and proteomic approaches to protein target identification.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/metabolism , Proteomics , Gene Expression , Humans , Neoplasms/drug therapy , Neoplasms/geneticsABSTRACT
With the sequencing of the human genome and the development of new genomic technologies, biomedical discovery has been transformed. The applications of these new approaches are ever-expanding from disease classification, to identification of new targets, to outcome prediction. A logical next step is the integration of genomic approaches into small molecule discovery. This review will focus on the application of genomics to compound discovery, with an emphasis on the hematological malignancies. It will focus on the use of genomic tools to discover cancer targets and the development and application of both cell-based and in silico gene expression-based approaches to small molecule discovery.
Subject(s)
Drug Discovery , Genome, Human , Genomics , Gene Expression , HumansSubject(s)
Cell Transformation, Neoplastic/genetics , DNA-Binding Proteins/physiology , Gene Expression Regulation, Leukemic , Leukemia/genetics , Neoplasm Proteins/physiology , Proto-Oncogene Proteins , Repressor Proteins , Transcription Factors/physiology , Adult , Amino Acid Sequence , Child , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 12/ultrastructure , Cloning, Molecular , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/genetics , Gene Deletion , Genes, abl , Helix-Loop-Helix Motifs/genetics , Humans , Leukemia/pathology , Leukemia, Myelomonocytic, Chronic/genetics , Leukemia, Myelomonocytic, Chronic/pathology , Models, Genetic , Molecular Sequence Data , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/physiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Proto-Oncogene Proteins c-ets , Receptor, Platelet-Derived Growth Factor beta , Receptors, Platelet-Derived Growth Factor/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/genetics , Translocation, Genetic , ETS Translocation Variant 6 ProteinABSTRACT
Polymerase chain reaction-based screening of childhood acute lymphoblastic leukemia (ALL) samples showed that a TEL/AML1 fusion transcript was detected in 27% of all cases, representing the most common known gene rearrangement in childhood cancer. The TEL/AML1 fusion results from a t(12;21)(p13;q22) chromosomal translocation, but was undetectable at the routine cytogenetic level. TEL/AML1-positive patients had exclusively B-lineage ALL, and most patients were between the ages of 2 and 9 years at diagnosis. Only 3/89 (3.4%) adult ALL patients were TEL/AML1-positive. Most importantly, TEL/AML1-positive children had a significantly lower rate of relapse compared with TEL/AML1-negative patients (0/22 v 16/54, P = .004). Co-immunoprecipitation experiments demonstrated that TEL/AML-1 formed homodimers in vitro, and heterodimerized with the normal TEL protein when the two proteins were expressed together. The elucidation of the precise mechanism of transformation by TEL/AML1 and the role of TEL/AML1 testing in the treatment of childhood ALL will require additional studies.
Subject(s)
Biomarkers, Tumor , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 21/genetics , Neoplasm Proteins/chemistry , Oncogene Proteins, Fusion , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Translocation, Genetic , Adolescent , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/chemistry , Child , Child, Preschool , Chromosomes, Human, Pair 12/ultrastructure , Chromosomes, Human, Pair 21/ultrastructure , Cloning, Molecular , Core Binding Factor Alpha 2 Subunit , DNA, Neoplasm/genetics , Dimerization , Female , Gene Deletion , Humans , Infant , Life Tables , Male , Neoplasm Proteins/analysis , Neoplasm Proteins/genetics , Polymerase Chain Reaction , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/mortality , Prognosis , Proto-Oncogenes , Survival Analysis , Treatment OutcomeABSTRACT
We have shown previously that loss of heterozygosity at chromosome band 12p13 is among the most frequent genetic abnormalities identified in acute lymphoblastic leukemia (ALL) of childhood. Two known genes map within the critically deleted region of 12p: TEL, the gene encoding a new member of the ETS family of transcription factors, which is rearranged in a variety of hematological malignancies; and KIP1, the gene encoding the cyclin-dependent kinase inhibitor p27. Both genes are, therefore, excellent candidate tumor suppressor genes. In this report, we determined the exon organization of the TEL gene and performed mutational analysis of TEL and KIP1 in 33 childhood ALL patients known to have loss of heterozygosity at this locus. No mutations in either TEL or KIP1 were found; this suggest that neither TEL nor KIP1 is the critical 12p tumor suppressor gene in childhood ALL.
Subject(s)
Cell Cycle Proteins , Chromosomes, Human, Pair 12/genetics , Exons/genetics , Gene Deletion , Genes, Tumor Suppressor/genetics , Microtubule-Associated Proteins/genetics , Oncogene Proteins , Point Mutation/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins , Base Sequence , Child , Cyclin-Dependent Kinase Inhibitor p27 , DNA Mutational Analysis , Humans , Molecular Sequence Data , Oligonucleotide Probes/genetics , Polymorphism, Single-Stranded Conformational , Proto-Oncogene Proteins c-etsSubject(s)
DNA-Binding Proteins/genetics , Leukemia, Myeloid/genetics , Leukemia, Myelomonocytic, Chronic/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins , Repressor Proteins , Transcription Factors/genetics , Cell Transformation, Neoplastic , Chromosomes, Human, Pair 12 , Chromosomes, Human, Pair 21 , Chromosomes, Human, Pair 5 , Chromosomes, Human, Pair 9 , Core Binding Factor Alpha 2 Subunit , Humans , Neoplasm Proteins/genetics , Proto-Oncogene Proteins c-ets , Receptors, Platelet-Derived Growth Factor/genetics , Translocation, Genetic , ETS Translocation Variant 6 ProteinABSTRACT
TEL is a new member of the ETS family of transcription factors which is rearranged in a number of hematologic malignancies with translocations involving chromosome band 12p13. In some cases, both TEL alleles are affected, resulting in loss of wild-type TEL function in the leukemic cells. In addition, 5% of children with acute lymphoblastic leukemia (ALL) have 12p12-p13 deletions, suggesting that a tumor suppressor gene resides on 12p. These observations led us to consider whether TEL loss of function may contribute to the pathogenesis of ALL. In this report we show that the TEL gene maps between the polymorphic markers D12S89 and D12S98, and we use these flanking markers to screen paired diagnosis and remission samples from 81 children with ALL for loss of heterozygosity (LOH) at the TEL gene locus. Fifteen percent of informative patients showed TEL LOH which was not evident on cytogenetic analysis. Detailed examination of patients with LOH at this locus showed that the critically deleted region included two candidate tumor suppressor genes: TEL and KIP1, the gene encoding the cyclin-dependent kinase inhibitor p27. These studies show that LOH at the TEL locus is a frequent finding in childhood ALL.
