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1.
Blood ; 139(5): 748-760, 2022 02 03.
Article in English | MEDLINE | ID: mdl-34587248

ABSTRACT

Acute lymphoblastic leukemia (ALL) harboring the IgH-CRLF2 rearrangement (IgH-CRLF2-r) exhibits poor clinical outcomes and is the most common subtype of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). While multiple chemotherapeutic regimens, including ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. To identify molecules/pathways relevant for IgH-CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of ruxolitinib using 2 IgH-CRLF2-r ALL lines that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled, for the first time, highly efficient transduction of human B cells. While single-guide RNAs (sgRNAs) targeting CRLF2, IL7RA, or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B, or STAT3 did not, suggesting that STAT signaling is largely dispensable for IgH-CRLF2-r ALL cell survival. We show that regulators of RAS signaling are critical for cell fitness and ruxolitinib sensitivity and that CRKL depletion enhances ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that blocks CRKL phosphorylation, effectively killed RAS WT IgH-CRLF2-r ALL cells in vitro and in vivo, either alone or combined with ruxolitinib. We further show that combining gilteritinib with trametinib, a MEK1/2 inhibitor, is an effective means to target IgH-CRLF2-r ALL cells regardless of RAS mutational status. Our study delineates molecules/pathways relevant for CRLF2-r ALL pathogenesis and could suggest rationally designed combination therapies appropriate for disease subtypes.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptors, Cytokine/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , CRISPR-Cas Systems , Cell Line, Tumor , Gene Rearrangement/drug effects , Humans , Mice , Nitriles/pharmacology , Philadelphia Chromosome , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Signal Transduction/drug effects
2.
Biochem Biophys Res Commun ; 590: 49-54, 2022 01 29.
Article in English | MEDLINE | ID: mdl-34971957

ABSTRACT

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.


Subject(s)
Antineoplastic Agents/pharmacology , E1A-Associated p300 Protein/antagonists & inhibitors , Gene Rearrangement , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , E1A-Associated p300 Protein/metabolism , Gene Expression Regulation, Leukemic/drug effects , Gene Rearrangement/drug effects , Humans , Mice , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Survival Analysis , Xenograft Model Antitumor Assays
3.
Biochem Biophys Res Commun ; 572: 191-196, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34375929

ABSTRACT

Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H2O2)-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H2O2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H2O2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated.


Subject(s)
Chromosomes/genetics , Gene Rearrangement/genetics , Homologous Recombination/genetics , Cells, Cultured , Chromosomes/drug effects , Chromosomes/metabolism , DNA Breaks, Double-Stranded/drug effects , DNA Repair , Gene Rearrangement/drug effects , Humans , Hydrogen Peroxide/pharmacology
4.
Int J Mol Sci ; 22(2)2021 Jan 14.
Article in English | MEDLINE | ID: mdl-33466757

ABSTRACT

Chromosomal rearrangements comprise unbalanced structural variations resulting in gain or loss of DNA copy numbers, as well as balanced events including translocation and inversion that are copy number neutral, both of which contribute to phenotypic evolution in organisms. The exquisite genetic assay and gene editing tools available for the model organism Saccharomyces cerevisiae facilitate deep exploration of the mechanisms underlying chromosomal rearrangements. We discuss here the pathways and influential factors of chromosomal rearrangements in S. cerevisiae. Several methods have been developed to generate on-demand chromosomal rearrangements and map the breakpoints of rearrangement events. Finally, we highlight the contributions of chromosomal rearrangements to drive phenotypic evolution in various S. cerevisiae strains. Given the evolutionary conservation of DNA replication and recombination in organisms, the knowledge gathered in the small genome of yeast can be extended to the genomes of higher eukaryotes.


Subject(s)
Chromosome Inversion/genetics , Chromosomes, Fungal/genetics , Gene Rearrangement/genetics , Saccharomyces cerevisiae/genetics , Translocation, Genetic/genetics , Antibiotics, Antineoplastic , Bleomycin/pharmacology , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , Gene Rearrangement/drug effects , Gene Rearrangement/radiation effects , Models, Genetic , Radiation, Ionizing
5.
Cancer Sci ; 111(5): 1851-1855, 2020 May.
Article in English | MEDLINE | ID: mdl-32216001

ABSTRACT

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy-related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide-treated or non-treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide-treated cells, variable-sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non-treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia-associated gene rearrangements, resulting in the low prevalence of leukemia development.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Cytokines/pharmacology , Gene Rearrangement/drug effects , Hematopoietic Stem Cells/drug effects , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Aged , Cell Survival/drug effects , Cells, Cultured , Etoposide/pharmacology , Hematopoietic Stem Cells/metabolism , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Middle Aged , Peripheral Blood Stem Cells/drug effects , Peripheral Blood Stem Cells/metabolism , Topoisomerase II Inhibitors/pharmacology
6.
Biochem Biophys Res Commun ; 501(2): 527-533, 2018 06 22.
Article in English | MEDLINE | ID: mdl-29738763

ABSTRACT

The ROS1 fusion gene has been identified in approximately 1% of non-small cell lung cancer (NSCLC) cases. Several clinical studies have highlighted ROS1 as a promising therapeutic target because crizotinib, a multi-targeted drug against ROS1, ALK, and the MET proto-oncogene, has elicited remarkable responses in ROS1-rearrangements NSCLC. However, acquired resistance mediated by ROS1 kinase domain mutations has been identified and a system to assess ROS1 inhibitors for these resistant mutations is necessary for the promotion of drug development. Publicly available NSCLC cell lines harboring the ROS1 fusion gene are limited to only HCC78 cells carrying SLC34A2-ROS1. This cell line exhibits resistance to ROS1 inhibitors through activation of the EGFR pathway, although the cells were established from ROS1-TKI naïve pleural effusion. Here, we demonstrate that 3D culture with gellan gum can restore the ROS1 oncogene dependence of HCC78 cells by upregulating the expression of the ROS1 fusion gene and reducing the activity of the EGFR pathway. Moreover, we established the HCC78xe3 cell line, a subclone of the HCC78 cell line, by repeated in vitro cultures from tumor xenografts and created xenograft tumors three times using in vitro cultured cells. This eventually enabled us to engraft and stably grow the cells in vivo, and subsequently evaluate various ROS1 inhibitors against HCC78xe3 cells by overexpressing crizotinib-resistant mutations in the ROS1 kinase domain including G2032R and D2033 N. We newly found that lorlatinib, a next generation ROS1/ALK inhibitor, remain the activity against D2033 N mutation. Furthermore, we demonstrated that HCC78xe3 cells expressing SLC34A2-ROS1 G2032R, and D2033 N, but not wild type (WT) cells, were resistant to crizotinib in vivo. Taken together, our data suggested that 3D cultures of HCC78 might reflect the features in patients and this new system will be a useful tool for evaluating ROS1 inhibitors.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Culture Media/pharmacology , Polysaccharides, Bacterial/pharmacology , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIb/genetics , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Culture Techniques/methods , Cell Line, Tumor , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Rearrangement/drug effects , Humans , Mice, Inbred BALB C , Mice, Nude , Proto-Oncogene Mas
7.
Lancet Oncol ; 18(12): 1590-1599, 2017 12.
Article in English | MEDLINE | ID: mdl-29074098

ABSTRACT

BACKGROUND: Most patients with anaplastic lymphoma kinase (ALK)-rearranged or ROS proto-oncogene 1 (ROS1)-rearranged non-small-cell lung cancer (NSCLC) are sensitive to tyrosine kinase inhibitor (TKI) therapy, but resistance invariably develops, commonly within the CNS. This study aimed to analyse the safety, efficacy, and pharmacokinetic properties of lorlatinib, a novel, highly potent, selective, and brain-penetrant ALK and ROS1 TKI with preclinical activity against most known resistance mutations, in patients with advanced ALK-positive or ROS1-positive NSCLC. METHODS: In this international multicentre, open-label, single-arm, first-in-man phase 1 dose-escalation study, eligible patients had advanced ALK-positive or ROS1-positive NSCLC and were older than 18 years, with an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Lorlatinib was administered orally to patients at doses ranging from 10 mg to 200 mg once daily or 35 mg to 100 mg twice daily, with a minimum of three patients receiving each dose. For some patients, tumour biopsy was done before lorlatinib treatment to identify ALK resistance mutations. Safety was assessed in patients who received at least one dose of lorlatinib; efficacy was assessed in the intention-to-treat population (patients who received at least one dose of study treatment and had either ALK or ROS1 rearrangement). The primary endpoint was dose-limiting toxicities during cycle 1 according to investigator assessment; secondary endpoints included safety, pharmacokinetics, and overall response. This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865. FINDINGS: Between Jan 22, 2014, and July 10, 2015, 54 patients received at least one dose of lorlatinib, including 41 (77%) with ALK-positive and 12 (23%) with ROS1-positive NSCLC; one patient had unconfirmed ALK and ROS1 status. 28 (52%) patients had received two or more TKIs, and 39 (72%) patients had CNS metastases. The most common treatment-related adverse events among the 54 patients were hypercholesterolaemia (39 [72%] of 54 patients), hypertriglyceridaemia (21 [39%] of 54 patients), peripheral neuropathy (21 [39%] of 54 patients), and peripheral oedema (21 [39%] of 54 patients). One dose-limiting toxicity occurred at 200 mg (the patient did not take at least 16 of 21 prescribed total daily doses in cycle 1 because of toxicities attributable to study drug, which were grade 2 neurocognitive adverse events comprising slowed speech and mentation and word-finding difficulty). No maximum tolerated dose was identified. The recommended phase 2 dose was selected as 100 mg once daily. For ALK-positive patients, the proportion of patients who achieved an objective response was 19 (46%) of 41 patients (95% CI 31-63); for those who had received two or more TKIs, the proportion of patients with an objective response was 11 (42%) of 26 patients (23-63). In ROS1-positive patients, including seven crizotinib-pretreated patients, an objective response was achieved by six (50%) of 12 patients (95% CI 21-79). INTERPRETATION: In this phase 1, dose-escalation study, lorlatinib showed both systemic and intracranial activity in patients with advanced ALK-positive or ROS1-positive NSCLC, most of whom had CNS metastases and had previously had two or more TKI treatments fail. Therefore, lorlatinib might be an effective therapeutic strategy for patients with ALK-positive NSCLC who have become resistant to currently available TKIs, including second-generation ALK TKIs, and is being investigated in a phase 3 randomised controlled trial comparing lorlatinib to crizotinib (ClinicalTrials.gov, NCT03052608). FUNDING: Pfizer.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Gene Rearrangement/drug effects , Lactams, Macrocyclic/therapeutic use , Lung Neoplasms/drug therapy , Protein-Tyrosine Kinases/drug effects , Proto-Oncogene Proteins/drug effects , Receptor Protein-Tyrosine Kinases/drug effects , Adult , Aged , Aminopyridines , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Disease-Free Survival , Dose-Response Relationship, Drug , Drug Administration Schedule , Humans , Internationality , Lactams , Lactams, Macrocyclic/adverse effects , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Maximum Tolerated Dose , Middle Aged , Patient Selection , Prognosis , Prospective Studies , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Mas , Proto-Oncogene Proteins/genetics , Pyrazoles , Receptor Protein-Tyrosine Kinases/genetics , Risk Assessment , Survival Analysis , Treatment Outcome
10.
Br J Cancer ; 109(9): 2347-55, 2013 Oct 29.
Article in English | MEDLINE | ID: mdl-24104963

ABSTRACT

BACKGROUND: Genomic rearrangements at the fragile site FRA1E may disrupt the dihydropyrimidine dehydrogenase gene (DPYD) which is involved in 5-fluorouracil (5-FU) catabolism. In triple-negative breast cancer (TNBC), a subtype of breast cancer frequently deficient in DNA repair, we have investigated the susceptibility to acquire copy number variations (CNVs) in DPYD and evaluated their impact on standard adjuvant treatment. METHODS: DPYD CNVs were analysed in 106 TNBC tumour specimens using multiplex ligation-dependent probe amplification (MLPA) analysis. Dihydropyrimidine dehydrogenase (DPD) expression was determined by immunohistochemistry in 146 tumour tissues. RESULTS: In TNBC, we detected 43 (41%) tumour specimens with genomic deletions and/or duplications within DPYD which were associated with higher histological grade (P=0.006) and with rearrangements in the DNA repair gene BRCA1 (P=0.007). Immunohistochemical analysis revealed low, moderate and high DPD expression in 64%, 29% and 7% of all TNBCs, and in 40%, 53% and 7% of TNBCs with DPYD CNVs, respectively. Irrespective of DPD protein levels, the presence of CNVs was significantly related to longer time to progression in patients who had received 5-FU- and/or anthracycline-based polychemotherapy (hazard ratio=0.26 (95% CI: 0.07-0.91), log-rank P=0.023; adjusted for tumour stage: P=0.037). CONCLUSION: Genomic rearrangements in DPYD, rather than aberrant DPD protein levels, reflect a distinct tumour profile associated with prolonged time to progression upon first-line chemotherapy in TNBC.


Subject(s)
DNA Copy Number Variations , Dihydrouracil Dehydrogenase (NADP)/genetics , Neoplasm Recurrence, Local/genetics , Triple Negative Breast Neoplasms/genetics , Antimetabolites, Antineoplastic/therapeutic use , BRCA1 Protein/genetics , Chromosome Fragile Sites/drug effects , Down-Regulation/drug effects , Female , Fluorouracil/therapeutic use , Gene Deletion , Gene Duplication/drug effects , Gene Duplication/genetics , Gene Rearrangement/drug effects , Humans , Middle Aged , Neoplasm Recurrence, Local/enzymology , Neoplasm Recurrence, Local/pathology , Prognosis , Radiography , Triple Negative Breast Neoplasms/diagnostic imaging , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/enzymology
12.
Blood ; 115(23): 4798-809, 2010 Jun 10.
Article in English | MEDLINE | ID: mdl-20215641

ABSTRACT

Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P < .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)-PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.


Subject(s)
Azacitidine/analogs & derivatives , CpG Islands , DNA Methylation/drug effects , Enzyme Inhibitors/pharmacology , Myeloid-Lymphoid Leukemia Protein/biosynthesis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Promoter Regions, Genetic , Azacitidine/pharmacology , Azacitidine/therapeutic use , Cell Line, Tumor , Child , Child, Preschool , Cohort Studies , DNA Methylation/genetics , DNA Modification Methylases/antagonists & inhibitors , DNA Modification Methylases/metabolism , Decitabine , Enzyme Inhibitors/therapeutic use , Female , Gene Rearrangement/drug effects , Gene Rearrangement/genetics , Gene Silencing/drug effects , Genome-Wide Association Study , Histone-Lysine N-Methyltransferase , Humans , Infant , Male , Myeloid-Lymphoid Leukemia Protein/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
14.
Blood Cancer J ; 12(1): 5, 2022 01 11.
Article in English | MEDLINE | ID: mdl-35017466

ABSTRACT

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.


Subject(s)
Antineoplastic Agents/pharmacology , Histone-Lysine N-Methyltransferase/genetics , Leukemia, Myeloid, Acute/drug therapy , Myeloid-Lymphoid Leukemia Protein/genetics , Nucleophosmin/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Aminopyridines/pharmacology , Benzimidazoles/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Gene Expression Regulation, Leukemic/drug effects , Gene Rearrangement/drug effects , Humans , Leukemia, Myeloid, Acute/genetics , Mutation/drug effects , Proto-Oncogene Proteins/genetics , Sulfonamides/pharmacology
15.
Int J Lab Hematol ; 43(3): 441-449, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33615710

ABSTRACT

INTRODUCTION: To investigate the effects and mechanism of action of upregulated CRLF2 expression resulting from different aberrations in the CRLF2 gene (CRLF2, CRLF2 + IK6, P2RY8-CRLF2 and CRLF2 F232C) in the B cell ALL cell line Nalm6. METHODS: Cell proliferation was measured using cell counting kit-8. Transcriptome sequencing technology (RNA-seq) was used to compare changes in gene expression resulting from different aberrations in CRLF2. High-throughput drug sensitivity testing was used to determine the drug sensitivity of cells. RESULTS: All four aberrations in CRLF2 upregulated CRLF2 expression and promoted the proliferation of Nalm6 cells. The RNA-seq results showed the upregulation of genes in the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and the downregulation of genes in the cell cycle pathway in the CRLF2 F232C-overexpressing cells. Western blotting showed that the expression of p-STAT5 protein was significantly higher in the CRLF2 F232C-overexpressing cells. Cells with aberrations in CRLF2 were more resistant to cyclophosphamide and drugs commonly used during treatment than cells in the vector group. The half-maximal inhibitory concentration (IC50 or GI50 ) of dexamethasone was significantly higher in the CRLF2 F232C-overexpressing cell line. CONCLUSIONS: The overexpression of CRLF2, CRLF2 + IK6, P2RY8-CRLF2 and CRLF2 F232C promotes the proliferation of Nalm6 cells, activates the JAK/STAT signalling pathway and leads to a reduction in sensitivity towards various chemotherapeutic drugs.


Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptors, Cytokine/genetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Gene Expression Regulation, Leukemic/drug effects , Gene Rearrangement/drug effects , Humans , Oncogene Proteins, Fusion/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Transcriptome/drug effects
16.
Cancer Lett ; 522: 119-128, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34534615

ABSTRACT

Anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) have improved clinical outcomes in non-small cell lung cancer (NSCLC) harboring ALK- rearrangements. However, a small population of tumor cells survives due to adaptive resistance under drug pressure and ultimately acquires drug resistance. Thus, it is necessary to elucidate the mechanisms underlying the prevention of drug resistance to improve the prognosis of patients with ALK-rearranged NSCLC. We identified novel adaptive resistance, generated through c-Jun N-terminal kinase (JNK)/c-Jun signaling, to initial ALK-TKIs-alectinib and brigatinib-in ALK-rearranged NSCLC. Inhibition of JNK/c-Jun axis showed suppression of growth and promotion of apoptosis induced by ALK-TKIs in drug-tolerant cells. JNK inhibition, in combination with the use of ALK-TKIs, increased cell apoptosis through repression of the Bcl-xL proteins, compared with ALK-TKI monotherapy. Importantly, combination therapy targeting JNK and ALK significantly delayed the regrowth following cessation of these treatments. Together, our results demonstrated that JNK pathway activation plays a pivotal role in the intrinsic resistance to ALK-TKIs and the emergence of ALK-TKI-tolerant cells in ALK-rearranged NSCLC, thus indicating that optimal inhibition of tolerant signals combined with ALK-TKIs may potentially improve the outcome of ALK-rearranged NSCLC.


Subject(s)
Anaplastic Lymphoma Kinase/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , JNK Mitogen-Activated Protein Kinases/genetics , Protein Kinase Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Carbazoles/pharmacology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Rearrangement/drug effects , Heterografts , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mice , Microarray Analysis , Organophosphorus Compounds/pharmacology , Piperidines/pharmacology , Proteome/genetics , Pyrimidines/pharmacology , Signal Transduction/drug effects , bcl-X Protein/genetics
17.
Nat Commun ; 11(1): 74, 2020 01 03.
Article in English | MEDLINE | ID: mdl-31900393

ABSTRACT

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Anaplastic Lymphoma Kinase/genetics , Apoptosis/drug effects , Carbazoles/administration & dosage , Gene Rearrangement/drug effects , Lung Neoplasms/drug therapy , Neoplasm Recurrence, Local/drug therapy , Piperidines/administration & dosage , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Anaplastic Lymphoma Kinase/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/physiopathology , Male , Mice , Mice, Inbred BALB C , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/physiopathology , Protein Kinase Inhibitors/administration & dosage , Transcription Factors/genetics , YAP-Signaling Proteins
18.
Anticancer Res ; 39(1): 413-420, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30591488

ABSTRACT

BACKGROUND: Patients with adenocarcinoma of the lung are routinely screened for anaplastic lymphoma kinase (ALK) rearrangement because they can be treated by ALK-specific targeted therapy. The clinical and molecular characteristics of large-cell neuroendocrine carcinoma (LCNEC) associated with ALK rearrangement are still unclear. Herein, we assessed the ALK status in a series of patients with LCNEC by testing methods commonly used for adenocarcinoma. MATERIALS AND METHODS: ALK expression was first examined by immunohistochemistry. For a positively stained tumor, molecular analyses were then conducted. The ALK fusion partner found in a patient with ALK rearrangement was further identified by direct DNA sequencing. Patient clinicopathological features were also analyzed, focusing on the ALK rearrangement-positive case. RESULTS: Immunohistochemistry of seven patients identified strong ALK expression in one case of stage IV LCNEC. Molecular analysis identified a novel rearranged gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 17 to ALK exon 20. The patient was treated with ALK-specific inhibitors, crizotinib and later, alectinib, and has remained alive for more than 24 months without disease progression. Three of the remaining six patients without ALK rearrangement had stage IV cancer and received cytotoxic chemotherapies. Their average overall survival was 5.4 months. CONCLUSION: To our knowledge, this is the first report of a KIF5B-ALK fusion gene in LCNEC. The patient was successfully treated with ALK inhibitors, suggesting that sensitivity to ALK inhibitor may define a specific LCNEC subtype. We propose that screening for ALK rearrangement in patients with LCNEC may assist in selecting potential candidates for targeted therapy.


Subject(s)
Anaplastic Lymphoma Kinase/genetics , Carcinoma, Large Cell/genetics , Carcinoma, Neuroendocrine/genetics , Oncogene Proteins, Fusion/genetics , Adult , Anaplastic Lymphoma Kinase/antagonists & inhibitors , Carcinoma, Large Cell/drug therapy , Carcinoma, Large Cell/pathology , Carcinoma, Neuroendocrine/drug therapy , Carcinoma, Neuroendocrine/pathology , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Rearrangement/drug effects , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Staging , Protein Kinase Inhibitors/administration & dosage
19.
Cancer Cell ; 36(6): 660-673.e11, 2019 12 09.
Article in English | MEDLINE | ID: mdl-31821784

ABSTRACT

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.


Subject(s)
Chromatin/drug effects , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Proto-Oncogene Proteins/drug effects , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromatin/genetics , Gene Expression Regulation, Leukemic/genetics , Gene Rearrangement/drug effects , Gene Rearrangement/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Mice , Proto-Oncogene Proteins/genetics , Transcription Factors/drug effects , Transcription Factors/genetics
20.
J Hematol Oncol ; 12(1): 66, 2019 06 28.
Article in English | MEDLINE | ID: mdl-31253180

ABSTRACT

INTRODUCTION: The chromosomal rearrangements of the mixed-lineage leukemia gene MLL (KMT2A) have been extensively characterized as a potent oncogenic driver in leukemia. For its oncogenic function, most MLL-fusion proteins exploit the multienzyme super elongation complex leading to elevated expression of MLL target genes. High expression of MLL target genes overwrites the normal hematopoietic differentiation program, resulting in undifferentiated blasts characterized by the capacity to self-renew. Although extensive resources devoted to increased understanding of therapeutic targets to overcome de-differentiation in ALL/AML, the inter-dependencies of targets are still not well described. The majority of inhibitors potentially interfering with MLL-fusion protein driven transformation have been characterized in individual studies, which so far hindered their direct cross-comparison. METHODS: In our study, we characterized head-to-head clinical stage inhibitors for BET, DHODH, DOT1L as well as two novel inhibitors for CDK9 and the Menin-MLL interaction with a focus on differentiation induction. We profiled those inhibitors for global gene expression effects in a large cell line panel and examined cellular responses such as inhibition of proliferation, apoptosis induction, cell cycle arrest, surface marker expression, morphological phenotype changes, and phagocytosis as functional differentiation readout. We also verified the combination potential of those inhibitors on proliferation and differentiation level. RESULTS: Our analysis revealed significant differences in differentiation induction and in modulating MLL-fusion target gene expression. We observed Menin-MLL and DOT1L inhibitors act very specifically on MLL-fused leukemia cell lines, whereas inhibitors of BET, DHODH and P-TEFb have strong effects beyond MLL-fusions. Significant differentiation effects were detected for Menin-MLL, DOT1L, and DHODH inhibitors, whereas BET and CDK9 inhibitors primarily induced apoptosis in AML/ALL cancer models. For the first time, we explored combination potential of the abovementioned inhibitors with regards to overcoming the differentiation blockage. CONCLUSION: Our findings show substantial diversity in the molecular activities of those inhibitors and provide valuable insights into the further developmental potential as single agents or in combinations in MLL-fused leukemia.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Histone-Lysine N-Methyltransferase/genetics , Leukemia/drug therapy , Myeloid-Lymphoid Leukemia Protein/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Gene Rearrangement/drug effects , Histone-Lysine N-Methyltransferase/metabolism , Humans , Leukemia/genetics , Leukemia/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein Interaction Maps/drug effects , Proto-Oncogene Proteins/metabolism
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