scholarly journals Downregulation of Mir-146a-5p, Mir-99b-5p, Mir-143-3p, Mir-10a-5p and Mir-151a-3p Associated with PI3K/AKT, p53, NF-Kb, and Fanconi Anemia/BRCA Signaling Pathways Are Observed in Imatinib-Resistant Chronic Myeloid Leukemia Patients without Detectable BCR-ABL kinase Domain Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3060-3060
Author(s):  
Ernie Yap ◽  
Zainul Abidin Norziha ◽  
Alfred Simbun ◽  
Nor Rafeah Tumian ◽  
Soon Keng Cheong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Signaling Pathways ◽  
Dna Damage Response ◽  
Myeloid Leukemia ◽  
Kinase Domain ◽  
Akt Pathway ◽  
Putative Gene ◽  
Abl Kinase ◽  
Damage Response

Abstract Introduction Chronic myeloid leukemia (CML) patients who do not achieve landmark responses are considered imatinib (IM)-resistant. IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, although many IM-resistant patients do not harbor KD mutations. The pathogenesis of this phenomenon is unclear. Aberrations in BCR-ABL independent downstream signaling pathways, namely PI3K/AKT, p53, NF-kB and Fanconi anaemia (FA)/BRCA, have been implicated. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and are notoriously promiscuous, with one miRNA regulating many mRNAs. In recognition of this, we sought to identify dysregulated miRNAs associated with the above pathways in patients with IM-resistant CML. Methods Eight patients with chronic phase CML who demonstrated primary resistance to IM and tested negative for KD mutations via Sanger sequencing were enrolled. Two healthy volunteers constituted the control group. Peripheral blood samples were taken and miRNA extracted from the white cells. MiRNA profiling was performed using miRNA sequencing (miRNA-seq) of the human microRNAome. MiRNA expression analyses (miEA) were performed on 4 sets of miRNAs associated with 4 different signaling pathways derived from publicly available databases: 237 miRNAs for PI3K/AKT, 223 miRNAs for p53, 221 miRNAs for NF-kB, and 126 miRNAs for FA/BRCA. MiRNA expression levels of the patients were compared with normal controls to obtain the differential expression of the miRNAs. MiRNAs that occur in all 4 pathways were further investigated in silico for their putative gene targets (Tarbase v7.0). Gene targets which were commonly linked with 3 different miRNAs were identified. Results MiRNA-seq yielded a total of 790 miRNAs. MiEA showed downregulation of 5 miRNAs in all 4 signaling pathways above: miR-146a-5p, miR-99b-5p, miR-143-3p and miR-10a-5p, miR-151a-3p. MiR-146a-5p and miR-99b-5p were the first and second most downregulated miRNAs in all 4 pathways. Putative gene targets that were commonly modulated by 3 miRNAs were: CDK6 (miR-143-3p, miR-10a-5p, miR-151a-3p), MDM2 (miR-146a-5p, miR-143-3p, miR-10a-5p), PTGS2 (miR-146a-5p, miR-143-3p, miR-10a-5p) and REV3L (miR-146a-5p, miR-143-3p, miR-151a-3p), as shown in Table 1. Conclusions The PI3K/AKT pathway is activated in response to IM-exposure to IM-naïve cells, implying a role in IM resistance. As cell cycle modulators, both p53 and NF-kB pathways are regulated within the PI3K/AKT pathway via AKT. FANCD2 is a central FA/BRCA pathway regulator and its transcription is maintained by AKT/mTOR via enhancement of NF-kB activity. The complexity of the interactions above has impeded efforts in identifying a unifying target in counteracting IM resistance. Thus, it is of significance that our results showed 5 differentially expressed miRNAs that consistently featured in the above signaling pathways, 4 of which remarkably influence PI3K/AKT and p53. In our study, PI3K/AKT and p53 pathways are inextricably linked with the same 4 miRNAs (miR-146a-5p, miR-143-3p, miR-10a-5p and miR-151a-3p) targeting the same 2 genes (MDM2 and CDK6) in these 2 pathways. MDM2 is an ubiquitin ligase that regulates the stability of p53a. AKT facilitates the functions of MDM2 to promote p53 ubiquitination. Conversely, inhibition of MDM2 leads to activation of p53 in response to cellular stresses. CDK6 is a cell cycle regulator and its overexpression influences the accumulation of pro-apoptotic p53 proteins, postulating a role in DNA damage response. Both PTGS2 and REV3L genes were commonly targeted by 3 miRNAs, within the NF-kB and FA/BRCA pathways respectively. PTGS2 overexpression has been shown in gastrointestinal adenocarcinomas; it induces carcinoma cell migration and invasion via activation of PI3K/AKT signaling. REV3L is the catalytic subunit of DNA polymerase ζ and plays a central role in DNA damage tolerance and chemoresistance towards DNA damaging agents. Notably, the NF-kB and FA/BRCA pathways were juxtaposed to the PI3K/AKT/p53 system via shared miRNA regulators. This provokes interesting questions regarding IM-resistance: are anti-apoptotic signaling and DNA damage response mechanisms driven simultaneously by these miRNAs? Can these miRNAs be potential biomarkers and therapeutic targets in IM-resistant CML? More studies are needed to further validate our findings. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dissecting the Molecular Mechanisms of Aneuploidy in Acute Myeloid Leukemia By Next Generation Sequencing

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1028-1028
Author(s):  
Giorgia Simonetti ◽  
Antonella Padella ◽  
Anna Ferrari ◽  
Viviana Guadagnuolo ◽  
Elisa Zago ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Chromosome Organization ◽  
Single Nucleotide ◽  
Damage Response ◽  
Generation Sequencing ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is a heterogeneous malignancy characterized by the expansion of myeloid precursor cells with limited or abnormal differentiation capacity. A relatively common event in AML is represented by chromosome gain or loss. Numerical chromosome abnormalities, which define aneuploidy, have a detrimental effect in primary non-malignant cells, since they dramatically reduce cellular fitness. However, evidence suggests that they have a causative role in tumorigenesis and are well tolerated in transformed cells belonging to the myeloid lineage. Aim of the study is to elucidate the pathogenic mechanisms causing and sustaining aneuploidy in AML in order to find novel potential therapeutic targets. A panel of genetic alterations was analyzed on 886 AML cases at Seràgnoli Institute in Bologna between 2002 and 2013. Among them, 31 samples were subjected to whole exome sequencing (WES, Illumina Hiseq2000). Raw data were processed with WES Pipeline web tool for variants detection. Gene expression profiling (GEP, Affymetrix) was performed on bone marrow cells from 49 AML patients at diagnosis with more than 80% blast cells, including 22 aneuploid cases (carrying monosomy, trisomy or a monosomal karyotype) and 27 cases with normal karyotype. The aneuploid status was confirmed by single nucleotide polymorphism (SNP) array. WES analysis of 13 aneuploid and 12 euploid AML cases revealed a significantly higher median value of genetic variants and mutated genes in aneuploid compared with euploid samples (aneuploid vs. euploid: median of variants, 30 vs. 20 (p=0.02) including nonsynonimous single nucleotide variants, frameshift insertions and deletions, stopgains; median of mutated genes, 25 vs. 17 (p=0.05); details will be presented at the meeting). Noticeably, by gene ontology analysis of mutated genes in the aneuploid cohort we observed a strong enrichment in genes regulating cell cycle, including chromosome organization (p=5.4x10-4) and mitotic sister chromatid cohesion (p=6.98x10-4), and chromatin modification (p=1.3x10-4), with most of the variants being not annotated in the COSMIC database. Euploid samples were enriched for mutations affecting genes involved in cytoskeleton (p=1.6x10-3) and metabolic activities (p=1.9x10-3). A number of genes mutated in the aneuploid cases belong to the APCCdc20 complex and localize on chromosomes generally spared by aneuploidy, supporting the key role of the identified aberrations in the molecular mechanisms leading to numerical chromosome abnormalities. Among several mutations predicted as “drivers” by DOTS-Finder tool (CCDC144NL, DNMT3A, GXYLT1, MESP1, TPRX1,TPTE, ZNF717), we defined some candidates involved in cell cycle regulation and DNA replication. Functional analysis are ongoing. Furthermore, a tumor suppressor function was associated with mutated genes involved in the DNA repair process. In our WES analysis, we identified a subgroup of genes linked to DNA damage response, including TP53, which are preferentially mutated in the aneuploid cohort. Since P53 is a limiting-factor in aneuploidy-induced tumorigenesis, we analyzed the mutational status in a larger cohort of AML patients by Next Generation sequencing (NGS) and Sanger sequencing. Interestingly, we identified TP53 mutations in 15/58 aneuploid vs. 1/36 euploid cases (p=3.8x10-3). Finally, differential expression of genes involved in DNA damage and integrity checkpoints was identified by GEP of aneuploid and euploid AML samples. Previous evidence showed that loss of the spindle checkpoint gene BUB1B induces aneuploidy and predisposes to tumorigenesis. Our data, obtained by integrated NGS and GEP approaches, support a causal link between mutations in a panel of genes involved in cell cycle control/chromosome organization and aneuploidy in AML. Genetic and transcriptional alterations of genes regulating DNA damage response were detected in our AML cohort, suggesting novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid condition and consequently, of leukemogenesis. The results indicate that the identified genomic aberrations likely drive chromosome gain and/or loss in AML by cooperating with alterations affecting different pathways, in order to overcome the unfitness barrier induced by aneuploidy. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

scholarly journals ATM: Main Features, Signaling Pathways, and Its Diverse Roles in DNA Damage Response, Tumor Suppression, and Cancer Development

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 845
Author(s):  
Liem Minh Phan ◽  
Abdol-Hossein Rezaeian
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Arrest ◽  
Signaling Pathways ◽  
Dna Damage Response ◽  
Cancer Development ◽  
Cellular Processes ◽  
Cycle Arrest ◽  
Damage Response ◽  
Cancer Cell Survival

ATM is among of the most critical initiators and coordinators of the DNA-damage response. ATM canonical and non-canonical signaling pathways involve hundreds of downstream targets that control many important cellular processes such as DNA damage repair, apoptosis, cell cycle arrest, metabolism, proliferation, oxidative sensing, among others. Of note, ATM is often considered a major tumor suppressor because of its ability to induce apoptosis and cell cycle arrest. However, in some advanced stage tumor cells, ATM signaling is increased and confers remarkable advantages for cancer cell survival, resistance to radiation and chemotherapy, biosynthesis, proliferation, and metastasis. This review focuses on addressing major characteristics, signaling pathways and especially the diverse roles of ATM in cellular homeostasis and cancer development.

Distinct Signaling Profiles of Gemtuzumab Ozogamicin Responsiveness and Refractoriness in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2745-2745
Author(s):  
David B. Rosen ◽  
James A Cordeiro ◽  
David M. Soper ◽  
Ying-Wen Huang ◽  
Donna E. Hogge ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Lines ◽  
Dna Damage Response ◽  
Myeloid Leukemia ◽  
Gemtuzumab Ozogamicin ◽  
Employment Equity ◽  
Damage Response ◽  
Equity Ownership

Abstract Abstract 2745 Poster Board II-721 Background: Gemtuzumab Ozogamicin (GO, Mylotarg), a humanized CD33 monoclonal antibody linked to calicheamicin was approved by the US FDA for use as a monotherapy in patients older than 60 years with relapsed acute myeloid leukemia (AML) unfit to tolerate standard salvage therapy. GO is internalized rapidly after infusion, and calicheamicin, a potent enediyene, is subsequently released and acts as a cytotoxic agent by causing double strand DNA breaks. Currently GO is in multiple clinical trials as a single agent or in combination with other therapies for both induction and consolidation treatment of various clinical subgroups of AML. However, the mechanisms of action and resistance of GO are incompletely understood and it is unclear which patient subgroups benefit from GO-based therapy. Single cell network profiling (SCNP) has shown promise as a methodology wherein multiple signaling networks are measured after treatment with an exogenous modulator such as a growth factor, cytokine or therapeutic agent and the identified signaling profiles can be used as clinical and therapeutic enablement tools. Objectives: SCNP using multiparameter flow cytometry was used to identify intracellular pathways that were associated with responsiveness or refractoriness to in vitro GO exposure in both cancer cell lines and primary AML samples. Methods: Signaling pathways emphasizing DNA damage response, cell cycle, apoptosis and drug transporter activity were measured by SCNP after in vitro exposure of cell lines and AML primary samples to clinically relevant concentrations of GO. Samples were processed for cytometry by paraformaldehyde /methanol fixation and permeabilzation followed by incubation with fluorochrome-conjugated antibody cocktails that recognize cell surface proteins to delineate cell subsets and intracellular signaling molecules. Results: In cell lines, responsiveness to in vitro GO exposure was defined as a) induction of DNA Damage as measured by increased p-ATM, p-Chk2 and p-H2AX, b) cell cycle arrest at G2/M as measured by increased cyclin B1 and DNA content & c) induction of apoptosis as measured by cleaved PARP and viability dyes. Of note, inhibition of drug transporter activity in 2 MDR-1+ cell lines did not restore GO responsiveness, suggesting the presence of additional relevant resistance mechanisms in these cell lines. In primary AML diagnostic samples, DNA damage and apoptosis pathway readouts were able to identify responsiveness or refractoriness to GO exposure. In the GO responsive profile, induction of both DNA damage responses and apoptosis were seen. Within the refractory samples, two distinct profiles were observed: a) robust and early induction of DNA damage response without apoptosis and 2) delayed and attenuated DNA damage response without apoptosis. Conclusions: Characterization of intracellular Cell Cycle, DNA Damage, and Apoptosis networks in single cells after GO exposure distinguishes GO responsive from refractory AML cells. Further, these pathway signatures provide information about mechanisms of refractoriness. (e.g. a block between a successful DNA damage response and initiation of apoptosis versus a block in the initial induction of DNA damage after GO exposure). The ability of the same profiles to predict clinical responses to the drug will be tested in future studies. Disclosures: Rosen: Nodality, Inc.: Employment, Equity Ownership. Cordeiro:Nodality Inc.: Employment, Equity Ownership. Soper:Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Cesano:Nodality Inc.: Employment, Equity Ownership. Fantl:Nodality Inc.: Employment, Equity Ownership.

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