Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR+. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The 2-nitroimidazolyl prodrug (1f) of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected NTR+ cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally in turn blocking colony formation. This system may be applied in future targeting approaches to reach tissue- or organ-type-specific inhibition of LSD1.<br>

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Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells

10.26434/chemrxiv.10012937 ◽

2019 ◽

Author(s):

Manfred Jung ◽

Eva-Maria Herrlinger ◽

Mirjam Hau ◽

Desiree M. Redhaber ◽

Gabriele Greve ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Specific Inhibition ◽

Cellular Activity ◽

Wild Type ◽

Site Specific ◽

Lysine Specific Demethylase ◽

Organ Type ◽

Promising Therapeutic Target ◽

Acute Myeloid

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In vitro activity of the flt3-inhibitor su5614 and standard cytotoxic agents in tumour cells from patients with wild type and mutated flt3 acute myeloid leukaemia

Leukemia Research ◽

10.1016/j.leukres.2005.02.017 ◽

2005 ◽

Vol 29 (9) ◽

pp. 1079-1081 ◽

Cited By ~ 7

Author(s):

Anna Åleskog ◽

Martin Höglund ◽

Jenny Pettersson ◽

Monica Hermansson ◽

Rolf Larsson ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Tumour Cells ◽

Cytotoxic Agents ◽

Vitro Activity ◽

In Vitro Activity ◽

Wild Type ◽

Flt3 Inhibitor ◽

Acute Myeloid

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Preferential transcription of the mutated allele in NPM1 mutated acute myeloid leukaemia

Scientific Reports ◽

10.1038/s41598-020-73782-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

G. D. Bailey ◽

L. Doolan ◽

A. Baskar ◽

L. C. Smith ◽

C. H. Seedhouse

Keyword(s):

Acute Myeloid Leukemia ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Myeloid Leukemia ◽

Splice Variants ◽

Wild Type ◽

Significant Bias ◽

Acute Myeloid

Abstract Nucleophosmin is commonly both over-expressed and mutated in acute myeloid leukemia (AML). NPM1 mutations are always heterozygous. In addition, NPM1 has a number of different splice variants with the major variant encoded by exons 1–9 and 11–12 (NPM1.1). Further variants include NPM1.2 which lacks exons 8 and 10 and NPM1.3 which comprises exons 1–10 (and so lacks the region of sequence mutated in AML). In this study we quantified the expression of these three variants in 108 AML patient samples with and without NPM1 mutations and also assessed the level of expression from the wild-type and mutant alleles in variants NPM1.1 and NPM1.2. The results show that NPM1.1 is the most commonly expressed variant, however transcripts from wild-type and mutated alleles do not occur at equal levels, with a significant bias toward the mutated allele. Considering the involvement of mutant nucleophosmin in the progression and maintenance of AML, a bias towards mutated transcripts could have a significant impact on disease maintenance.

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Genomic profiling of acute myeloid leukaemia associated with ataxia telangiectasia identifies a complex karyotype with wild‐type TP53 and mutant KRAS, G3BP1 and IL7R

Pediatric Blood & Cancer ◽

10.1002/pbc.28354 ◽

2020 ◽

Vol 67 (9) ◽

Author(s):

Mae A. Goldgraben ◽

Eleanor Fewings ◽

Alexey Larionov ◽

James Scarth ◽

James Redman ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Ataxia Telangiectasia ◽

Genomic Profiling ◽

Complex Karyotype ◽

Wild Type ◽

Acute Myeloid

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Knockdown of MicroRNA-10a in Acute Myeloid Leukaemia Cells Bearing the Nucleophosmin1 Mutation Causes Cell Death and Reduced Clonogenicity

Blood ◽

10.1182/blood.v118.21.1367.1367 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1367-1367

Author(s):

Adam J Bryant ◽

Catalina A Palma ◽

Mark Lutherborrow ◽

Vivek Jayaswal ◽

Yee Hwa Yang ◽

...

Keyword(s):

Cell Death ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Line ◽

Caspase 3 ◽

Luciferase Reporter ◽

Unknown Etiology ◽

Wild Type ◽

Over Expression ◽

Acute Myeloid

Abstract Abstract 1367 Acute Myeloid Leukaemia (AML) with a mutation in the Nucleophosmin1 gene (NPM1c+) accounts for one of the largest subtypes of AML, with an unknown etiology. MicroRNA dysregulation has now been implicated in the oncogenesis of many cancers including AML. We sought to investigate the role of microRNAs in the initiation and development of AML with the NPM1c+ mutation. MicroRNA profiling of bone marrow samples from 28 AML patients and confirmation by qRT-PCR demonstrated a unique microRNA signature in AML-NPM1c+ samples dominated by miR-10a over-expression of 19.6-fold compared to Nucleophosmin1 wild type (NPM1) samples. Functional assessments were performed in the human OCI-AML3 cell line, which is the only cell line to harbour NPM1c+. miR-10a repression was induced by transfection with miRCURY LNA microRNA knockdown probes (Exiqon). Cell growth (MTS) assay demonstrated a significant decrease of 19% in miR-10a knockdown cells compared to the Scrambled control. AnnexinV and Caspase 3 assays assessed the effect of miR-10a knockdown on apoptosis. miR-10a knockdown increased the proportion of AnnexinV positive events when compared to control treated cells by 34.9% and 39.3% at 24 and 48 hours respectively, but had no effect on Caspase 3 expression. Proliferation (BrdU uptake) assays did not show a change, however, clonogenic assays demonstrated a 26.1% decrease in colony number in miR-10a knockdown cells compared to the control. Potential mechanisms were elucidated by determining miR-10a mRNA targets in silico and confirmed by luciferase reporter assays. These included ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C. In this study, we have demonstrated that miR-10a was highly differentially expressed between AML-NPM1c+ cells compared to leukaemic cells bearing wild type NPM1. Knockdown of miR-10a in OCI-AML3 cells resulted in increased cell death as detected by AnnexinV binding (but not Caspase 3, indicating an effect independent of the classical apoptotic pathways) and reduced clonogenic capacity. These effects are thought to occur through miR-10a mediated modulation of ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C, all of which are associated with neoplastic transformation. Taken together, our results suggest that aberrant miR-10a over-expression in AML-NPM1c+ patients promotes cell survival. Disclosures: No relevant conflicts of interest to declare.

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Hydroxyurea synergizes with valproic acid in wild-type p53 acute myeloid leukaemia

Oncotarget ◽

10.18632/oncotarget.6991 ◽

2016 ◽

Vol 7 (7) ◽

pp. 8105-8118 ◽

Cited By ~ 11

Author(s):

Calum Leitch ◽

Tereza Osdal ◽

Vibeke Andresen ◽

Maren Molland ◽

Silje Kristiansen ◽

...

Keyword(s):

Valproic Acid ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Wild Type ◽

Wild Type P53 ◽

Acute Myeloid

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Identification and Analysis of Oncogenic Pathways in Deletion 20q Acute Myeloid Leukaemia

Blood ◽

10.1182/blood.v120.21.1324.1324 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1324-1324

Author(s):

Matthew Ku ◽

Nisha Narayan ◽

Meaghan Wall ◽

Ruth N. MacKinnon ◽

Lynda J Campbell ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Tyrosine Phosphatase ◽

Wild Type ◽

Src Tyrosine Kinase ◽

Chromosome 20 ◽

Stat3 Phosphorylation ◽

Acute Myeloid

Abstract Abstract 1324 Deletion of the long arm of chromosome 20 [del(20q)] is a common recurrent chromosomal abnormality in acute myeloid leukaemia (AML). It is a key step in AML development and a better understanding of the associated molecular events is important. The abnormal chromosome 20 in del(20q) AML has been shown to have lost a “Common Deleted Region” (CDR) that contains Protein Tyrosine Phosphatase Receptor T (PTPRT), a tyrosine phosphatase that is mutated in many human cancers such as AML. We have previously reported (MacKinnon et al, Genes, Chromosomes and Cancer 2010) that del(20q) also harbours an amplified “Common Retained Region,” (CRR) which contains Haemopoietic Cell Kinase (HCK). HCK is anoncogenic Src tyrosine kinase and its aberrant activation has been shown to contribute to the pathogenesis of some haematological malignancies. We hypothesize that the amplification of HCK in the CRR cooperates with the loss of PTPRT in the CDR to cause AML. Our model proposes that AML occurs either through direct interaction between HCK and PTPRT, or through aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3), a cytoplasmic second messenger that is important in cellular signalling. Constitutively activated STAT3 has been shown to be oncogenic in several malignancies, including AML. STAT3 is a direct target of both HCK and PTPRT. It is phosphorylated (hence activated) by HCK, and dephosphorylated (hence inactivated) by PTPRT. This provides a downstream leukaemogenic pathway for our model. The ultimate aim of our experiments is to prove this hypothesis using mouse models. Murine haemopoietic stem cells (HSC) were isolated from the bone marrows of wild type C57BL/6 (WT) and PTPRT-null mice by Fluorescence Activated Cell Sorting for Lineage negative, C-kit and Sca-1 positive (LKS+) cells. Retroviral constructs of HCK were generated by cloning it into the retroviral vector pMSCViresEGFP(MIG), with GFP as reporter. Murine HSC were transduced with either retroviral HCK or MIG vector control and Phoenix cell system was used for retroviral packaging. Experiments using isolated LKS+ HSC were performed to examine for features of AML. Examination of bone marrow cells from del(20q) AML patients by quantitative PCR revealed an increase in HCK mRNA expression and a reduction in PTPRT expression. Wild type (WT) and PTPRT-null murine HSC transduced with either MIG or HCK were cultured in methylcellulose media. Colony forming units (CFU) were enumerated on day7 and day12. We found that both WT and PTPRT-null HSC transduced with HCK showed a significant increase in colony numbers compared to MIG transduced HSC. Furthermore, the fold increment in colony number was higher in the PTPRT-null genotype as shown in figure 1. Moreover, an intracellular anti-phosphoSTAT3 assay was performed to assess STAT3 phosphorylation levels in the transduced HSC. It demonstrated that in both WT and PTPRT-null HSC that have been transduced with HCK, STAT3 hyperphosphorylation, and hence overactivation, occured. This response was again more exaggerated in the PTPRT-null HSC, as seen in figure 2. We are currently transplanting transduced LKS+ HSC (either MIG or HCK) into lethally irradiated murine recipients to assess AML formation in a reconstitution study. The recipient mice will be assessed for evidence of engraftment and subsequent AML. The preliminary data reveals a likely new oncogenic-signalling cascade: that HCK amplification and loss of PTPRT in del(20q) AML may cooperate to cause AML directly, or by aberrant activity of hyperphosphorylated STAT3. Disclosures: No relevant conflicts of interest to declare.

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TP53mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance

10.1101/404475 ◽

2018 ◽

Cited By ~ 1

Author(s):

Martin Michaelis ◽

Constanze Schneider ◽

Florian Rothweiler ◽

Tamara Rothenburger ◽

Marco Mernberger ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Lines ◽

Cancer Cell ◽

Drug Sensitivity ◽

De Novo ◽

Wild Type ◽

Inhibitor Resistance ◽

Mdm2 Inhibitor ◽

Acute Myeloid

AbstractBackground:MDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AMLTP53wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.Methods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. TheTP53status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.Results:All MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existingTP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varyingTP53mutations or wild typeTP53, indicating the induction ofde novo TP53mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.Conclusion:We introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existingTP53-mutant subpopulations or inducede novo TP53mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.

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Survival Outcomes in Patients with FLT3-Itd Positive Acute Myeloid Leukaemia Relative to Biological Stratification: Smaller FLT3-Itd Clone Size Predict Better Overall Survival

Blood ◽

10.1182/blood.v120.21.1460.1460 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1460-1460 ◽

Cited By ~ 1

Author(s):

Justin Ching Ting Loke ◽

Susanna Akiki ◽

Joanne Ewing ◽

Syed W Bokhari ◽

Deepak Chandra ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Conflicts Of Interest ◽

Wild Type Allele ◽

Wild Type ◽

Relative Expression ◽

Type Allele ◽

The Impact ◽

Acute Myeloid

Abstract Abstract 1460 Background: FLT3 internal tandem duplication (itd) mutations are found in 25% of adult patients with acute myeloid leukaemia (AML) and are associated with an adverse prognosis. This mutation results in constitutive activation of downstream pathways. Distinct biological subgroups can be identified based on FLT3-itd mutation type: clones with heterozygous FLT3-itd mutated/wildtype; homozygous mutated FLT3 allele; FLT3 heterozygous biallelic mutant and clones with evidence of overexpression of FLT3-itd. There is evidence to suggest that these mechanisms are important in the clonal evolution of AML cells. We sought to investigate their clinical impact. Method: Itd mutation analysis within exon 14 and/or exon 15 of the FLT3 gene was carried out at diagnosis by PCR of genomic DNA (gDNA) and cDNA for all new AML referrals in the region over 8 years. PCR products were identified and sized using fluorescent based fragment analysis. Allelic ratio (AR) and expression ratio (ER) (mutation: wild type ratio in gDNA and cDNA respectively) was determined from the relative peak heights. High relative expression was defined as 10 fold difference of ER/AR. Copy neutral loss of heterozygosity for the wild type allele resulting in homozygosity of the FLT3-itd mutation (acquired isodisomy (AID)) was determined by analysis of microsatellite markers along chromosome 13. Overall survival (OS) (time of diagnosis to death), event free survival (EFS) (time from diagnosis to induction failure, relapse or death) was calculated. Survival rates were estimated by the Kaplan-Meier method. Differences between the survival distributions were compared with the log-rank test. Results: 177 patients positive for the FLT3-itd mutation were identified. Median follow-up for patients alive was 3.4 years. A separate group of 49 patients tested negative for this mutation during this period had better OS and EFS (p=0.02) compared to the patients who were FLT3-itd positive (median survival 1715 and 307 days respectively). Patients who were FLT3-itd positive had statistically significant (p<0.05) differences in outcomes based on age, presenting white cell count, treatment intensity and cytogenetic risk. The characteristics of this group of patients are described below. Patients with lower AR (less than/equal to 0.3) as compared to higher AR (greater than 0.3) had an improved OS (p=0.018) and EFS (p=0.02). The impact of AR on OS had borderline significance (p=0.05) when only patients treated with intensive chemotherapy were considered. AID provides true evidence of FLT3 mutant homozygosity and was detected in 15 (142 tested) patients. In 38 patients who relapsed and had samples at these stages, 5 had developed AID, but were heterozygous (mutant/wildtype) at diagnosis. 6 patients with multiple FLT3-itd products may comprise patients who have multiple different mutant/wildtype clones but may include patients with a second, independent FLT3-itd mutation resulting in biallelic heterozygous mutations, although this cannot be confirmed. A high relative expression level of FLT3-itd was seen in 12 patients. The clinical significance of these findings is uncertain due to the small numbers. Conclusion: The impact of FLT3-itd mutation and other known prognostic factors has been confirmed in a heterogeneous, real life cohort of patients. An AR over 1 provides firm evidence of loss of the wild type allele (9 patients). AID also occurs at an AR of less than 1 because of the presence of normal cells or due to preferential amplification of the wildtype allele. Direct testing for AID is a more sensitive measure of FLT3 mutant homozygosity (detected in 14% of tested patients). The development of AID in patients who relapse may be an important mechanism by which an AML clone gains a further advantage. Lower AR was associated with improved survival. In the context of a high blast count (median bone marrow blast 80%), this implies having a small sub-clone of FLT3-itd positive cells is advantageous to having a larger FLT3-itd clone in their population of AML cells. Disclosures: No relevant conflicts of interest to declare.

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