scholarly journals Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Tamara Rothenburger ◽  
Dominique Thomas ◽  
Yannick Schreiber ◽  
Paul R. Wratil ◽  
Tamara Pflantz ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Nucleoside Analogue ◽  
Acquired Resistance ◽  
Nucleoside Analogues ◽  
Cross Resistance ◽  
Enzymatic Assays ◽  
Anti Cancer ◽  
Acute Myeloid

Abstract Background SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. Methods CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. Results Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. Conclusion Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

scholarly journals Inhibition of ATR in Combination with Nucleoside Analogues Eradicates Acute Myeloid Leukaemia in an Orthotopic Murine Xenograft Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4031-4031
Author(s):  
Sarah E Fordham ◽  
Helen J Blair ◽  
Claire J Elstob ◽  
Deepali Pal ◽  
Nicola Curtin ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukaemia ◽  
Disease Control ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Nucleoside Analogues ◽  
Post Injection ◽  
Inhibitory Effects ◽  
Early Disease ◽  
Acute Myeloid

Abstract The ataxia telangiectasia and RAD3-related (ATR) protein kinase is a component of the cellular DNA damage response pathway and promotes cell survival by signalling repair of collapsed replication forks generated by replication stress. We hypothesised that inhibition of ATR potentiates the anti-leukaemic activity of chain terminating nucleoside analogues used in the treatment of acute myeloid leukaemia (AML). We used VE-821 and its derivative VX-970 (Vertex Pharmaceuticals, Abingdon, UK) as potent and specific inhibitors of ATR kinase activity to examine the effects of ATR inhibition in AML cell lines, primary AML cells and AML xenografts. Co-treatment with 1mM VE-821 did not consistently potentiate the anti-proliferative effects of cytarabine, clofarabine or fludarabine in a panel of AML cell lines. However, there was consistent potentiation of hydroxyurea and gemcitabine in all 7 AML cell lines tested. Treatment with hydroxyurea, which induces replication stress via depletion of dNTPs, resulted in phosphorylation of CHK1, a downstream target of ATR. CHK1 phosphorylation was attenuated when 1mM VE-821 was co-administered with hydroxyurea. Exposure of cells to gemcitabine or hydroxyurea slowed transit through S phase, which was pronounced in combination with VE-821. HL-60 AML cell clones expressing either a constitutively active or inducible shRNA construct targeting ATR had reduced ATR protein expression compared to control cells and were significantly more sensitive to the anti-proliferative effects of gemcitabine and hydroxyurea, but not to cytarabine, clofarabine or fludarabine. The growth inhibitory effects of hydroxyurea and gemcitabine were also significantly potentiated by VE-821 in primary AML patient samples, which included three adult patients with de novo AML and a paediatric patient with therapy-related AML. In contrast, ATR inhibition did not potentiate the inhibitory effects of hydroxyurea or gemcitabine in primary bone marrow cells from healthy donors ex vivo. We next sought to determine whether ATR inhibition potentiated hydroxyurea and gemcitabine in an orthotopic mouse model of AML. MV4-11 AML cells engineered to express firefly luciferase (MV4-11 pSLIEW) were intrafemorally transplanted into immunodeficient Rag2-/- gc-/- mice. Bioluminescent imaging via IVIS Spectrum (PerkinElmer, Buckinghamshire, UK) demonstrated localised femoral engraftment first detectable 4-5 days post-injection, with luciferase signal developing in other parts of the body (liver, ovaries) between days 15 and 18 in untreated mice. Treatment was initiated 7 days post-injection when disease was localised to the femur and prior to emergence of disseminated luciferase signal. Single agent hydroxyurea (250mg per kg, IP days 0-4 and 7-11) conferred some early disease control compared to controls as determined by luciferase total body flux measured on day 14, but this was not statistically significant (p=0.18) and did not affect overall survival (mean 35 days for controls and 37 days for hydroxyurea, p=0.47). Monotherapy with VX-970 (60 mg per kg, orally on days 0-4 and 7-11) also conferred early disease control compared to vehicle-treated mice (p=0.18), and resulted in significantly longer overall survival (mean 40 days, p=0.017). Combination treatment with hydroxyurea and VX-970 did not result in more effective early disease control or improved overall survival compared to monotherapy with either agent. Treatment with gemcitabine monotherapy (100 mg per kg, intraperitoneal injection on days 0, 3, 7 and 10) conferred significant early disease control (p=0.002) and significantly improved overall survival compared to controls (mean survival 73 days, p<0.001). Nevertheless, leukemic cells persisted and the cause of death in all mice randomised to this arm was relapsed disseminated disease. Treatment with gemcitabine and VX-970 in combination also conferred very significant early disease control (p=0.002), which eradicated luciferase signal by day 21 in all 6 mice randomised to this arm (Figure 1), and also conferred significantly improved overall survival compared to controls (p<0.001) or mice treated with gemcitabine monotherapy (p=0.001), with four mice remaining disease free at day 126. No significant weight loss or toxicity was observed in any of the treatment arms. Taken together, these data suggest ATR inhibition as a promising therapeutic strategy in AML. Disclosures Pollard: Vertex Pharmaceuticals (Europe) Ltd.: Employment.

scholarly journals Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines

2004 ◽  
Vol 126 (5) ◽  
pp. 726-735 ◽  
Author(s):  
Dai Watanabe ◽  
Sachiko Ezoe ◽  
Minoru Fujimoto ◽  
Akihiro Kimura ◽  
Yoshiyuki Saito ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Gene Silencing ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Haematopoietic Cell ◽  
Acute Myeloid

Functional Alterations of Lin−CD34+CD38+ Progenitors in Chronic Myelomonocytic Leukaemia and on Progression to Acute Leukaemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4119-4119
Author(s):  
Qian Sun ◽  
Chi-Chiu So ◽  
Sze-Fai Yip ◽  
Thomas S.K. Wan ◽  
Edmond Shiu Kwan Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Acquired Resistance ◽  
Differentiation Potential ◽  
Cd34 Cells ◽  
Cell Disorder ◽  
Chronic Myelomonocytic Leukaemia ◽  
Acute Myeloid

Abstract Chronic myelomonocytic leukaemia (CMML) is a clonal bone marrow stem cell disorder based on the presence of trilineage involvement, the association of myelodysplastic and myeloproliferative features and its ability to transform into acute myeloid leukaemia. The objectives of our study are to identify the cell population and its functional characteristics involved in evolution from CMML phase to acute myeloid leukaemia. We analysed Lin−CD34+ stem/progenitor population and performed cell proliferation, apoptotic assays, self-renewal ability and differentiation potential studies in purified populations of Lin−CD34+CD38− stem cells and Lin−CD34+CD38+ committed progenitors from peripheral blood of 16 patients with CMML and in six of the 16 after transformation to acute myeloid leukaemia (AML-t). We observed an expansion of the stem cell/progenitor pool (Lin−CD34+ cells) in AML-t comprising mainly of Lin−CD34+CD38+ committed progenitors within Lin−CD34+ cells. The Lin−CD34+CD38+ committed progenitors displayed highly proliferative activity in CMML and in AML-t; and additionally acquired resistance to apotosis and myeloid colony self-renewing ability in AML-t. Impairment of dendritic cell (DC) differentiation was observed with complete block in AML-t. Our findings suggest Lin−CD34+CD38+ committed progenitors instead of Lin−CD34+CD38− stem cells could be the target(s) of secondary genetic lesions underpinning progression from CMML to AML. These results have implications for the further study of the biology of leukaemic transformation and the design of new strategies for the effective treatment of CMML.

2′–Cyano–2′–Deoxyarabinofuranosylcytosine Is Active in Acute Myeloid Leukaemia and Acts in Synergy with Cytarabine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4153-4153 ◽  
Author(s):  
Elisabeth J Walsby ◽  
Chiara Ghiggi ◽  
Ruth H Mackay ◽  
Simon R Green ◽  
Steven Knapper ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Prolonged Exposure ◽  
Nucleoside Transporters ◽  
Deoxycytidine Kinase ◽  
Dna Breaks ◽  
Cytotoxic Effects ◽  
Acute Myeloid

Abstract Abstract 4153 2′–Cyano–2′–deoxyarabinofuranosylcytosine (CNDAC) is the metabolic product of sapacitabine following hydrolysis of the palmitoyl sidechain from the pyrimidine analog primarily by plasma, gut and liver amidases. CNDAC is in turn phosphorylated into the active triphosphate form (CNDACTP) by deoxycytidine kinase (dCK). CNDACTP is incorporated into DNA resulting in single stranded DNA breaks during replication and inducing cell cycle arrest. Previously the cytotoxic effects of CNDAC have also been associated with intracellular accumulation of CNDAC triphosphate and chain termination. CNDAC and sapacitabine have overlapping cytotoxic effects. Acute myeloid leukaemia (AML) cell lines NB4 and HL-60 had an LD50 of 0.24μM (± 0.24) for CNDAC and 0.23μM (± 0.21) for cytarabine (AraC) following 24 hours treatment. Primary AML blasts isolated from patients at diagnosis (n = 15) had a higher mean LD50 (25.22μM ± 19.41) for CNDAC and AraC (8.09μM ± 8.93). This is thought to be due to the requirement of cells to be actively cycling in order to be susceptible to these agents. CNDAC induces apoptosis in NB4 and HL-60 cell lines with significant increases in the percentage of cells with increased Annexin V/propidium iodide staining at concentrations of 1.0μM and above (P < 0.04) and significant caspase-3 activation at concentrations of 0.1μM and above (P < 0.05). Treatment with CNDAC also results in a significant concentration-dependent accumulation in the G2 phase of the cell cycle after 24 hours in NB4 and HL-60 cells (P = 0.003 and 0.011 respectively). Synergy was observed in the AML cell lines when CNDAC was combined with AraC at a ratio of 2:1 The mean combination index for CNDAC and AraC was 0.67 (± 0.21). The activity of deoxycytidine kinase (dCK) was blocked by the addition of excess deoxycytidine, under these conditions the effects of CNDAC were abrogated (P < 0.05) in NB4 and HL-60 cells suggesting that CNDAC requires phosphorylation by dCK for its activation in the cells. The nucleoside transporters hENT 1 and 2 and hCNT3 transport a range of nucleoside analogues through the cell membrane into cells, the use of hENT inhibitors led to a 2.5 fold increase in the LD50 for CNDAC (P = 0.028) over 48 hours. This prolonged exposure to CNDAC could have resulted in some passive uptake of CNDAC into the cells potentially explaining why the agent retained some cell killing activity. Equivalent results have been obtained with dCK and hENT inhibitors in other cell lines indicating that there is a general requirement for these enzymes for CNDAC activity. Interestingly, when cells are treated with the parent drug sapacitabine in the presence of excess deoxycytidine the cytotoxicity is reduced, but when cells are treated in the presence of hENT inhibitors, sapacitabine's cytotoxicity is improved. This suggests that the presence of the palmitoyl side-chain allows membrane permeability even in the absence of the traditional nucleoside transporters. Disclosures: Green: Cyclacel Ltd: Employment.

scholarly journals Metabolomic Profiling of Drug Responses in Acute Myeloid Leukaemia Cell Lines

PLoS ONE ◽  
2009 ◽  
Vol 4 (1) ◽  
pp. e4251 ◽  
Author(s):  
Stefano Tiziani ◽  
Alessia Lodi ◽  
Farhat L. Khanim ◽  
Mark R. Viant ◽  
Christopher M. Bunce ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Leukaemia Cell ◽  
Drug Responses ◽  
Metabolomic Profiling ◽  
Acute Myeloid Leukaemia Cell ◽  
Acute Myeloid

Annexin II cell surface and mRNA expression in human acute myeloid leukaemia cell lines

2005 ◽  
Vol 115 (1-2) ◽  
pp. 109-114 ◽  
Author(s):  
Shane A. Olwill ◽  
Hugh McGlynn ◽  
William S. Gilmore ◽  
H. Denis Alexander
Keyword(s):  
Cell Surface ◽  
Acute Myeloid Leukaemia ◽  
Mrna Expression ◽  
Myeloid Leukaemia ◽  
Cell Lines ◽  
Annexin Ii ◽  
Leukaemia Cell ◽  
Acute Myeloid Leukaemia Cell ◽  
Acute Myeloid

The BH3 Mimetic, ABT-737, Overcomes Stromal-Mediated Pro-Survival Signals and Synergizes with PHA-767491, a Dual Cdc7/CDK9 Inhibitor, In Acute Myeloid Leukaemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1841-1841
Author(s):  
Ruth C Morrell ◽  
Eva Szegezdi ◽  
Anna Halpin-McCormick ◽  
Karen Cawley ◽  
Afshin Samali ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
In Vivo Studies ◽  
Relative Reduction ◽  
Induced Apoptosis ◽  
Cdk9 Inhibitor ◽  
Acute Myeloid

Abstract Abstract 1841 ABT-737 is a small molecule inhibitor of Bcl-2 and Bcl-xL with reported activity in pre-clinical in-vitro and in-vivo studies of acute myeloid leukaemia(AML) but to date no data has been reported on its activity in an AML co-culture model. To address this, we examined the effects of co-culture of AML cell lines (MOLM-13, ML-1, KG-1, OCI-AML2) with HS5 cells, a human stromal cell line, on sensitivity to Ara-C and ABT-737. All cell lines cultured in the presence of HS-5 stroma demonstrated a significant reduction in Ara-C-induced apoptosis (% relative reduction - OCI-AML2:80%; ML1:65%; MOLM-13:53%; KG-1:55%) as compared to cells cultured in suspension in normal complete media, with the effect on expression of Bcl-2 family members being currently under evaluation. In contrast, in the presence of ABT-737, HS-5 co-culture did not provide any protective effect whatsoever to AML cells, with IC50 ranging from 0.1 to 0.3μM in the cell lines noted above, regardless of the presence of stroma. OCI-AML3, an AML cell line known to express high levels of Mcl-1 was resistant to ABT-737 in both normal suspension cultures and co-culture. Indeed Mcl-1, an important pro-survival protein in haematopoietic cells is thought to be a key factor promoting resistance to ABT-737 and it has recently been reported that transcriptional upregulation of Mcl-1 may follow exposure to ABT-737. Thus, the combination of ABT-737 with strategies to deplete Mcl-1 is particularly attractive. Cdk9 inhibition is such a strategy. Since Cdk9 phosphorylates RNA polymerase II affecting the rate of transcription, inhibition leads to a depletion of proteins with short half-lives, such as Mcl-1. Here we report that resistance of OCI-AML3 cells to ABT-737-induced apoptosis can be overcome by combination with PHA-767491, a novel dual Cdc7/CDK9 inhibitor. OCI-AML3 cells were treated with increasing concentrations of ABT-737, PHA-767491 or both. Co-administration resulted in a strong synergistic apoptosis-inducing effect as assessed by AnnexinV staining, with combination indices, as calculated by Chou et Talalay, for a range of doses of both drugs of <1 (range 0.3–0.9). Importantly, the sensitising effect of PHA-767491 was seen only at concentrations (≥ 2μM) that resulted in significant downregulation of Mcl-1 protein expression, implicating Mcl-1 downregulation as a possible cause of synergy. We are currently investigating the precise role of Mcl-1 in this regard. In conclusion, taken together, these studies support that ABT-737, possibly in combination with agents to deplete Mcl-1, represents a promising therapeutic strategy for AML and warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

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