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

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.

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Incidence and reasons for late failure after allogeneic haematopoietic cell transplantation following BuCy2 in acute myeloid leukaemia

British Journal of Haematology ◽

10.1111/j.1365-2141.2009.07947.x ◽

2010 ◽

Vol 148 (4) ◽

pp. 623-626 ◽

Cited By ~ 2

Author(s):

Shubham Pant ◽

Mehdi Hamadani ◽

Anthony J. Dodds ◽

Jeffrey Szer ◽

Pamela A. Crilley ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Transplantation ◽

Haematopoietic Cell ◽

Late Failure ◽

Allogeneic Haematopoietic Cell Transplantation ◽

Acute Myeloid

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Gene Transfer to Primary Acute Myeloid Leukaemia Blasts and Myeloid Leukaemia Cell Lines

Cytokines Cellular & Molecular Therapy ◽

10.1080/mccm.6.3.127.134 ◽

2000 ◽

Vol 6 (3) ◽

pp. 127-134 ◽

Cited By ~ 15

Author(s):

Patrick Huw Roddie ◽

Trevor Paterson ◽

Marc Leighton Turner

Keyword(s):

Gene Transfer ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Lines ◽

Leukaemia Cell ◽

Acute Myeloid

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Second allogeneic haematopoietic cell transplantation using HLA‐matched unrelated versus T‐cell replete haploidentical donor and survival in relapsed acute myeloid leukaemia

British Journal of Haematology ◽

10.1111/bjh.17426 ◽

2021 ◽

Author(s):

Mohamed A. Kharfan‐Dabaja ◽

Myriam Labopin ◽

Eolia Brissot ◽

Nicolaus Kroger ◽

Jürgen Finke ◽

...

Keyword(s):

T Cell ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Cell Transplantation ◽

Haematopoietic Cell ◽

Allogeneic Haematopoietic Cell Transplantation ◽

Acute Myeloid

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

Blood ◽

10.1182/blood.v114.22.4153.4153 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4153-4153 ◽

Cited By ~ 1

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.

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