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. 2017 Dec 19;9(2):2268-2278.
doi: 10.18632/oncotarget.23405. eCollection 2018 Jan 5.

HPRT1 activity loss is associated with resistance to thiopurine in ALL

Fan Yang #  1   2   3 , Houshun Fang #  2   3 , Dan Wang #  2   3 , Yao Chen  2   3 , Yonggong Zhai  1 , Bin-Bing S Zhou  2   3   4 , Hui Li  2   3   4
Affiliations

HPRT1 activity loss is associated with resistance to thiopurine in ALL

Fan Yang et al. Oncotarget. .

Abstract

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Thiopurine is a widely used drug in the maintaining treatment of ALL. After a period of chemotherapy, 20% of pediatric patients and over 50% of adult patients will relapse. To investigate the mechanisms of drug resistance in vitro, we established the thiopurine resistant cell lines Reh-6MPR (6-MP Resistant cell) and Reh-6TGR (6-TG Resistant cell) by stepwise selection of the ALL cell line Reh. Cell viability assay revealed that 6MPR and 6TGR cells were almost 1000-fold more resistant to thiopurine comparing with the control Reh cells, and thiopurine conversion was significantly impaired in the resistant cells. Mechanistically, a same novel hypoxanthine phosphoribosyl transferase 1 (HPRT1) mutation c.495_496insA (p.V165fs) was found by whole exome sequencing in both resistant cells. The HPRT1 mutation dramaticly decreased the production of [13C5,15N4]-IMP from [13C5,15N4]-hypoxanthine (HX), showed a loss-of-funciton mechanism. Notably, re-expression the wildtype HPRT1 in Reh-6MPR cell can reverse the drug resistance and thiopurine conversion in Reh-6MPR cells. These results highlight the importance of HPRT1's activity in thiopurine resistance.

Keywords: HPRT1 mutation; drug resistance; leukemia; purine metabolism; thiopurine.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Establishment of the thiopurine resistant cell lines Reh-6MPR and Reh-6TGR
(A, B, C and D) Drug sensitivity of 6-MP, 6-TG, MTX and AraC were analyzed with Cell TiterGlo assay. (E, F) Apoptosis induced by 6-MP and 6-TG was analyzed with annexin V-PE apoptosis detection Kit. Data are expressed as mean ± SD. **P<0.01 compared with ctrl Reh group. (G) Western blot of DDR markers (PARP-cleaved, P-CHK2 and γ-H2AX).
Figure 2
Figure 2. Thiopurine metabolism in Reh cells and resistant cells
(A) The diagram of thiopurine metabolism. (B, C) The accumulations of 6-MP and 6-TG were measured by LC-MS in ctrl Reh cells and resistant cells. Data are expressed as mean ± SD. *P<0.05 compared with ctrl Reh group. (D, E) Drug metabolites (TIMP and TGMP) were measured by LC-MS in ctrl Reh cells and resistant cells. Data are expressed as mean ± SD. **P<0.01 compared with ctrl Reh group.
Figure 3
Figure 3. Purine metabolism in Reh cells and resistant cells
(A) Identification HPRT1 mutation in resistant cells by Sanger sequencing. (B) After incubation with [13C5,15N4]-hypoxanthine for 4h, labeled-IMP was measured by LC-MS in Reh cells and resistant cells. Data are expressed as mean ± SD. ***P<0.001 compared with ctrl Reh group. (C, D, E, F) The metabolites (IMP, AICAR, Inosine and HX) were measured by LC-MS in Reh cells and resistant cells. Data are expressed as mean ± SD. **P<0.01 compared with ctrl Reh group. (G) The expression of MDR1 was detected by using Real-time quantitative RT-PCR assay. Data are expressed as mean ± SD.
Figure 4
Figure 4. HPRT1-wt can reverse the resistance in Reh-6MPR cells
(A) The expression levels of HRPT1 were detected by western blot. (B) 6-MP and 6-TG IC50 values of Reh-6MPR cells and HPRT1 re-expression cells. Data are expressed as mean ± SD. ***P<0.001 compared with ctrl Reh group. (C, D, E, F) The TIMP, TGMP, labeled-IMP and hypoxanthine were measured in Reh-6MPR cells and HPRT1 re-expression cells by LC-MS. Data are expressed as mean ± SD. **P<0.01 compared with ctrl Reh group.
Figure 5
Figure 5. Knockdown HPRT1 induce thiopurine resistance
(A) Western blot of HPRT1 in the knockdown cells. (B) 6-MP and 6-TG IC50 values of HPRT1 knockdown cells. Data are expressed as mean ± SD. ***P<0.001 compared with ctrl Reh group. (C) The expression levels of endogenous and exogenous HRPT1 were detected by western blot. (D) 6-MP and 6-TG IC50 values of knockdown cells with HPRT1 re-expression. Data are expressed as mean ± SD. ***P<0.001 compared with ctrl Reh group. (E, F, G, H) The TIMP, TGMP, labeled-IMP and hypoxanthine were measured in HPRT1 knockdown cells and HPRT1 re-expression cells by LC-MS. Data are expressed as mean ± SD. ***P<0.001, **P<0.05 compared with ctrl Reh group.
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