Alkylation of nucleobases by 2-chloro- N,N-diethylethanamine hydrochloride (CDEAH) sensitizes PARP1-deficient tumors

Abstract

Targeting BRCA1- and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1- and xeroderma pigmentosum A-deficient cells. CDEAH is a monofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi.

Graphical Abstract

Figure 1.

CDEAH selectively kills XPA- and PARP1-deficient cells. (A) Structure of CDEAH. (B) CDEAH induces DNA replication stress. HEK293T cells stably expressing luciferase-fused ATAD5 were grown in 96-well plates at a density of 15,000 cells per well and treated with 5-FUrd as a positive control or CDEAH for 24 h. The luciferase activity was measured using the ONE-Glo luciferase reagent. (C) CDEAH kills XPA- and PARP1-deficient HAP1 cells better than WT. HAP1 cells deficient in XPA, PARP1, 53BP1, XRCC4 and RAD52 or WT were cultured in 96-well plates at a density of 5,000 cells per well and exposed to 20 μM CDEAH for 48 h. Cell viability was determined using Cell Titer-Glo reagent. (D) Cell survival response to dose-dependent CDEAH treatment. Indicated cells were grown in 96-well plates and treated with various doses of CDEAH for 48 h. Cell viability was determined using Cell Titer-Glo reagent. (E) Cell survival in response to various doses of CDEAH with PARPi (olaparib). Indicated cells were grown in 96-well plates and treated with different doses of CDEAH with a fixed indicated dose of olaparib for 6 days. Cell viability was determined using Cell Titer-Blue reagent. (F) Cell survival in response to different doses of olaparib with CDEAH. Cells were grown in 96-well plates and treated with various doses of olaparib with a fixed indicated dose of CDEAH for 6 days. Cell viability was determined using Cell Titer-Blue reagent. (G) CDEAH kills XPA- and CSB-deficient U2OS cells better than WT. U2OS cells deficient in XPA, XPC, CSB or WT were grown in 96-well plates at a density of 3,000 cells per well and treated with CDEAH for 48 h. Cell viability was determined using Cell Titer-Glo reagent. (H) Co-treatment of CDEAH and olaparib shows the effect of CDEAH on lowering the dosage of olaparib to specifically kill BRCA2-deficient cells. IC₅₀ was calculated by nonlinear regression (curve fit) using GraphPad Prism (version 9.0.0). Data are presented as mean ± SEM.

Figure 2.

Characterization of alkylated nucleobases by CDEAH. (A) Alkylating reactions with different nucleobases by CDEAH. (B) Representative UPLC–HRAM-PRM traces of DEAE-purines from CDEAH-treated CTDNA. After incubation with CDEAH for 24 h, alkylated purines were released by thermal hydrolysis and enriched for analysis as described in the ‘Materials and Methods’ section. The black traces represent UPLC–HRAM-PRM analysis of synthesized DEAE-guanine (panel 1, m/z= 251.1615) and DEAE-adenine (panel 3, m/z= 235.1662) standards. The red traces represent UPLC–HRAM-PRM of DEAE-guanine (panel 2) and DEAE-adenine (panel 4) detected from 1.2 μg depurinated CTDNA. (C) Representative UPLC–HRAM-PRM traces of DEAE-pyrimidines enzymatically released from 5 μg CDEAH-treated CTDNA. The top panel represents DEAE-dC (m/z= 327.2027) and the bottom panel represents DEAE-dT (m/z= 342.2023).

Scheme 1.

Reaction of adenine with CDEAH.

Scheme 2.

Reaction of guanine with CDEAH.

Scheme 3.

Reaction of thymine with CDEAH.

Scheme 4.

Reactions of cytosine with CDEAH.

Figure 3.

CDEAH induces more DNA double-strand breaks (DSBs) in PARP1-deficient cells. (A) Cell cycle of HCT116 after CDEAH treatment. HCT116 WT and PARP1-deficient cells were incubated with different doses of CDEAH for 24 h and the relative percentage of cell cycle stages was calculated by FlowJo software. (B) DSB occurrence caused by CDEAH treatment was confirmed by γ-H2AX. HCT116 WT or PARP1-deficient cells were incubated with 80 μM CDEAH for 24 h and indicated protein level was determined in whole-cell extracts. (C) CDEAH treatment enhances DNA damage in HCT116 PARP1-deficient cells. The tail moment in the CometChip^® assay was calculated using the Comet analysis software (Trevigen). (D) SCE analysis in HCT116 WT and PARP1-deficient cells. SCEs were imaged by a BX53 microscope. At least 20 metaphases per each condition were analyzed. (E) Abnormal chromosomes were analyzed in HCT116 WT and PARP1-deficient cells. Abnormal chromosomes were imaged by a BX53 microscope. At least 20 metaphases per each condition were analyzed. (F) Graph displaying the number of SCEs in one metaphase in different conditions. (G) Cells that have >25 breaks in one metaphase were analyzed from panel (E). (H) CDEAH treatment causes more apoptotic cell death in PARP1-deficient cells. Apoptotic cell death was quantified using an Annexin V Alexa Fluor™ 488 conjugate and analyzed by flow cytometry. Data are presented as mean ± SEM.

Figure 4.

CDEAH inhibits the growth of PARP1-deficient xenograft tumors in nude mice. (A) Scheme of in vivo xenograft experiment. Four million cells of either WT HCT116 or PARP1-deficient HCT116 cells were subcutaneously injected into seven-week-old male nude mice. When the tumor size reached ∼200 mm³, vehicle (PBS) or CDEAH (6 mg/kg) was injected intratumorally every 3 days for 16 days. The mice were euthanized, followed by the indicated analyses. (B) Representative orthotopic xenografts of each indicated group (n = 3). (C) Tumor volume change was measured every 3 days for 16 days during drug treatment [n = 5, WT/PBS (black circle); 5, WT/CDEAH (gray square); 4, PARP1-deficient/PBS (gray triangle); 6, PARP1-deficient/CDEAH (pale gray inverted triangle)]. (D) Representative images of H&E, TUNEL and γ-H2AX staining of each dissected xenograft tumor. Data are presented as mean ± SEM.