Many drugs require extensive metabolism en route to their targets. High-resolution visualization of prodrug metabolism should therefore utilize analogs containing a small modification that does not interfere with its metabolism or mode of action. In addition to serving as mechanistic probes, such analogs provide candidates for theranostics when applied in both therapeutic and diagnostic modalities. Here a traceable mimic of the widely used anticancer prodrug cytarabine (ara-C) was generated by converting a single hydroxyl group to azide, giving "AzC." This compound exhibited the same biological profile as ara-C in cell cultures and zebrafish larvae. Using azide-alkyne "click" reactions, we uncovered an apparent contradiction: drug-resistant cells incorporated relatively large quantities of AzC into their genomes and entered S-phase arrest, whereas drug-sensitive cells incorporated only small quantities of AzC. Fluorescence microscopy was used to elucidate structural features associated with drug resistance by characterizing the architectures of stalled DNA replication foci containing AzC, EdU, γH2AX, and proliferating cell nuclear antigen (PCNA). Three-color superresolution imaging revealed replication foci containing one, two, or three partially resolved replication forks. Upon removing AzC from the media, resumption of DNA synthesis and completion of the cell cycle occurred before complete removal of AzC from genomes in vitro and in vivo. These results revealed an important mechanism for the low toxicity of ara-C toward normal tissues and drug-resistant cancer cells, where its efficient incorporation into DNA gives rise to highly stable, stalled replication forks that limit further incorporation of the drug, yet allow for the resumption of DNA synthesis and cellular division following treatment.
Nucleosides and their analogues constitute
an essential family
of anticancer drugs. DNA has been the presumptive target of the front-line
prodrug for acute myeloid leukemia (AML), cytarabine (ara-C), since
the 1980s. Here, the biomolecular targeting of ara-C was evaluated
in primary white blood cells using the ara-C mimic “AzC”
and azide–alkyne “click” reactions. Fluorescent
staining and microscopy revealed that metabolic incorporation of AzC
into primary white blood cells was unexpectedly enhanced by the DNA
polymerase inhibitor aphidicholine. According to RNaseH digestion
and pull-down-and-release experiments, AzC was incorporated into short
RNA fragments bound to DNA in peripheral blood monocytes (PBMCs) collected
from all six healthy human donors tested. Samples from 22 AML patients
(French–American–British classes M4 and M5) exhibited
much more heterogeneity, with 27% incorporating AzC into RNA and 55%
into DNA. The overall survival of AML patients whose samples incorporated
AzC into RNA was approximately 3-fold higher as compared to that of
the DNA cohort (p ≤ 0.056, χ2 = 3.65). These results suggest that the RNA primers of DNA synthesis
are clinically favorable targets of ara-C, and that variable incorporation
of nucleoside drugs into DNA versus RNA may enable future patient
stratification into treatment-specific subgroups.
For nearly 50 years, translational research studies aimed at improving chemotherapy-induced killing of cancer cells have focused on the induction of apoptosis. Here we show that a PARP-1-mediated programmed cell death mechanism "parthanatos" is associated with the successful, front-line treatment of a common cancer. Peripheral blood mononuclear cells (PBMCs) from healthy human donors (10 of 10 tested), as well as primary cancer cells from approximately 50% of acute myeloid leukemia (AML) patients (n = 18 of 39 tested, French-American-British (FAB) subtypes M4 and M5) exhibited two distinctive features of parthanatos upon treatment with a front-line drug combination of cytarabine and an anthracycline. Statistically significant improvements in survival rates were observed in the parthanatos positive versus parthanatos negative AML patient groups (HR = 0.22-0.38, p = 0.002-0.05). Near-median expression of PARP1 mRNA was associated with a 50% longer survival time (HR = 0.66, p = 0.01), and the poly [ADP-ribose] polymerase (PARP) inhibitor Olaparib exhibited antagonistic activities against ara-C and idarubicin in primary blood monocytes from healthy donors as well as primary cancer isolates from ~50% of AML patients. Together these results suggest that PARP activity is a prognostic biomarker for AML subtypes M4 and M5 and support the relevance of parthanatos in curative chemotherapy of AML.
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