DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

Abstract

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations

“…Venugopal et al showed increased sensitivity to medicamentous replication stress in cells expressing DNMT3A R882 , due to impaired PARP1 recruitment and dampened DNA damage repair. Consistently, primary DNMT3A WT AML cells were sensitive to pre-treatment with PARP inhibitor olaparib, while DNMT3A mut samples remained unaffected (1). These data suggest that hematopoietic cells with DNMT3A mutations may exhibit PARPi resistance and warrant investigation of PARP inhibitors for managing non-DNMT3A-driven CH to reduce the risk of progression to hematological malignancies and of adverse outcomes in other diseases.…”

“…Our previous studies observed more efficient engagement of the p53 signaling cascade and increased apoptosis in cells with DNMT3A R882 mutations in response to cytarabine (1). Pharmacologic stabilization and potentiation of p53 by pre-treatment with MDM2 inhibitor nutlin-3a was effective in further augmenting cytarabine-induced cell killing.…”

“…Development of such approaches that synergize with this treatment strategy requires identification of tractable molecular mechanisms that execute cytarabine-mediated cell killing. We have zeroed in on impaired recruitment of poly(ADP-ribose) polymerase 1 (PARP1) to damaged DNA, which is necessary for DNA repair initiation as key to cytarabine efficacy in DNMT3A-mutant AML (1). The resultant DNA repair defect and ensuing accumulation of DNA damage triggers activation of the p53 pathway, which is further unleashed by p53-potentiating pharmacologics (1).…”

“…We have zeroed in on impaired recruitment of poly(ADP-ribose) polymerase 1 (PARP1) to damaged DNA, which is necessary for DNA repair initiation as key to cytarabine efficacy in DNMT3A-mutant AML (1). The resultant DNA repair defect and ensuing accumulation of DNA damage triggers activation of the p53 pathway, which is further unleashed by p53-potentiating pharmacologics (1). Here, we outline DNA repair pathways responsible for the removal of chain-terminating aberrant nucleotides, discuss pharmacological inhibition of PARP as a mechanistically-informed strategy to augment cytarabine sensitivity in AML with wild-type DNMT3A, while weighing up the utility of p53 potentiators as a means to boost cytarabine efficacy in the presence of mutant DNMT3A.…”