HSP90 inhibition depletes DNA repair proteins to sensitize acute myelogenous leukemia to nucleoside analog chemotherapeutics

Lai, Tzung Huei; Mitchell, Shaneice; Wu, Pei Jung; Orwick, Shelley; Liu, Chaomei; Ravikrishnan, Janani; Woyach, Jennifer A.; Mims, Alice S.; Plunkett, William; Puduvalli, Vinay K.; Byrd, John C.; Lapalombella, Rosa; Sampath, Deepa

doi:10.1080/10428194.2019.1571197

Cited by 5 publications

(5 citation statements)

References 12 publications

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“…Importantly, HSP90i by NW457 significantly reduced the clonogenic survival upon irradiation in both cell lines ( Figure 1E ), confirming our hypothesis that multi-target interference with DDR function by HSP90i at per se non-toxic doses suffices to sensitize resistant GBM cells to irradiation. Similar findings were very recently reported for other cancer entities ( 62 , 63 ). Morphologically, the mode of cell death underlying reduced clonogenic survival upon HSP90i plus radiation was a highly disruptive, necrotic one which occurred after several rounds of aberrant mitosis and intermediate states of highly aneuploid cells with multiple and/or giant nuclei ( Supplementary Movie File 1 ) ( 64 ).…”

Section: Resultssupporting

confidence: 92%

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

et al. 2021

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Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

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Section: Resultssupporting

confidence: 92%

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

et al. 2021

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“…While previous studies have attributed specific DNA damage repair pathways as defining therapeutic responses to replication stress-inducing nucleoside analogs 33,34,[67][68][69][70] , the exact mechanism(s) that underlie impaired resolution of cytarabine-induced DNA lesions in cells with mutant DNMT3A remain uncertain. Interestingly, other than a slight upregulation of genes annotated to be repressed during UV response indicative of a possible disruption of nucleotide excision repair 42 (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Once cytarabine is converted into its active metabolite ara-CTP, it is incorporated into nascent DNA during replication where it is a poor substrate for chain extension 32 . This results in chain termination, replication fork stalling, and DNA damage response (DDR) often leading to p53-dependent apoptosis [33][34][35][36] . Singlestranded DNA breaks (SSBs) resulting from stalled replication forks actuate ATR-dependent CHK1 phosphorylation necessary to preserve replication fork integrity 37 .…”

Section: Introductionmentioning

confidence: 99%

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

Venugopal

Nowialis

Feng

et al. 2021

Preprint

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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.

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“…Such distinctive sensitivity to cytarabine arises following a defect in recovery from replication fork arrest that leads to accumulation of persistent, unrepaired DNA damage observed in cells with mutant DNMT3A. While previous studies have attributed specific DNA damage repair pathways as defining therapeutic responses to replication stress-inducing nucleoside analogs (28,(56)(57)(58)(59), the exact mechanism(s) that underlie impaired resolution of cytarabine-induced DNA lesions in cells with mutant DNMT3A remain uncertain. Interestingly, other than a slight upregulation of genes annotated to be repressed during UV response indicative of a possible disruption of nucleotide excision repair (34) (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Venugopal

Feng

Nowialis

et al. 2021

Clinical Cancer Research

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Purpose: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Experimental Design: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. Results: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Conclusions: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with 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 replication stress. See related commentary by Viny, p. 573

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HSP90 inhibition depletes DNA repair proteins to sensitize acute myelogenous leukemia to nucleoside analog chemotherapeutics

Cited by 5 publications

References 12 publications

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond

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

DNMT3A Harboring Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

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