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

Open AccessPublished:March 16, 2022DOI:https://doi.org/10.1016/j.exphem.2022.03.008

      Highlights

      • Chain-terminating nucleotides such as cytarabine are removed by NER, resection of reversed replication forks, or HR.
      • DNMT3AR882 predisposes to replication stress and sensitizes to replication-stalling chemotherapeutics such as cytarabine.
      • PARP1 recruitment defect disrupts DNA damage repair in cells with mutant DNMT3A, rendering cells insensitive to PARPis.
      • Combination with p53 potentiator/MDM2 inhibitor nutlin-3a augments cytarabine sensitivity with DNMT3AR882.
      Cytarabine and other chain-terminating nucleoside analogs that damage replication forks in rapidly proliferating cells are a cornerstone of leukemia chemotherapy, yet the outcomes remain unsatisfactory because of resistance and toxicity. Better understanding of DNA damage repair and downstream effector mechanisms in different disease subtypes can guide combination strategies that sensitize leukemia cells to cytarabine without increasing side effects. We have previously found that mutations in DNMT3A, one of the most commonly mutated genes in acute myeloid leukemia and associated with poor prognosis, predisposed cells to DNA damage and cell killing by cytarabine, cladribine, and other nucleoside analogs, which coincided with PARP1 dysfunction and DNA repair defect (Venugopal K, Feng Y, Nowialis P, et al. Clin Cancer Res 2022;28:756–769). In this article, we first overview DNA repair mechanisms that remove aberrant chain-terminating nucleotides as determinants of sensitivity or resistance to cytarabine and other nucleoside analogs. Next, we discuss PARP inhibition as a rational strategy to increase cytarabine efficacy in cells without DNMT3A mutations, while considering the implications of PARP inhibitor resistance for promoting clonal hematopoiesis. Finally, we examine the utility of p53 potentiators to boost leukemia cell killing by cytarabine in the context of mutant DNMT3A. Systematic profiling of DNA damage repair proficiency has the potential to uncover subtype-specific therapeutic dependencies in AML.
      Acute myeloid leukemia (AML) is an aggressive malignancy of the blood system and the most common acute leukemia in adults [
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      ]. Improvements in chemotherapy combined with a limited number of targeted approaches, thanks to better understanding of the disease mechanisms, have significantly extended survival in younger patients. Yet, the outcomes in advanced-age AML patients (≥65 years, constituting more than half of all cases) remain dismal. Although studies have reported survival benefit of induction chemotherapy dose intensification [
      • Patel JP
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      • Figueroa ME
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      Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.
      ,
      • Sehgal AR
      • Gimotty PA
      • Zhao J
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      DNMT3A mutational status affects the results of dose-escalated induction therapy in acute myelogenous leukemia.
      ], most patients are unable to tolerate aggressive treatment because of comorbidities and frailty [
      • Tamamyan G
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      Frontline treatment of acute myeloid leukemia in adults.
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      ]. A recent clinical study evaluating a novel cladribine- and cytarabine-based low-intensity regimen pointed toward a potential survival benefit specifically in patients with DNMT3A mutations [
      • Kadia TM
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      • Ravandi F
      • et al.
      Cladribine and low-dose cytarabine alternating with decitabine as front-line therapy for elderly patients with acute myeloid leukaemia: a phase 2 single-arm trial.
      ], which motivated our effort to identify a molecular mechanism underpinning this sensitivity [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ].
      Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo AML (20%–35%) and are associated with poor prognosis because of relative resistance to anthracycline-based induction regimens, high rates of minimal residual disease (MRD) positivity, and increased risk of relapse [
      • Patel JP
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      Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.
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      DNMT3A mutational status affects the results of dose-escalated induction therapy in acute myelogenous leukemia.
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      ]. While in myelodysplastic syndromes (MDS) and in clonal hematopoiesis (CH) most DNMT3A mutations are consistent with a loss-of-function (LOF, represented by nonsense, splice, and frameshift alterations), in AML its mutational profile is dominated by hotspot mutations at arginine 882 (R882, 50% and up to 75% of all DNMT3A mutations, with about a half of remaining mutations being LOF), which leads to decreased enzymatic activity and may have additional functions and confer differential therapeutic response [
      • Russler-Germain DA
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      The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by blocking its ability to form active tetramers.
      ,
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      ,
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      Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns.
      ,
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      ]. Although dose-dense anthracyclines can overcome resistance and improve outcomes in younger patients (<65 years), the survival advantage is lost in older patients because of unacceptable treatment-related mortality [
      • Tamamyan G
      • Kadia T
      • Ravandi F
      • et al.
      Frontline treatment of acute myeloid leukemia in adults.
      ,
      • Patel JP
      • Gonen M
      • Figueroa ME
      • et al.
      Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.
      ,
      • Sehgal AR
      • Gimotty PA
      • Zhao J
      • et al.
      DNMT3A mutational status affects the results of dose-escalated induction therapy in acute myelogenous leukemia.
      ]. This prompted exploration of lower-intensity approaches that often combine multiple nucleoside analogs such as cytarabine and cladribine [
      • Kadia TM
      • Cortes J
      • Ravandi F
      • et al.
      Cladribine and low-dose cytarabine alternating with decitabine as front-line therapy for elderly patients with acute myeloid leukaemia: a phase 2 single-arm trial.
      ,
      • Budziszewska BK
      • Salomon-Perzynski A
      • Pruszczyk K
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      Cladribine combined with low-dose cytarabine as frontline treatment for unfit elderly acute myeloid leukemia patients: Results from a prospective multicenter study of Polish Adult Leukemia Group (PALG).
      ,
      • Kadia TM
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      • Borthakur G
      • et al.
      Long-term results of low-intensity chemotherapy with clofarabine or cladribine combined with low-dose cytarabine alternating with decitabine in older patients with newly diagnosed acute myeloid leukemia.
      ], which work by inducing unrestrained replication stress while overwhelming DNA damage repair mechanisms.
      The success of personalized cancer therapy depends on detailed understanding of the molecular mechanisms driving each disease subtype and treatment response. Today, cytarabine (ara-C) and similar replication chain-terminating nucleoside analogs remain a core of AML treatment, whether alone or in combination [
      • Stone RM
      • Mandrekar SJ
      • Sanford BL
      • et al.
      Midostaurin plus chemotherapy for acute myeloid leukemia with a FLT3 mutation.
      ], with low-dose ara-C being a commonly used regimen in advanced-age patients [
      • Tamamyan G
      • Kadia T
      • Ravandi F
      • et al.
      Frontline treatment of acute myeloid leukemia in adults.
      ,
      • Lowenberg B
      • Pabst T
      • Vellenga E
      • et al.
      Cytarabine dose for acute myeloid leukemia.
      ,
      • Burnett AK
      • Russell N
      • Hills RK
      • et al.
      A randomised comparison of the novel nucleoside analogue sapacitabine with low-dose cytarabine in older patients with acute myeloid leukaemia.
      ]. However, combinatorial strategies to further increase cytarabine effect and/or decrease toxicity have not been investigated. 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 [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]. The resultant DNA repair defect and ensuing accumulation of DNA damage trigger activation of the p53 pathway, which is further unleashed by p53-potentiating pharmacologics [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]. Here, we outline DNA repair pathways responsible for the removal of chain-terminating aberrant nucleotides and 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. These combinations potentially offer lower-toxicity approaches suitable for older/unfit patients, a subpopulation with limited treatment options. We also examine the link between PARP inhibitor therapy and emergence of clonal hematopoiesis (CH) in the context of DNMT3A mutations.

      MECHANISMS OF DNA REPAIR AFTER CYTARABINE-INDUCED DAMAGE: PARP1 DYSFUNCTION IN CELLS EXPRESSING MUTANT DNMT3A

      Cytarabine (ara-C) has been the cornerstone of AML treatment since the 1960s [
      • Lamba JK.
      Genetic factors influencing cytarabine therapy.
      ,
      • Kantarjian H
      • Ravandi F
      • O'Brien S
      • et al.
      Intensive chemotherapy does not benefit most older patients (age 70 years or older) with acute myeloid leukemia.
      ,
      • Kantarjian H.
      Acute myeloid leukemia—major progress over four decades and glimpses into the future.
      ]. The main antineoplastic mechanism of action of cytarabine and the similar pyrimidine and purine nucleoside analogs gemcitabine, cladribine, and fludarabine is through incorporation of these non-extendable antimetabolites into the nascent DNA during DNA replication. As a result, these drugs interfere with DNA synthesis and inhibit replicative DNA polymerases, leading to replication fork stalling, strand breaks, and ultimately cell death [
      • Ewald B
      • Sampath D
      • Plunkett W.
      Nucleoside analogs: molecular mechanisms signaling cell death.
      ,
      • Sampath D
      • Rao VA
      • Plunkett W.
      Mechanisms of apoptosis induction by nucleoside analogs.
      ,
      • Berti M
      • Cortez D
      • Lopes M
      The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.
      ]. Genetic variation in the components of nucleoside analog cellular uptake, activation, and metabolism has been implicated in regulating intracellular drug concentrations [
      • Lamba JK.
      Genetic factors influencing cytarabine therapy.
      ], prompting development of dose-intensified regimens. Unfortunately, even with high-dose induction, a substantial subset of patients fail to respond, and many more experience relapse [
      • Tamamyan G
      • Kadia T
      • Ravandi F
      • et al.
      Frontline treatment of acute myeloid leukemia in adults.
      ,
      • Lowenberg B
      • Pabst T
      • Vellenga E
      • et al.
      Cytarabine dose for acute myeloid leukemia.
      ,
      • Kantarjian H
      • Ravandi F
      • O'Brien S
      • et al.
      Intensive chemotherapy does not benefit most older patients (age 70 years or older) with acute myeloid leukemia.
      ,
      • Kantarjian H.
      Acute myeloid leukemia—major progress over four decades and glimpses into the future.
      ], suggesting additional mechanisms of primary resistance. Intensification of DNA repair capacity is appreciated as a key resistance mechanism to DNA-damaging chemotherapy, yet the evidence of its contribution to the resolution of cytarabine-induced lesions is fragmented. More importantly, unlike repair of the damaged template strand, which has been comprehensively studied [
      • Berti M
      • Cortez D
      • Lopes M
      The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.
      ,
      • Zeman MK
      • Cimprich KA.
      Causes and consequences of replication stress.
      ], the mechanisms underlying removal of aberrant chain-terminating nucleotides from the nascent strand during DNA replication remain speculative.
      Incorporation of cytarabine or similar chain-terminating nucleoside analogs into nascent DNA effectively results in generation of single-stand breaks (SSBs) and activation of the ATR-CHK1-mediated intra-S-phase checkpoint; the cell attempts to repair the blockage and restore functional replication forks (Figure 1A, steps 1 and 2). One potential mechanism mediating removal of aberrant nucleotides is nucleotide excision repair (NER) coupled with repriming [
      • Berti M
      • Cortez D
      • Lopes M
      The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.
      ]. In support of this, a small clinical study found that single-nucleotide polymorphisms in NER pathway genes XPC and XPD were associated with differences in overall survival in AML patients treated with standard cytarabine-based induction chemotherapy [
      • Strom SS
      • Estey E
      • Outschoorn UM
      • Garcia-Manero G.
      Acute myeloid leukemia outcome: role of nucleotide excision repair polymorphisms in intermediate risk patients.
      ]. Alternatively, the fork may undergo regression, forming a “chicken foot” structure, followed by resection and partial fork degradation by exonucleases, most notably MRE11 and EXO1 [
      • Berti M
      • Cortez D
      • Lopes M
      The plasticity of DNA replication forks in response to clinically relevant genotoxic stress.
      ]. A critical role for MRE11 3′-to-5′ exonuclease activity in removing gemcitabine blockage from stalled replication forks was recently reported in several eukaryotic systems [
      • Boeckemeier L
      • Kraehenbuehl R
      • Keszthelyi A
      • et al.
      Mre11 exonuclease activity removes the chain-terminating nucleoside analogue gemcitabine from the nascent strand during DNA replication.
      ]. Should the rate of DNA damage repair provided by these mechanisms be inadequate (both of which are PARP dependent), then SSBs are converted to double-strand breaks (DSBs) through fork scission and breakage, activating the ATM/CHK2 signaling node (Figure 1A, step 3). DNA ends are then processed in preparation for strand invasion mediated by RAD51 and homologous recombination repair (HR). Indeed, DSBs induced by another antileukemia nucleoside analog, CNDAC, were reported to be repaired exclusively through HR rather than nonhomologous end joining (NHEJ) [
      • Liu X
      • Wang Y
      • Benaissa S
      • et al.
      Homologous recombination as a resistance mechanism to replication-induced double-strand breaks caused by the antileukemia agent CNDAC.
      ]. Finally, failure to repair DSBs is highly lethal to cells through p53-dependent apoptosis or mitotic catastrophe (Figure 1A, step 4).
      Figure 1
      Figure 1Cells with mutant DNMT3A exhibit specific DNA damage repair defects after cytarabine treatment. (A) DNA damage pathways involved in the repair of cytarabine-induced replication termination and fork stalling. Cytarabine and similar nucleoside analogs such as fludarabine, cladribine, and gemcitabine are incorporated into nascent DNA, leading to chain termination (1). Growing stretches of single-stranded DNA are bound by RPA, which triggers assembly and activation of the ATR/CHK1-dependent checkpoint (2) to facilitate repair mechanism(s), such as (i) repriming and removal of the nonextendable nucleotides by nucleotide excision repair (NER), or (ii) fork reversal and partial degradation by exonucleases EXO1 and/or MRE11. If single-stand break (SSB) repair is unsuccessful, fork breakage ensues (3). Resultant DNA double-stranded breaks (DSBs) activate ATM/CHK2-dependent checkpoint and DNA damage repair through homologous recombination (homologous recombination repair [HR]). Excessive levels of DNA damage lead to activation of p53-dependent apoptosis (4). (B) Cells expressing mutant DNMT3A are proficient in engaging DSB repair pathways. U2OS cells lentivirally expressing wild-type and R882C mutant forms of DNMT3A, or empty vector control, exhibit equal ability to form RAD51 (marker of HR) and 53BP1 (marker of NHEJ) foci after 24 hours of treatment with 10 μmol/L cytarabine, visualized by immunofluorescence staining. (C) Expression of mutant DNMT3A attenuates recruitment of XPA, a key player in the NER pathway, and of MRE11 exonuclease, involved in processing of reversed forks, to the chromatin from soluble nuclear fraction after 12 hours of treatment with 10 μmol/L cytarabine, compared with cells overexpressing wild-type DNMT3A or empty vector control. All experimental procedures for (B) and (C) were as described in Venugopal et al.
      [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]
      .
      We have previously found accentuated sensitivity to cytarabine in cells expressing R882-mutant DNMT3A in vitro and in vivo, wherein high levels of replication stress and DNA damage markers coincided with impaired recruitment of PARP1 to chromatin and decreased DNA repair capacity [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]. However, the specific DNA repair pathway differentially inactivated by expression of mutant DNMT3A was not identified. Examination of the DSB repair pathways revealed that cells with mutant DNMT3A were proficient in engaging both HR and NHEJ, as seen by efficient RAD51 and 53BP1 focus formation after cytarabine treatment compared with controls (Figure 1B). In contrast, chromatin recruitment of a critical NER pathway component, XPA, was strongly promoted by wild-type but not mutant DNMT3A, while chromatin binding of MRE11 was abrogated in the presence of DNMT3Amut (Figure 1C). Thus, MRE11 is a double-edged sword that has been reported to excessively degrade replication forks in the context of HR deficiency such as in Fanconi anemia (FA) or in cells with BRCA1/2 deficiency [
      • Okamoto Y
      • Abe M
      • Mu A
      • et al.
      SLFN11 promotes stalled fork degradation that underlies the phenotype in Fanconi anemia cells.
      ] yet plays a protective role in clearing up replication fork blockage [
      • Boeckemeier L
      • Kraehenbuehl R
      • Keszthelyi A
      • et al.
      Mre11 exonuclease activity removes the chain-terminating nucleoside analogue gemcitabine from the nascent strand during DNA replication.
      ]. These observations point to impaired removal of chain-terminating nucleotides from stalled replication forks in DNMT3Amut cells through PARP1 dysfunction. As a result, DNMT3Amut cells become disproportionately dependent on DSB repair mechanisms, which, when overwhelmed, constitute a therapeutic vulnerability. Further, this mechanism aligns with the favorable response to a nucleoside analog combination regimen consisting of cladribine and low-dose cytarabine alternating with decitabine observed in a group of elderly AML patients with DNMT3A-mutated disease [
      • Kadia TM
      • Cortes J
      • Ravandi F
      • et al.
      Cladribine and low-dose cytarabine alternating with decitabine as front-line therapy for elderly patients with acute myeloid leukaemia: a phase 2 single-arm trial.
      ,
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]. At the same time, cells expressing mutant DNMT3A are insensitive to PARP inhibitors.

      MUTATIONAL PROFILES ILLUMINATE DETERMINANTS OF PARP INHIBITION SENSITIVITY AND RESISTANCE IN MYELOID MALIGNANCIES

      Since the U.S. Food and Drug Administration (FDA) approval of olaparib in 2014, PARP inhibitors (PARPis) have gained a prominent role in the treatment of breast, ovarian, and prostate cancers with BRCA1 and BRCA2 mutations that disrupt the HR pathway. Later, the clinical benefit of olaparib and other PARPis—rucaparib, niraparib, and talazoparib—was observed in tumors with HR deficiency yet without BRCA1/2 lesions, with clinical trials in multiple malignancies and more agents ongoing [
      • Dias MP
      • Moser SC
      • Ganesan S
      • Jonkers J.
      Understanding and overcoming resistance to PARP inhibitors in cancer therapy.
      ]. Recruitment of PARP1 to sites of DNA damage leads to its activation and autoPARylation, which acts as a scaffold for other DNA repair proteins to bind and initiate DNA repair [
      • Ray Chaudhuri A
      • Nussenzweig A
      The multifaceted roles of PARP1 in DNA repair and chromatin remodelling.
      ]. Because of their mechanism of action, which relies mainly on disrupting various pathways of SSB DNA repair (Figure 1A) and direct trapping of PARP1 on DNA lesions [
      • Pommier Y
      • O'Connor MJ
      • de Bono J.
      Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action.
      ], PARP inhibitors have shown promise as a single agent and in combination with DNA-damaging chemotherapy, beyond being synthetic lethal with BRCA1/2 mutations and other HR deficiencies [
      • Dias MP
      • Moser SC
      • Ganesan S
      • Jonkers J.
      Understanding and overcoming resistance to PARP inhibitors in cancer therapy.
      ,
      • Kontandreopoulou CN
      • Diamantopoulos PT
      • Tiblalexi D
      • Giannakopoulou N
      • Viniou NA.
      PARP1 as a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome.
      ].
      Several types of DNA damage are associated with replication stress. Replication fork stalling can be attributed to DNA–protein crosslinks forming bulky adducts, abnormal DNA secondary structures or topology, RNA–DNA hybrids (R-loops), or, in rapidly proliferating cancer cells, scarcity of histones or deoxyribonucleotide triphosphates. Further, impediments to fork progression lead to uncoupling of DNA polymerase from helicase, generating extended stretches of single-stranded DNA (ssDNA). If not adequately protected by RPA in the case of depletion of the RPA pool, this results in scission of naked ssDNA and formation of SSBs [
      • Toledo LI
      • Altmeyer M
      • Rask MB
      • et al.
      ATR prohibits replication catastrophe by preventing global exhaustion of RPA.
      ]. As repair of these DNA lesions is PARP1 dependent, it presents a therapeutic opportunity for the use of PARPis.
      In AML and MDS specifically, current efforts are directed at identification of mutationally defined disease subgroups that may benefit from addition of PARPis as part of their management [
      • Kontandreopoulou CN
      • Diamantopoulos PT
      • Tiblalexi D
      • Giannakopoulou N
      • Viniou NA.
      PARP1 as a therapeutic target in acute myeloid leukemia and myelodysplastic syndrome.
      ]. To this end, a tour-de-force study by Tothova et al. [
      • Tothova Z
      • Valton AL
      • Gorelov RA
      • et al.
      Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.
      ] identified PARP1-mediated DNA damage repair mechanisms as a dependency in STAG2-mutant cells critical for balancing out elevated replication stress experienced by these cells. As a result, AML and MDS with cohesin mutations were uniquely sensitive to the PARPi talazoparib [
      • Tothova Z
      • Valton AL
      • Gorelov RA
      • et al.
      Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.
      ]. Similarly, accumulation of R-loops induced by spliceosome mutations was a source of preferential sensitivity to PARP inhibitors in myeloid malignancies [
      • Nguyen DH
      • Liu ZS
      • Sinha S
      • et al.
      Spliceosome mutant myeloid malignancies are preferentially sensitive to PARP inhibition.
      ]. Earlier studies had found that, akin to solid tumors, myeloid malignancies with HR deficiencies may be more sensitive to PARP inhibition [
      • Pratz KW
      • Koh BD
      • Patel AG
      • et al.
      Poly(ADP-ribose) polymerase inhibitor hypersensitivity in aggressive myeloproliferative neoplasms.
      ]. For example, olaparib was effective against AMLs driven by AML1-ETO and PML-RARα as these fusion transcription factors repressed key HR genes [
      • Esposito MT
      • Zhao L
      • Fung TK
      • et al.
      Synthetic lethal targeting of oncogenic transcription factors in acute leukemia by PARP inhibitors.
      ]. Neo-oncometabolite 2-hydroxyglutarate (2-HG) produced by mutant IDH1 and IDH2 led to increased DNA damage, which was further accentuated by PARPis. Yet, this therapeutic sensitivity was extinguished by pharmacologic inhibition of mutant IDH [
      • Molenaar RJ
      • Radivoyevitch T
      • Nagata Y
      • et al.
      IDH1/2 mutations sensitize acute myeloid leukemia to PARP inhibition and this is reversed by IDH1/2-mutant inhibitors.
      ]. Conversely, the JAK2(V617F) mutation boosts the HR pathway in addition to potently driving myeloproliferative neoplasms (MPNs). This can be reversed by the JAK2 inhibitor ruxolitinib, leading to resensitization of MPN cells to PARPis [
      • Nieborowska-Skorska M
      • Maifrede S
      • Dasgupta Y
      • et al.
      Ruxolitinib-induced defects in DNA repair cause sensitivity to PARP inhibitors in myeloproliferative neoplasms.
      ].
      In circumstances where PARPis are ineffective as a single agent despite DNA repair defects, sensitivity to PARP inhibition can be unveiled by therapeutic interventions that induce or augment DNA damage. Thus, addition of cytotoxic drugs was reported to overcome resistance to PARP inhibition in AMLs driven by MLL fusions attributable to highly active HR [
      • Esposito MT
      • Zhao L
      • Fung TK
      • et al.
      Synthetic lethal targeting of oncogenic transcription factors in acute leukemia by PARP inhibitors.
      ,
      • Maifrede S
      • Martinez E
      • Nieborowska-Skorska M
      • et al.
      MLL-AF9 leukemias are sensitive to PARP1 inhibitors combined with cytotoxic drugs.
      ]. Hypomethylating agents (HMAs) covalently trap DNMTs on DNA forming bulky adducts, which requires PARP1 activity for repair and hence may synergize with PARPis [
      • Muvarak NE
      • Chowdhury K
      • Xia L
      • et al.
      Enhancing the cytotoxic effects of PARP inhibitors with DNA demethylating agents—a potential therapy for cancer.
      ]. Vitamin C, an essential TET2 cofactor, promotes PARPi sensitivity likely through increased demand on PARP-dependent base excision repair (BER), which removes products of TET2-catalyzed methylcytosine oxidation during active DNA demethylation [
      • Brabson JP
      • Leesang T
      • Fang B
      • et al.
      Vitamin C enhances PARPi efficacy for the treatment of AML.
      ].
      Venugopal et al. [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ] found that cells harboring R882 mutant DNMT3A were unable to effectively resolve replication fork damage induced by chain-terminating drugs such as cytarabine and fludarabine. This DNA repair defect coincided with impaired chromatin recruitment of PARP1, rendering it functionally inactive [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]. Consistently, DNMT3A-mutant cells derived no further benefit from pretreatment with the PARP inhibitor olaparib, suggesting PARPi resistance, while cells with wild-type DNMT3A were sensitized by the combination [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ] (Figure 2). In support of this, clinical studies found that efficacy of PARP inhibition correlated with functional levels of PARP1 measured by a novel tracer for microPET imaging, while lack of PARP1 conferred resistance to all PARPis in vitro [
      • Makvandi M
      • Pantel A
      • Schwartz L
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      A PET imaging agent for evaluating PARP-1 expression in ovarian cancer.
      ]. Although the effects of DNMT3A LOF were not investigated in this study, our finding of a lack of PARPi synergism in cells with leukemia-associated DNMT3A R882 mutations aligns well with the joint work of the Skorski and Challen groups indicating that AMLs with DNMT3A mutation or loss were highly resistant to olaparib because of repression of PARP1. On the other hand, TET2-deficient cells were sensitive to PARP inhibition owing to downregulation of several HR genes [
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      ]. Genetic background and choice of primary drug are paramount when devising synthetic lethal approaches for maximal clinical benefit. The possibility of promoting selection and expansion of PARPi-resistant premalignant and leukemic clones should be carefully considered given an emerging link between PARP inhibition and clonal hematopoiesis.
      Figure 2
      Figure 2Mechanism-based combinatorial targeting opportunities to augment cytarabine-induced replication stress in acute myeloid leukemias with and without DNMT3A mutations.

      CLONAL HEMATOPOIESIS DRIVEN BY DNMT3A MUTATIONS: COLLATERAL DAMAGE OF PARP INHIBITOR THERAPY?

      Somatic mutations accumulate in human cells over time, fueling clonal evolution. Each decade, a hematopoietic stem cell (HSC) acquires 100–200 random mutations in its genome, one or two of which affect protein-coding regions [
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      The origin and evolution of mutations in acute myeloid leukemia.
      ]. It is estimated that with 100,000–200,000 HSCs present in the bone marrow in early adulthood, on average 600,000 to 1,200,000 protein-coding mutations are accumulated in the HSC pool by the age of 60 [
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      The evolutionary dynamics and fitness landscape of clonal hematopoiesis.
      ]. Although most mutations are functionally inconsequential or even detrimental, select genetic changes may confer growth advantage allowing a handful of HSCs to outcompete their peers. Similar to other tissues such as esophagus and skin [
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      ,
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      ], age-related clonal expansion of HSCs is a common phenomenon, termed clonal hematopoiesis (CH) [
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      Clonal hematopoiesis in human aging and disease.
      ]. While emergence of small clones defined by variant allele frequency (VAF) <2% without overt hematologic abnormalities is ubiquitous, the presence of larger CH clones (VAF >10%) is associated with increased risk of blood malignancies [
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      ,
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      ,
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      ] and other adverse outcomes ranging from atherosclerosis to cancer to chronic kidney disease [
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      ,
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      ,
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      ,
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      ].
      More than 90% of CH mutations fall into one of three major categories: (1) epigenetic modifier genes, including DNMT3A, TET2, and ASXL1 (“DTA mutations”), which together define a lion's share of CH cases; (2) splicing factors SF3B1, SRSF2, and others; and (3) DNA damage response (DDR) genes such as PPM1D and TP53 [
      • Watson CJ
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      • et al.
      The evolutionary dynamics and fitness landscape of clonal hematopoiesis.
      ]. DNMT3A lesions dominate the mutation landscape, constituting up to a half of all genetic alterations identified in CH [
      • Jaiswal S
      • Fontanillas P
      • Flannick J
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      Age-related clonal hematopoiesis associated with adverse outcomes.
      ,
      • Genovese G
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      Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence.
      ,
      • Buscarlet M
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      DNMT3A and TET2 dominate clonal hematopoiesis and demonstrate benign phenotypes and different genetic predispositions.
      ,
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      Prevalence, predictors, and outcomes of clonal hematopoiesis in individuals aged >/=80 years.
      ]. Although the accumulation of mutations is a stochastic event, research to date has indicated that smoking, chronic inflammation, and previous exposure to chemotherapy or irradiation can promote context-dependent clonal expansion driven by specific genetic alterations [
      • Takahashi K
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      Preleukaemic clonal haemopoiesis and risk of therapy-related myeloid neoplasms: a case–control study.
      ,
      • Coombs CC
      • Zehir A
      • Devlin SM
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      Therapy-related clonal hematopoiesis in patients with non-hematologic cancers is common and associated with adverse clinical outcomes.
      ,
      • Bolton KL
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      • Gao T
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      Cancer therapy shapes the fitness landscape of clonal hematopoiesis.
      ,
      • Dawoud AAZ
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      Clonal myelopoiesis in the UK Biobank cohort: ASXL1 mutations are strongly associated with smoking.
      ,
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      Chronic infection drives Dnmt3a-loss-of-function clonal hematopoiesis via IFNgamma signaling.
      ,
      • Hsu JI
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      PPM1D mutations drive clonal hematopoiesis in response to cytotoxic chemotherapy.
      ]. Notably, recent clinical studies reported that previous anticancer therapy, including that with PARP inhibitors, selectively expands clones with mutations in the DDR-related genes PPM1D, TP53, and CHK2 and confers elevated risk of therapy-related myeloid neoplasia (t-MN) [
      • Bolton KL
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      Cancer therapy shapes the fitness landscape of clonal hematopoiesis.
      ,
      • Bolton KL
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      The impact of poly ADP ribose polymerase (PARP) inhibitors on clonal hematopoiesis.
      ,
      • Gillis NK
      • Ball M
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      Clonal haemopoiesis and therapy-related myeloid malignancies in elderly patients: a proof-of-concept, case–control study.
      ].
      To date, the overwhelming majority of research efforts have been dedicated to investigating the prevalence and implications of CH in various disease settings. In contrast, our knowledge of potential clinical tools for CH prevention and management is still in its infancy. Venugopal et al. [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ] reported increased sensitivity to medicamentous replication stress in cells expressing DNMT3AR882, as a result of impaired PARP1 recruitment and dampened DNA damage repair. Consistently, primary DNMT3AWT AML cells were sensitive to pretreatment with the PARP inhibitor olaparib, while DNMT3Amut samples remained unaffected. 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 adverse outcomes in other diseases. However, considering recent studies linking CH and t-MN to PARPi exposure [
      • Bolton KL
      • Ptashkin RN
      • Gao T
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      Cancer therapy shapes the fitness landscape of clonal hematopoiesis.
      ,
      • Bolton KL
      • Moukarzel LA
      • Ptashkin R
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      The impact of poly ADP ribose polymerase (PARP) inhibitors on clonal hematopoiesis.
      ,
      • Gillis NK
      • Ball M
      • Zhang Q
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      Clonal haemopoiesis and therapy-related myeloid malignancies in elderly patients: a proof-of-concept, case–control study.
      ], careful patient stratification will be paramount for balancing potential beneficial and detrimental consequences of this intervention. Indeed, a meta-analysis of more than 7,000 evaluable patients enrolled in 18 randomized placebo-controlled clinical trials of PARPis found significantly increased risk of MDS and AML during follow-up [
      • Morice PM
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      Myelodysplastic syndrome and acute myeloid leukaemia in patients treated with PARP inhibitors: A safety meta-analysis of randomised controlled trials and a retrospective study of the WHO pharmacovigilance database.
      ]. This concerning finding raises an important question: What strategies can be devised to target PARPi-resistant premalignant and leukemic clones?

      COMBINATIONS WITH P53 ACTIVATORS: AN EMERGING THERAPEUTIC OPPORTUNITY FOR DNMT3A-MUTATED MALIGNANCIES

      Despite being the most commonly inactivated tumor suppressor in human cancers [
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      ,
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      p53 mutations in cancer.
      ], the TP53 gene, which codes for the p53 protein, is rarely mutated in de novo AML [
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      TP53 mutations in newly diagnosed acute myeloid leukemia: clinicomolecular characteristics, response to therapy, and outcomes.
      ,
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      TP53 mutations in older adults with acute myeloid leukemia.
      ,
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      TP53 in hematological cancer: low incidence of mutations with significant clinical relevance.
      ]. Instead, many leukemias evade its tumor suppressor function by increased expression of the p53 negative regulators MDM2 and MDMX, which inactivate p53 through direct protein binding and ubiquitin-mediated degradation [
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      The human MDM-2 oncogene is overexpressed in leukemias.
      ,
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      High Mdm4 levels suppress p53 activity and enhance its half-life in acute myeloid leukaemia.
      ,
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      MDM2 antagonists induce p53-dependent apoptosis in AML: implications for leukemia therapy.
      ]. Retention of functional p53 in these neoplasms spurred efforts to develop targeted anticancer therapies that aim to reverse its negative regulation.
      Interest in compounds that impede p53 binding to MDM2 and/or MDMX and thus restore p53 activity has led to an explosion of small molecule drug candidates, as well as stapled peptides and proteolysis-targeting chimeras (PROTACs) [
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      ,
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      ,
      • Li Y
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      ,
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      ,
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      Recent progress and clinical development of inhibitors that block MDM4/p53 protein–protein interactions.
      ]. Additionally, taking aim at negative regulators of the p53 downstream effectors, such as BCL-2 inhibitor venetoclax, which in combination with hypomethylating agents is now considered the standard of care for AML and MDS patients ineligible for intensive chemotherapy [
      • DiNardo CD
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      ,
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      ], has shown great promise. For tumors that express mutant p53 protein, including gain-of-function isoforms, there is a class of therapies that target aberrant p53, either by inhibiting its activity or by refolding it to the correct conformation [
      • Hu J
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      ]. These and other strategies are reviewed in detail elsewhere [
      • Hu J
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      Targeting mutant p53 for cancer therapy: direct and indirect strategies.
      ,
      • Konopleva M
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      MDM2 inhibition: an important step forward in cancer therapy.
      ,
      • George B
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      TP53 in acute myeloid leukemia: molecular aspects and patterns of mutation.
      ].
      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 [
      • Venugopal K
      • Feng Y
      • Nowialis P
      • et al.
      DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks.
      ]. Pharmacologic stabilization and potentiation of p53 by pretreatment with the MDM2 inhibitor nutlin-3a were effective in further augmenting cytarabine-induced cell killing. In agreement with this, ex vivo drug dose–response studies conducted as part of the BeatAML clinical trial [
      • Tyner JW
      • Tognon CE
      • Bottomly D
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      Functional genomic landscape of acute myeloid leukaemia.
      ] found preferential sensitivity to the cytarabine/nutlin-3a combination specifically in AML specimens with DNMT3A R882 mutations. These findings are a strong indication that DNMT3AR882 may be a predictive biomarker for the efficacy of p53-potentiating therapy (Figure 2). The pressing need to identify mechanistically informed criteria for patient stratification is highlighted by the results of a recent double-blind placebo-controlled clinical trial evaluating the efficacy of idasanutlin, a derivative of nutlin-3a with improved druglike properties, in combination with cytarabine in patients with relapsed/refractory AML (ClinicalTrials.gov identifier: NCT02545283). Although the experimental arm revealed a favorable safety and toxicity profile (no patients were discontinued because of adverse events), the trial was terminated as meaningful superiority was not reached at time of interim analysis. The design of this study did not mandate molecular testing beyond the clinically actionable mutation-defined subpopulations (FLT3 and IDH1/2). Yet, the IDH-mutated subtype tended to exhibit superior rates of complete response and complete response with incomplete hematologic recovery (CR/CRi) at the end of induction. This indicates that more granular genetic characterization to separate disease subgroups wherein p53 potentiators are beneficial is warranted and feasible.
      Further studies are necessary to clarify molecular mechanisms underlying enhanced cytarabine/nutlin-3a response in DNMT3A-mutant cells. It has been reported that p53 allosterically represses methyltransferase activity of wild-type DNMT3A, and this inhibition is blocked by R882 hotspot mutation [
      • Sandoval JE
      • Reich NO.
      The R882H substitution in the human de novo DNA methyltransferase DNMT3A disrupts allosteric regulation by the tumor supressor p53.
      ]. Whether p53 can promote the protective effect of wild-type but not mutant DNMT3A in stabilizing damaged replication forks, or if wild-type and mutant DNMT3A are differentially involved in p53 downstream responses [
      • Zhang Y
      • Gao Y
      • Zhang G
      • et al.
      DNMT3a plays a role in switches between doxorubicin-induced senescence and apoptosis of colorectal cancer cells.
      ], remains to be determined, along with potential implications of DNMT3A LOF mutations. Changes in p53 axis in the context of mutant DNMT3A may merit separating AML patients with this mutation into a distinct treatment category that may particularly benefit from a combination treatment with cytotoxic chemotherapy and MDM2/MDMX inhibitors.

      CONCLUDING REMARKS AND TRANSLATIONAL OUTLOOK

      Detailed mechanistic understanding of the dependencies and synthetic–lethal interactions in genetically defined disease subtypes is crucial to improving treatment response rates in AML. This is an exciting and rapidly growing field, and many questions remain unanswered. How is DNA damage repaired depending on treatment and disease subtype? Which factors determine efficacy and should be used for patient stratification? Can mechanistically guided combination approaches offer lower toxicity in elderly and/or frail patients while maintaining the “intent to treat” of high-intensity regimens? What intervention strategies can be devised to manage clonal hematopoiesis and to reduce the risk of malignant progression, and to which patient subgroups will these apply? We propose that AML patients with wild-type DNMT3A may benefit from a combination of PARP inhibitors with low-intensity regimens of replication stalling nucleoside analogs in the absence of other negative predictive factors. Conversely, cases bearing the DNMT3A hotspot R882 mutation may benefit from MDM2/MDMX inhibitors (Figure 2). Better understanding of the mechanisms of DNA damage repair, together with personalized therapy based on the genetic landscape of the disease, will be crucial to improving outcomes in this challenging group of leukemia patients.

      Conflict of Interest Disclosure

      The authors do not have any conflicts of interest to declare in relation to this work.

      Acknowledgments

      The authors gratefully acknowledge support by the National Institutes of Health (R01 DK121831), the Ocala Royal Dames for Cancer Research, and the Thomas H. Maren Junior Investigator Fund (to OAG). We thank Lidia Kulemina (University of Florida Health Cancer Center) for critical reading of the manuscript and Cassandra Berntsen (Department of Pharmacology and Therapeutics, University of Florida) for editorial assistance.

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