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REVIEW ARTICLE
ACUTE MYELOID LEUKEMIA

Relapsed acute myeloid leukemia in children and adolescents:

current treatment options and future strategies
1 3✉
Sara Zarnegar-Lumley , Kenneth J. Caldwell2 and Jeffrey E. Rubnitz

© The Author(s), under exclusive licence to Springer Nature Limited 2022

Pediatric acute myeloid leukemia (AML) develops from clonal expansion of hematopoietic precursor cells and is characterized by
morphologic and cytomolecular heterogeneity. Although the past 40 years have seen significant improvements in overall survival,
the prevailing treatment challenges in pediatric AML are the prevention of relapse and the management of relapsed disease.
Approximately 25% of children and adolescents with AML suffer disease relapse and face a poor prognosis. Our greater
understanding of the genomic, epigenomic, metabolomic, and immunologic pathophysiology of relapsed AML allows for better
therapeutic strategies that are being developed for pediatric clinical trials. The development of biologically rational agents is critical
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as conventional chemotherapeutic salvage regimens are not effective for all patients and pose risk of organ toxicity in heavily
pretreated patients. Another major barrier to improvement in outcomes for relapsed pediatric AML is the historic lack of availability
and participation in clinical trials. There are ongoing efforts to launch multinational clinical trials of emerging therapies. The purpose
of this review is to summarize currently available and newly developed therapies for relapsed pediatric AML.

Leukemia (2022) 36:1951–1960; https://doi.org/10.1038/s41375-022-01619-9

INTRODUCTION evolution occurred in about 30% of AML patients who relapsed

Approximately 25% of children with acute myeloid leukemia
(AML) suffer relapse of their disease, and despite aggressive
chemotherapy and hematopoietic cell transplantation (HCT), only
about 40% of these patients become long-term survivors. The
outcome of children with relapsed AML has only slightly improved
over the past 20 years, likely related to improvements in
supportive care and better outcomes after HCT [1]. This slow
progress is partly related to the lack of enrollment of such patients
on clinical trials. We previously estimated that from 2006 to 2016,
less than 25% of children with relapsed AML in the US and Canada
were treated on clinical trials [2], and a literature review identified
only a single report of a randomized trial for children with
relapsed AML [3, 4]. In addition, the lack of improvement can be
attributed to a shortage of new agents, a paucity of targeted
therapies, and the slow development of immunotherapy. We
predict, however, that recent advances in our understanding of
the biology and genomics of relapsed AML, the expansion of
therapies directed at leukemia-selective abnormalities, and the
organization of international collaborative clinical trials will soon
lead to novel therapies and improved outcomes.
BIOLOGY AT RELAPSE
In the 1970s, conventional banding techniques on paired samples
from diagnosis and relapse demonstrated that karyotypic
[5]. Approximately 30 years later, whole-genome sequencing of
eight adult AML patients at diagnosis and at relapse demonstrated
clonal evolution in all cases [6]. In three cases, the founding
(dominant) clone at diagnosis gained mutations and evolved into
the relapse clone. In the other five cases, a subclone of the
dominant clone gained mutations and expanded to become the
relapse clone. In both scenarios, it is likely that at least a subset of
the mutations acquired at relapse contributed to chemotherapy
resistance. Another mechanism by which clonal evolution
contributes to relapse is human leukocyte antigen (HLA)
haplotype loss after HCT [7, 8]. In the first report of this
phenomenon of immune escape, the leukemic blasts of five
patients who relapsed after haploidentical HCT had lost the HLA
haplotype that differed from the donor haplotype though
uniparental disomy of chromosome 6p, including the HLA region
[7]. A similar mechanism of immune escape has been reported
after matched unrelated HCT [9].
Among children with relapsed AML, karyotypic evolution is
observed in ~50% of cases, similar to the findings that have been
reported in adults [10]. Until recently, however, little was known
about the genomics of relapsed childhood AML. As part of the
Therapeutically Applicable Research to Generate Effective Treat-
ment AML initiative of the National Cancer Institute (NCI) and the
Children’s Oncology Group (COG), whole-exome sequencing was
performed from diagnostic, remission, and relapse samples for 20

1
Division of Pediatric Hematology and Oncology, Vanderbilt University Medical Center, Nashville, TN, USA. 2Cancer and Blood Disorders Institute, Johns Hopkins All Children’s
Hospital, St. Petersburg, FL, USA. 3Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA. ✉email: Jeffrey.Rubnitz@stjude.org

Received: 31 March 2022 Revised: 18 May 2022 Accepted: 26 May 2022

Published online: 6 June 2022
 S. Zarnegar-Lumley et al.
1952
pediatric patients, excluding those with high-risk cytogenetics
[11]. This group identified significant clonal heterogeneity
between diagnostic and relapse specimens with resolution of
diagnostic somatic mutations and emergence of new mutations at
relapse. They also noted change in the variant allele frequency
(VAF) at the two time points; a somatic mutation with a higher VAF
at diagnosis was more likely to persist at relapse. Similar to the
clonal evolution of somatic mutations, divergence was observed
for copy number alterations. Overall, this work highlights the
heterogeneity and evolving genomic profile of pediatric AML at
relapse. It should be noted, however, that the simple presence of a
new mutation at the time of relapse does not necessary imply that
the mutation confers drug resistance or is a therapeutic target. In
addition, some mutations may be subclonal, in which case
targeting those mutations may not be effective.
Researchers at St. Jude Children’s Research Hospital (St. Jude)
recently performed RNA sequencing, whole-genome sequencing,
and target-capture sequencing on 136 relapsed cases [12]. This
relapse cohort was characterized by fusions that are known to be
associated with a high risk of relapse, including KMT2A fusions
(n = 36) [13], NUP98 fusions (n = 18) [14], GLIS fusions (n = 3) [15],
PICALM-MLLT10 (n = 5) [16], FUS-ERG (n = 4) [17], and DEK-NUP214
(n = 4) [18]. In addition, in-frame exon 13 tandem duplications
(TD) in upstream binding transcription factor (UBTF) were
identified in 12 (8.8%) cases. Because UBTF-TDs have rarely been
reported in pediatric AML, 417 pediatric AML cases from previous
studies were re-analyzed and 15 additional cases with UBTF-TDs
were detected. In multivariable analysis, UBTF-TD was associated
with a poor outcome, even in patients with FLT3-ITD, suggesting
that UBTF-TD is the driver mutation in patients whose blasts
harbor FLT3-ITD without known driver mutations. Importantly,
although first characterized in a relapsed cohort, this study
indicates that UBTF-TD is more prevalent at diagnosis than
previously recognized and identifies a novel subtype of childhood
AML that is associated with a high risk of relapse.
Leukemia stem cells (LSCs) are a key contributor to the biology
of AML relapse and the focus of enhanced risk stratification and
new therapies. First identified as rare subpopulations responsible
for engraftment in xenotransplantation assays [19, 20], LSCs
maintain the properties of self-renewal and cell cycle quiescence
and can evade standard chemotherapeutic agents, thus allowing
for disease persistence and relapse [21]. Stemness in AML has
been characterized by unique immunophenotypic signatures,
designated LSC17 and pLSC6, that have been proposed as
surrogate biomarkers for adverse prognosis [22–24]. Other
biomarkers of stemness have emerged as potential predictors of
relapse and targets for therapy including calcitonin receptor-like
(CALCRL) [25]. More recently, better understanding of LSC
machinery has allowed for investigation into metabolic and
epigenetic differences between LSCs and normal hematopoietic
stem cells (HSCs) allowing for exploitation of differences in
emerging therapeutics [21].
Epigenetic dysregulation has been implicated in the pathophy-
siology of pediatric AML [26]. Pathologic modification of DNA,
histones, and chromatin promote aberrant cell proliferation, self-
renewal, and differentiation block. These mechanisms are
essential for chemotherapy resistance and may underlie AML
relapse. Therapeutic epigenetic reprogramming has the potential
to restore gene expression of silenced tumor suppressor genes
and enhance chemo-sensitivity of AML blasts.
Retrospective studies
Reports published over the past 20 years indicate that the
prognosis of children with relapsed AML has remained unaccep-
tably low. Among 106 children with AML who relapsed after
treatment on the Leucemie Aique Myeloide Enfant 89/91 protocol
from 1988–1998, the 5-year overall survival rate was 33%. In this
study, late relapse, defined as relapse greater than 1 year from the
attainment of remission, was associated with better outcome [27].
An analysis of 379 children with AML who relapsed after treatment
on the AML-Berlin/Frankfurt/Muenster (BFM) 87, 93, and 98
protocols from 1987–2003 revealed a 5-year survival rate of 23%
overall and 31% for patients treated on the most recent protocol
(AML-BFM 98). Late relapse, no HCT in first remission, and the
presence of core-binding-factor (CBF) leukemia were all associated
with better survival [28]. Similarly, a retrospective study of 99
children with relapsed AML treated at Therapeutic Advances in
Childhood Leukemia & Lymphoma (TACL) institutions between
1995–2004 revealed a 5-year survival rate of 29% [29]. A slightly
higher survival rate of 37% was reported for the 71 patients who
relapsed after treatment on the Japanese AML99 trial (2000–2002).
In multivariate analysis, early relapse and the presence of FLT3-ITD
were significantly associated with worse outcome [30]. A nearly
identical survival rate of 36% was reported in an analysis of 111
patients who relapsed after treatment on the subsequent Japanese
Pediatric Leukemia-Lymphoma Study Group AML-05 trial
(2006–2010). Again, multivariate analysis identified early relapse
and the presence of FLT3-ITD as negative prognostic factors [31].
Likewise, the 5-year survival rate among 208 children who relapsed
after treatment on the Nordic Society for Paediatric Haematology
and Oncology (NOPHO)-AML93 or NOPHO-AML 2004 (1993–2012)
trials was 39%. As in all forementioned studies, the strongest
predictor of survival after relapse was time to relapse. Late relapse,
no HCT in first remission, and the presence of CBF leukemia were
all associated with better survival [32]. In a retrospective analysis of
patients in the most recent treatment era, investigators from the
BFM and COG reported survival rates of 42% and 35% respectively,
with high-risk cytomolecular features, poor initial induction
response, and early relapse predictive of worse outcome [1]. In
this analysis, there was a trend toward improved survival rates in
recent years, possibly related to a greater number of such patients
undergoing HCT from alternative donor sources or proceeding to
HCT prior to count recovery. In fact, this report demonstrated that
full hematopoietic recovery after salvage therapy was not
predictive of survival [1]. In addition, improvements in supportive
care, including the availability of more potent antifungal agents,
likely contributed to the improvement in outcome.
Although the outcome after second relapse is poor, a subset of
these patients can be cured even without novel interventions. For
example, in an analysis of 73 patients who suffered second
relapses after treatment on AML-BFM trials from 2004–2017, the
5-year survival was 15%. Whereas patients with early second
relapses and those who failed to achieve a third remission had
dismal outcomes, 31% of those who underwent subsequent HCT
survived [33]. A recent review of 157 patients on NOPHO-DB SHIP
consortium trials who experienced a first relapse that was
refractory (n = 98) or a second relapse (n = 59) indicated similar
outcomes, with 1-year and 5-year survival rates of 22% and 14%,
respectively [34]. At St. Jude, outcomes among AML patients who
relapsed after undergoing HCT have increased over time, with a
second HCT being the only curative modality [35]. Thus, patients
who suffer second relapses may be candidates for intensive
chemotherapy or second transplants, options that may hamper
the enrollment of such patients on phase 1 trials.
Completed trials
New combinations of cytotoxic chemotherapy. Until recently, the
mainstay of relapse treatment had been reinduction therapy with
new combinations of conventional cytotoxic chemotherapy with a
goal of achieving remission prior to HCT. Many combinations have
shown activity, but few have led to significantly better outcomes.
A Children’s Cancer Group study that enrolled children with
relapsed or refractory (R/R) AML showed that reinduction with
mitoxantrone and cytarabine was effective with reasonable
toxicity [36]. Clofarabine, a nucleoside analog that induced a
response rate of 30% as single agent [37], has been studied in a
Leukemia (2022) 36:1951 – 1960

variety of combinations. Among 49 children with R/R AML who
received clofarabine and cytarabine on the COG study AAML0523,
the overall response rate was 48% [38]. Similar response rates
were reported after treatment with the combinations of clofar-
abine, high-dose cytarabine and liposomal daunorubicin [39],
clofarabine, cyclophosphamide and etoposide [40], and clofar-
abine, topotecan, vinorelbine, and thiotepa [41]. Other combina-
tions of conventional therapy that have been studied include
cladribine and topotecan [42] and topotecan, vinorelbine,
thiotepa, dexamethasone, and gemcitabine [43].
New formulations of cytotoxic chemotherapy. Several clinical trials
have focused on new formulations of cytotoxic chemotherapy
rather than on novel agents. In the only published randomized
clinical trial for relapsed pediatric AML, 394 patients were randomly
assigned to receive fludarabine, cytarabine, and GCSF (FLAG) or
FLAG plus liposomal daunorubicin [4]. The complete remission (CR)
rates were 69 and 59% for patients who were treated with FLAG
plus liposomal daunorubicin vs. FLAG alone, but survival rates were
similar across treatment groups. However, treatment with liposo-
mal daunorubicin was associated with improved survival among
patients with CBF leukemia [4]. More recently, investigators from
the COG reported that Vyxeos (formerly CPX-351), a liposomal
formulation of daunorubicin and cytarabine, was safe and active in
children with relapsed AML, with a CR rate of 54% and an minimal
residual disease (MRD) negative rate of 84% among patients who
achieved CR or CR with incomplete platelet recovery (CRp) [44]. It
should be noted, however, that the results of aforementioned trials
should not be directly compared, as the number of patients in the
latter trial was quite small (n = 38) and the confidence intervals
large (e.g., CR + CRp rate = 68.3%, CI, 52.9–78.0%). Nevertheless,
these encouraging results have led to a randomized trial
(AAML1831, NCT: 04293562) of Vyxeos and gemtuzumab ozoga-
micin (GO) vs. conventional daunorubicin, cytarabine, and GO in
children with newly diagnosed AML.
Novel and targeted agents. The results of phase 1 or 2 studies of a
variety of agents, including valspodar [45], lenalidomide [46],
bortezomib [47], plerixafor [48], selinexor [49], panobinostat [50],
azacitidine [51], and venetoclax [52] have been reported.
Valspodar, an inhibitor of potent P-glycoprotein, was tested in
combination with mitoxantrone and etoposide in pediatric
patients with R/R leukemia. The regimen was safe, but blasts
from only one patient demonstrated inhibition of P-glycoprotein
and no patients with AML responded [45]. Similarly, in a phase
2 study of lenalidomide in 17 pediatric patients with R/R AML, only
1 patient responded [46]. Notably, the responding patient had a
complex karyotype that included del(5q), consistent with reports
that lenalidomide is active in this genetic subtype of AML in
adults. A phase 2 study of bortezomib combined with either
idarubicin and cytarabine or cytarabine and etoposide in patients
with R/R AML demonstrated that both regimens were tolerable.
However, low CR rates led to closure of both study arms during
the first stage of the two-stage design [47]. In a phase 1 trial of the
CXCR4 antagonist plerixafor in combination with cytarabine and
etoposide, treatment with plerixafor resulted in mobilization of
leukemia blasts into the blood in 14 of 16 patients. However,
among the 13 patients with relapsed AML, only 2 achieved CR
[48]. The disappointing response rates observed in these studies
of valspodar, lenalidomide, bortezomib, and plerixafor suggest
that they should not be studied further, except in specific
subgroups of patients who are likely to benefit.
Epigenetic priming with histone deacetylase (HDAC) inhibitors
or DNA methyltransferase (DNMT) inhibitors has been used in
attempts to sensitize leukemic blasts to standard chemotherapy. A
phase 1 study in which 17 children with R/R AML received the
HDAC inhibitor panobinostat before, and in combination with,
fludarabine and cytarabine showed that blasts from 7 of 9 patients
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S. Zarnegar-Lumley et al.
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demonstrated increases in acetylation. In addition, this regimen
was safe and 8 patients achieved CR, of whom 6 had negative
MRD, suggesting that priming with HDAC inhibitors should be
further studied in patients with AML [50]. Using a similar design,
the TACL consortium conducted a phase 1 study in which the
DNMT inhibitor azacitidine was given prior to fludarabine and
cytarabine in children with R/R leukemia [51]. This regimen was
well-tolerated and 7 of 12 patients achieved CR, suggesting that it,
too, be studied further. The TACL consortium recently completed a
trial that combined these two strategies of epigenetic reprogram-
ming in which pediatric patients with R/R AML received decitabine
and vorinostat prior to conventional therapy. In this study, 19 of 35
of patients achieved CR or CR with incomplete count recovery
(CRi) and 17 achieved MRD negativity [53]. Based on the
encouraging results of the aforementioned studies, epigenetic
priming is now being tested in a phase 2 trial in children with
newly diagnosed AML at St. Jude (NCT03164057).
Overexpression of the antiapoptotic proteins BCL2, BCL-XL, and
MCL1, which sequester pro-apoptotic BH3-only proteins and
prevent activation of the death effector proteins BAX and BAK,
is associated with resistance to chemotherapy and represent
another therapeutic target to which potent inhibitors have been
developed [54, 55]. Venetoclax, a selective inhibitor of BCL2,
induces apoptosis in AML cell lines, primary samples, and mouse
xenograft models [56]. Clinical trials demonstrated the safety and
activity of venetoclax in combination with hypomethylating
agents or low-dose cytarabine in elderly patients with AML,
leading to FDA approval of these combinations [57–59]. A pilot
study (VENAML) demonstrated that venetoclax could be safely
combined with intensive chemotherapy in children with R/R AML
[52]. Overall responses were observed in 24 (69%) of 35 evaluable
patients, including 16 CR (11 MRD negative), 4 CRi (2 MRD
negative), and 4 partial response (PR). Among the 20 patients
treated at the recommended phase 2 dose 14 (70%) achieved CR
or CRi and 16 (80%) CR/CRi/PR. This trial has been amended to
include the combination of azacitidine, venetoclax, and high-dose
cytarabine and remains open to accrual (NCT03194932).
Exportin 1 (XPO1) is a nuclear exporter of tumor suppressor and
growth regulatory proteins that AML cells can co-opt to proliferate
and evade apoptosis. Selective inhibitors of nuclear export, such
as selinexor, bind XPO1, prevent nuclear export of cargo proteins,
and restore tumor suppressor function in AML. A phase I pediatric
study from St. Jude characterized the toxicity, pharmacokinetics,
and pharmacodynamics of selinexor combined with fludarabine
and cytarabine in children with R/R leukemia [49]. The results of
this trial demonstrated that selinexor was tolerable at doses up to
55 mg/m2 given twice weekly and that all patients who received
selinexor at ≥40 mg/m2 demonstrated XPO1 target inhibition. In
this group of heavily pretreated patients, 7 of 15 evaluable
patients achieved CR or CRi, including two patients who became
MRD negative after receiving only four doses of selinexor without
chemotherapy [49]. The striking single-agent activity of selinexor
in these patients indicates that there is a subgroup of patients
who may benefit from this agent, even in the absence of cytotoxic
chemotherapy. However, the lack of biomarkers to identify which
patients will respond to selinexor has slowed the further
development of this agent in pediatric AML.
Current and future targets and trials
The development of new agents for pediatric AML has been slow,
in part because of the low incidence of the disease and the
different spectrum of mutations in childhood AML compared to
AML in older adults. Prioritizing agents for clinical trials and
efficiently conducting such trials will require international
collaboration. The Leukemia and Lymphoma Society Pediatric
Acute Leukemia initiative is currently working with the European
Pediatric Acute Myeloid Leukemia consortium to coordinate
preclinical testing, prioritize targets, screen patients, and develop
 S. Zarnegar-Lumley et al.
1954
Table 1. Current and future studies.
Target Regimen ClinicalTrials.gov ID Study group Status
BCL2 Venetoclax + azacitidine + cytarabine NCT03194932 St. Jude Recruiting
BCL2 and CD33 Fludarabine/cytarabine/gemtuzumab with or NCT05183035 LLS PedAL Initiative, LLC Planned
without venetoclax
BCL2 and XPO1 Venetoclax + selinexor + fludarabine/cytarabine NCT04898894 St. Jude Recruiting
PD-1 Nivolumab + azacitidine NCT03825367 TACL Recruiting
IDH2 Enasidenib NCT04203316 COG Recruiting
MDM2 Idasanutlin + chemotherapy or venetoclax NCT04029688 Hoffmann-La Roche Recruiting
Menin SNDX-5613 + fludarabine/cytarabine NCT04065399 Syndax Pharmaceuticals Recruiting
Menin KO-539 + fludarabine/cytarabine Pending Kura Oncology Planned
E-selectin Uproleselan + fludarabine/cytarabine Pending LLS PedAL Initiative, LLC Planned
CD123 CD123 CAR T cells NCT04318678 St. Jude Recruiting
CD123 CD123 CAR T cells NCT04678336 University of Pennsylvania Recruiting
CD123 Flotetuzumab NCT04158739 COG/PEP-CTN Closed
CD123 IMGN632 Pending LLS PedAL Initiative, LLC Planned
CD123 Tagraxofusp IL3 Pending TACL Planned
CD33 CD33 CAR T cells NCT03971799 CIBMTR Recruiting
CD33 CD33 x CD3 bispecific T-cell-engager NCT05077423 COG/PEP-CTN Planned
Non-specific NK cells + DLI NCT03068819 Washington University Recruiting
CD38 Isatuximab + chemotherapy NCT03860844 Sanofi Recruiting
CD33 Vyxeos + gemtuzumab NCT04915612 M.D. Anderson Cancer Center Recruiting
XPO1 exportin 1, PD-1 programmed death-1, IDH2 isocitrate dehydrogenase 2, MDM2 mouse double minute 2, CAR chimeric antigen receptor, NK natural killer,
DLI donor lymphocyte infusion, LLS Leukemia & Lymphoma Society, PedAL pediatric acute leukemia, TACL Therapeutic Advances in Childhood Leukemia &
Lymphoma, COG Children’s Oncology Group, CIBMTR Center for International Blood and Marrow Transplant Research, PEP-CTN The Pediatric Early Phase Clinical
Trials Network.

Bispecific antibodies:
• CD3xCD123
Epigenetic priming • CD3xCD33
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Menin inhibitors mm mu
ro g ra no
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CD123 CART
LSD1 inhibitors r ep era PD-1 (Nivolumab) + azacitidine
n etic p eu
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Antibody-drug conjugate
Monoclonal antibody
Cell cycle & signaling

IMGN632
SYK inhibitors
Tagraxofsup
CDK4/6 inhibitors
Magrolimab
MEK inhibitors
Isatuximab
FLT3 inhibitors

Venteoclax + Azacitidine + HiDAC

al

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et FLA + GO ± Venetoclax
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Idasanutlin + Venetoclax
IDH inhibitors
Microenvironment ALRN-6924
Talazoparib
Pevonedistat
Uproselan
SYK: spleen tyrosine kinase, CDK: cyclin dependent kinase, MEK: mitogen-activated protein kinase kinase, FLT3: fms-like tyrosine kinase 3, HiDAC: high dose cytarabine, FLA: fludarabine + cytarabine, GO: gemtuzumab ozogamicin, CART: chimeric antigen receptor, PD-1: programmed death-1, LSD1: lysine-specific demethylase 1

Fig. 1 Current and future targets by therapeutic subclass. Therapeutic targets are indicated in the circle and potential therapies are listed in
the boxes. Abbreviations: SYK, spleen tyrosine kinase; CDK, cyclin dependent kinase; MEK, mitogen-activated protein kinase; FLT3, fms-like
tyrosine kinase 3; HiDAC, high-dose cytarabine; FLA, fludarabine + cytarabine; GO, gemtuzumab ozogamicin; CART, chimeric antigen receptor;
PD-1, programmed death-1; LSD1, lysine-specific demethylase 1.

clinical trials that will be conducted in North America, Europe, BCL2/BCL-XL/MCL1 inhibitors
Australia, and New Zealand [60]. A partial list of trials that are This excellent response rate observed in the VENAML trial
currently recruiting or planned is shown in Table 1. Current and described above has led to the development of an international
future targets are represented schematically by therapeutic randomized trial (NCT05183035) that will compare the outcome of
subclass in Fig. 1 and discussed below. patients with relapsed AML treated with fludarabine, cytarabine,

Leukemia (2022) 36:1951 – 1960


and GO to that of patients treated with the same agents plus
venetoclax. Although venetoclax is active in a variety of AML
subtypes, resistance can develop through changes in mitochon-
drial metabolism and structure, as well as through overexpression
and increased dependence on the antiapoptotic proteins BCL-XL
or MCL1 [61–64]. In patients with acute lymphoblastic leukemia
(ALL), dependence on BCL-XL or both BCL-XL and BCL2 suggests
that targeting both proteins may be an effective strategy [65].
Indeed, a recent study of navitoclax, a BCL-XL/BCL2 inhibitor,
combined with venetoclax, in children and adults with ALL
demonstrated that this combination is safe and active [65]. In
AML, overexpression of MCL1, rather than BCL-XL, is a more
common mechanism of venetoclax resistance [66]. Selective
inhibitors of MCL1 alone and in combination with venetoclax
are highly active in preclinical AML models [66–68]. However, the
role of MCL1 in cardiac, neuronal, hepatic, and intestinal cell
development and survival may limit the use of MCL1 inhibitors,
especially in pediatric patients [64].
Although selective MCL1 inhibitors are currently undergoing
safety testing in adults and are not yet ready to be studied in
children, MCL1 can potentially be targeted indirectly. In one study,
the combination of selinexor and venetoclax synergistically
induced apoptosis in AML samples through modulation of MCL1
by selinexor [69]. In this study, selinexor and venetoclax
reciprocally abrogated apoptosis resistance to the other agent
via selinexor-mediated inhibition of the binding of Bim to MCL1
and venetoclax-mediated inhibition of the binding of BCL2 to Bim.
In a similar report, venetoclax plus selinexor or eltanexor induced
apoptosis and reduced tumor growth in AML xenografts, effects
that were partly due to decreases in MCL1 expression [70]. In
addition, primary leukemia samples from venetoclax-resistant AML
patients responded to the combination of selinexor and veneto-
clax, providing further evidence that treatment with selinexor may
reverse venetoclax resistance. Based on these data, we recently
opened a phase I study to assess the safety and activity of
venetoclax and selinexor in combination with chemotherapy in
pediatric patients with R/R AML (NCT04898894). Other potential
approaches to MCL1 inhibition include the use of conventional
chemotherapy [71, 72] or inhibitors of cyclin dependent kinase
(CDK)9 [73, 74].
Given the key role of TP53 in the transcriptional activation of
apoptosis machinery, it is not surprising that loss or inactivation of
TP53 confers resistance to BCL2 inhibition by venetoclax [75, 76].
As predicted, clinical studies have indicated that TP53 loss is
associated with poor response to venetoclax or the development
of resistance [77, 78]. Elegant preclinical studies demonstrate that
simultaneous inhibition of BCL2 with venetoclax and activation of
TP53 with the MDM2 inhibitor idasanutlin induce synthetic
lethality and overcome resistance to BCL2 inhibition [76]. In
several models, activation of TP53 promoted MCL1 degradation
and thus abrogated BCL2 resistance, while BCL2 inhibition
overcame resistance to TP53 activation. Clinical trial
NCT04029688 is now evaluating the safety, pharmacokinetics,
and activity of idasanutlin in combination with venetoclax in
pediatric patients with R/R leukemia or solid tumors. In addition,
ALRN-6924, a duel MDM2/MDMX inhibitor, is being studied in
children with relapsed cancer in an ongoing phase I trial
(NCT03654716).
Menin inhibitors
KMT2A (formerly MLL) was initially cloned in 1991 as a result of
translocations involving 11q23 in AML and ALL and was
subsequently found to bind menin, the product of the MEN1
tumor suppressor gene [79]. The KMT2A oncoprotein is a chimeric
fusion of the N-terminus of KMT2A with one of more than 80
translocation partners, which have demonstrated variable ther-
apeutic response in pediatric AML, thereby leading to risk
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S. Zarnegar-Lumley et al.
1955
stratification of KMT2A fusions by translocation partner in upfront
pediatric AML trials. The KMT2A oncoprotein binds to DNA in a
chromatin-associated protein complexes which is required for
maintenance of the gene expression program required for
leukemic transformation. Proteins of therapeutic interest in these
complexes have included the histone methyltransferase, disruptor
of telomeric silencing 1 like (DOT1L), and more recently menin.
Whereas DOT1L enzymatic inhibition was promising in preclinical
experiments [80], translation to a clinical trial was not successful
due to only modest response [81]. Subsequent preclinical
investigation of the KMT2A-menin complex has led to intense
interest in therapeutic targeting of this interaction. Several
preclinical studies demonstrated that small-molecule inhibition
of KMT2A-menin induced remission and, in some cases, eradi-
cated disease, in KMT2A-rearranged and NPM1-mutated leukemia
[82–85]. Similarities in gene expression patterns, particularly
overexpression of HOXA9 and MEIS1, in other leukemia subtypes
suggests that additional subtypes may be amenable to menin
inhibition. Notably, a recent study indicated that NUP98-rear-
ranged AML, which is characterized by high expression of HOXA9
and MEIS1, is dependent on the KMT2A-menin interaction and is
sensitive to menin inhibition [86]. Based on these very strong
preclinical data, at least five menin inhibitors are in clinical
development (Syndax, NCT04065399; Kura, NCT04067336; Daiichi
Sankyo, NCT04752163; Janssen, NCT04811560; Biomea Fusion,
NCT05153330) [87].
SYK inhibitors
Integrated proteomic and genetic studies designed to identify the
mechanism by which epidermal growth factor receptor inhibitors
induce AML differentiation identified spleen tyrosine kinase (SYK)
as a potential therapeutic target in AML [88]. More recent studies
demonstrated that expression of the homeodomain-containing
transcription factors HOXA9 and MEIS1 is associated with SYK
dependence [89]. Mutations in NPM1 lead to activation and high
expression of HOXA9 and MEIS1, suggesting that targeting SYK is a
rational approach to the treatment of NPM1-mutated AML. The
results of a clinical trial of entospletinib (formerly GS-9973), a
selective inhibitor of SYK, combined with chemotherapy demon-
strated higher CR rates and OS among patients with NPM1
mutations and high HOXA9 and MEIS1 expression, further
supporting the role of SYK in NPM1-mutated AML [90]. Entosple-
tinib is now being tested in a phase 3 trial in adults with newly
diagnosed AML and should be considered in future trials for
children with relapsed NPM1-mutated AML.
LSD1 inhibitors
Lysine-specific demethylase 1 (LSD1) is a chromatin modifier that
is expressed in HSCs and AML blasts and plays a role in terminal
differentiation in normal hematopoiesis [91]. Pharmacologic LSD1
inhibition induces monocytic differentiation of AML blasts and
substantially impairs their self-renewal capacity [92]. Iadademstat,
a selective inhibitor of LSD1 was shown to be safe and active in a
phase I study of adult patients with R/R AML [93].
Proteosome inhibitors
Pevonedistat is a small-molecule inhibitor of the NEDD8-activating
enzyme. It is a proteasome inhibitor that specifically blocks
degradation of key proteins involved in cell proliferation and
survival. Preclinical studies demonstrate that pevonedistat in
cancer cell models leads to potent cell death [94]. Pevonedistat
has been studied in combination with azacytidine in adult patients
with treatment-naïve AML who were unfit for standard induction
chemotherapy. The combination was tolerable with 50% overall
response rate [34]. The COG has recently completed a feasibility
study of pevonedistat in combination with azacytidine, fludar-
abine, and cytarabine in pediatric R/R AML (NCT03813147).
 S. Zarnegar-Lumley et al.
1956
PARP inhibitors
Poly(ADP-ribose) polymerase 1 (PARP1) is a key mediator of DNA
repair, is activated in response to DNA damage, and is a potential
therapeutic target in AML [95]. PARP inhibitors were first
developed for the treatment of BRCA-mutated tumors based on
the demonstration of synthetic lethality in preclinical models and
several inhibitors are now FDA-approved for BRCA-mutated breast
and ovarian cancer [96, 97]. Preclinical data suggest that the
effects of PARP inhibitors may be potentiated by cytotoxic
chemotherapy or DNMT inhibitors, even in BRCA wild-type AML
models. For example, the addition of PARP inhibitors led to
increased double-strand breaks and synergistically enhanced the
effects of cytarabine and doxorubicin in an MLL-AF9 AML model.
However, it is not yet known if this effect is limited to specific AML
subtypes or is a general phenomenon [98]. A trial of talazoparib
plus chemotherapy for pediatric patients with R/R AML will open
in 2022.
E-selectin inhibitors
AML blasts commonly express E-selectin ligand, which enhances
adherence to the vasculature of the bone marrow niche. E-selectin-
mediated binding of leukemic blasts leads to sequestration of AML
blasts and LSC, and ultimately to chemoresistance [99]. Uprolese-
lan, an E-selectin antagonist, disrupts leukemic cell adhesion to the
bone marrow microenvironment. It was shown to be safe and
active when combined with chemotherapy in adults with R/R AML
[100]. Planning is underway for trial in pediatric relapsed AML.
Hedgehog pathway
The Hedgehog (Hh) signaling pathway plays an important role in
embryogenesis and stem cell maintenance and has been shown
to play a role in the development and expansion of LSCs. PTCH1,
SMO, and GLI are all components of the Hh pathway that can be
upregulated in patients with AML and therefore serve as potential
targets. Glasdegib is the first SMO inhibitor approved by the FDA
for treatment of AML in adults unsuitable for intensive
chemotherapy [101].
CDK inhibitors
CDK6 and CDK4 are key regulators of the cell cycle. CDK6 is highly
expressed in murine and human AML samples and loss of CDK6
attenuated leukemogenesis in some AML, specifically NUP98
fusion driven AML [102]. Palbociclib inhibits CDK4/CDK6, was
shown in preclinical models to be active in vitro and in vivo
against NUP98-rearranged AML, and is therefore a potential
therapy option for future studies [102]. A phase I/II study of
palbociclib and Vyxeos is ongoing in adults.
MEK inhibitors
Ras/MAPK pathway mutations are frequently activated in relapsed
AML. Trametinib, a MEK1/2 inhibitor has been studied in RAS-
mutated leukemia and demonstrated ex-vivo activity in primary
patient samples [103]. A phase I/II study performed in adults with
RAS-mutated AML showed that single-agent trametinib was
tolerated and had activity in some patients [104].
Other inhibitors
Mutations in isocitrate dehydrogenase (IDH) alter cellular meta-
bolism by formation of a neomorphic metabolite, 2-hydroxyglu-
tarate, that alters cellular epigenetics blocks differentiation. For
patients with IDH mutations, IDH inhibitors ivosidenib (IDH1) [105]
and enasidenib (IDH2) [106] have proven to be safe and active in
adults. Due to the rarity of these mutations in children with AML,
safety has not yet been established. Investigators from the COG
are currently testing enasidenib in patients with R/R IDH2-mutated
AML (NCT04203316).
Many FLT3 inhibitors are commercially available and are
considered standard of care for newly diagnosed and relapsed
patients with FLT3 activating mutations [107]. Although there are
no active trials of FLT3 inhibitors in pediatric patients with
relapsed AML in the US, quizartinib is being evaluated in children
with R/R FLT3-ITD-positive AML in Europe, while gilteritinib is
being investigated in the COG AAML1831 trial in newly diagnosed
children with this AML subtype.
Immunotherapy
The lack of known leukemia-specific antigens that are not
expressed on normal hematopoietic precursors has hampered
the development of immunotherapy for AML, which lags
significantly behind immunotherapy for ALL [108]. The targets
against which most current efforts are directed are CD33 and
CD123, both of which are expressed in ~90% of AML cases. Several
additional targets are the focus of preclinical and early phase trials.
CD123
CD123 is expressed in most AML cases, as well as many cases of
precursor B-cell ALL, some cases of T-cell ALL, and, most
importantly, AML LSCs. Although CD123 is expressed on normal
hematopoietic precursors, its lower expression on normal stem
cells compared to LSCs may offer a therapeutic window. High
CD123 expression in pediatric AML is associated with high-risk
KMT2A rearrangements and FLT3-ITD mutations and is indepen-
dently prognostic of inferior outcomes [109]. CD123-directed
therapies include flotetuzumab, a CD123 x CD3 bispecific
designed to target CD123+ blasts for recognition by CD3+
T cells [110, 111]; CD123-targeted chimeric antigen receptor (CAR)
T-cell therapy; IMGN632, in which a humanized anti-CD123
antibody is conjugated to indolino-benzodiazepine dimers [112];
and tagraxofusp (SL-401), a CD123-directed protein consisting of
human interleukin-3 fused to truncated diphtheria toxin [113].
Flotetuzumab has recently been investigated in pediatric patients
in the PEPN1812 trial (NCT04158739), while CD123-directed CAR T
therapy is being explored by the CATCHAML trial (NCT04318678)
at St. Jude and with a different product at the Children’s Hospital
of Philadelphia (CHOP) (NCT04678336). Clinical trials of IMGN632
and tagraxofusp are expected to open in 2022.
CD33
CD33 is commonly expressed on pediatric AML blasts and was an
early immunotherapy target, leading to the development of
antibody drug conjugate, GO. Increased expression of CD33 is
associated with FLT3-ITD and NPM1 mutations and was associated
with favorable response to GO [114]. The safety and activity of GO
against CD33+ AML has led to the development of CD33-directed
CAR T-cell therapy, including a pediatric trial (NCT03971799) that
is recruiting patients at CHOP and the NCI. Bispecific T-cell
engagers that target CD33 [115] have been developed and a
pediatric clinical trial will open to accrual this year (NCT05077423).
CD47
Over 10 years ago, Majeti et al. identified CD47 as a key LSC
marker that contributes to the pathogenesis of myeloid malig-
nancies by inhibiting phagocytosis through its binding to SIRP1-
alpha on macrophages [116, 117]. Their early studies demon-
strated that CD47 expression was preferentially higher in AML
stem cells compared to normal hematopoietic precursors and
higher levels were associated with a worse outcome. Importantly,
treatment of xenografted mice with an anti-CD47 antibody led to
phagocytosis of patient-derived AML stem cells and clearance of
leukemia [117]. These exciting findings led to the clinical
development of magrolimab, the first anti-CD47 antibody to enter
clinical trials [118]. Initial studies of magrolimab given as a single
agent to patients with AML or MDS were disappointing, possibly
because optimal activity will require blocking the CD47-mediated
anti-phagocytic signal with anti-CD47 antibodies and enhancing
pro-phagocytic signals with other agents. Preclinical studies
Leukemia (2022) 36:1951 – 1960

demonstrating synergy between magrolimab and azacitidine or
other chemotherapeutic agents support this hypothesis and have
led to several ongoing clinical trials in adults with AML or MDS
[119, 120]. The preliminary results of a trial of azacitidine,
venetoclax, and magrolimab in older or unfit adults with AML or
in adults with R/R AML (NCT04435691) were recently reported
(Daver et al., ASH 2021). Among 16 evaluable patients with newly
diagnosed AML, 15 (94%) achieved CR/CRi, with 7 patients
becoming MRD negative. The CR/CRi rates were 5 of 8 among
venetoclax-naive relapsed/refractory patients and 3 of 13 in
patients who had failed prior venetoclax-based therapy. Overall,
the therapy was well-tolerated, with no early mortality in the
cohort of newly diagnosed patients. A pediatric development plan
for magrolimab is being developed.
WT1
Wilms Tumor 1 (WT1) is a transcription factor which had limited
expression in normal tissue but is commonly overexpressed in
AML. It has not been shown to have independent prognostic
significance in pediatric AML; however, it co-occurs with other
high-risk mutations such as FLT3-ITD [121] and is enriched in
patients with chemo-refractory disease [122]. WT1 is an intracel-
lular protein making it challenging to target using traditional cell-
surface directed antibody therapy. For this reason, therapies have
been developed that recognize portions of the intracellular
peptides in the context of HLA, including CAR T cells [123],
genetically modified EBV specific T cells [124], and bispecific
antibodies [125].
CLL-1
C-type lectin-like molecule 1 (CLL-1) is expressed on LSCs and AML
blasts, but not on normal HSCs, making it a potential target in
AML. CAR T-cell therapy targeting CLL-1 is being studied in a
phase I/II trial, in which three of the first four patients achieved CR
[126].
CD70
AML blasts and LSCs express CD70, a type 2 transmembrane
protein [127]. Cusatuzumab, at CD70 monoclonal antibody, has
been tested in a phase I study and shown to be tolerated and
eliminate LSCs [128]. CD70-specific CAR T cells have been
developed and shown preclinically to have potent activity against
AML, sparing normal hematopoiesis [129].
Preclinical testing of several other potential targets, including
CD7, CD13, TIM3, NKG2D, CD64, mesothelin, and GRP78, has
yielded promising results [130]. It is possible that targeting more
than a single tumor antigen is necessary. It was observed that
CD33/CLEC12A are highly upregulated targets on pediatric AML
and can be preferentially targeted together [131]. It was also
noted that CD33/FLT3 are immunotargets specific to KMT2A-
mutated infant AML suggesting that combinatorial targeting may
be effective in this group [131]. Checkpoint inhibition is being
trialed by the TACL Consortium in a phase I/II study of nivolumab
in combination with azacytidine in childhood relapsed AML
(NCT03825367).
HCT has long been a mainstay in treatment of relapsed AML
with novel donor selection and graft manipulation being a major
area of research that is outside the scope of this review. Donor
lymphocyte infusions have been used for relapse after HCT in an
effort to enhance graft-versus-leukemia effect but have limited
success and can often come with a cost of graft-versus-host
disease (GVHD). Allogeneic NK cells have been given to children
with AML safely without causing GVHD [132]. Bednarski et al.
recently completed a phase I trial using donor derived NK cells to
treat pediatric AML that relapsed after HCT. They showed that
donor derived NK cells persisted up to 6 months and maintain
anti-leukemia activity highlighting the potential role for adoptive
NK cell immunotherapy in treating AML [133].
Leukemia (2022) 36:1951 – 1960
S. Zarnegar-Lumley et al.
1957
Palliative care
Finally, we should emphasize the critical importance of palliative
and supportive care to preserve quality of life for children and
adolescents with relapsed AML [134]. The promise of emerging
therapeutics should be balanced with the provision of symptom
alleviation, compassionate and truthful communication, psycho-
social support, and if necessary, end-of-life planning and hospice
care. As we move forward with early and advanced phase clinical
trials, integration of quality of life metrics and patient reported
outcome measures can provide powerful adjunctive understand-
ing of the impact of these therapies on patient quality of life [135].
Furthermore, the opportunity to expand clinical trials to multi-
center settings with broad geographic distribution can benefit
patients by limiting travel.
CONCLUSION
Relapsed pediatric AML poses a significant therapeutic challenge
with suboptimal outcomes from conventional chemotherapy
salvage regimens. Currently, many biologically rational therapeu-
tics are under development that leverage our enhanced under-
standing of the underpinnings of AML relapse. Overarching goals
of emerging relapse therapy include targeting of LSCs, therapeutic
reprogramming of epigenetic, metabolic, and cell survival path-
ways co-opted by leukemic blasts, and selective immunother-
apeutic targeting LSCs and AML blasts while sparing normal HSCs.
The pace of pediatric drug development is slow compared to that
in adults and can attributed to the low absolute numbers of
pediatric patients to enroll in randomized clinical trials, safety
concerns in pediatric patients despite their overall better
functional status and organ function compared to adults, and
often the requirement to test drugs in monotherapy, which is
unattractive in relapsed AML that can rapidly outpace a
monotherapy approach. With increased interest and resources
being put toward international collaborative clinical trials of
biologically rational therapies, there is the promise of increased
clinical trial participation with optimism for improved outcomes.
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AUTHOR CONTRIBUTIONS
SZ-L, KJC, and JER contributed to the preparation of the manuscript and reviewed
and approved the final manuscript.
FUNDING
JER was supported by American Lebanese Syrian Associated Charities and National
Institutes of Health CA21765.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Correspondence and requests for materials should be addressed to Jeffrey E.
Rubnitz.
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