Leukemia (2013) 27, 1451–1460 & 2013 Macmillan Publishers Limited All rights reserved 0887-6924/13 www.nature.com/leu

REVIEW Novel therapies for children with acute myeloid leukaemia

AS Moore1,2,3, PR Kearns4,5, S Knapper6, ADJ Pearson3,5 and CM Zwaan5,7

Significant improvements in survival for children with acute myeloid leukaemia (AML) have been made over the past three decades, with overall survival rates now approximately 60–70%. However, these gains can be largely attributed to more intensive use of conventional cytotoxics made possible by advances in supportive care, and although over 90% of children achieve remission with frontline therapy, approximately one third in current protocols relapse. Furthermore, late effects of therapy cause significant morbidity for many survivors. Novel therapies are therefore desperately needed. Early-phase paediatric trials of several new agents such as clofarabine, and gemtuzumab ozogamicin have shown encouraging results in recent years. Due to the relatively low incidence of AML in childhood, the success of paediatric early-phase clinical trials is largely dependent upon collaborative design by international cooperative study groups. Successfully incorporating novel therapies into frontline therapy remains a challenge, but the potential for significant improvement in the duration and quality of survival for children with AML is high.

Leukemia (2013) 27, 1451–1460; doi:10.1038/leu.2013.106 Keywords: acute myeloid leukaemia; AML; children; novel therapeutics; inhibitors;

INTRODUCTION with only one third of children with relapsed AML becoming long- 8,9 (AML) is a heterogeneous class of term survivors. leukaemias with an age-related incidence.1 AML is relatively rare Although the past 30 years has seen major improvements in in children, accounting for 15–20% of paediatric leukaemias, but overall survival for childhood AML, additional gains are unlikely to causes a disproportionate number of childhood cancer deaths. For be achieved by simply intensifying conventional cytotoxic children less than 15 years of age overall survival (OS) rates are chemotherapy further.6 Chemotherapy such as and now approximately 60–70%.2–5 Although there has been a steady is limited not only by acute toxicity, but also late and gradual improvement in survival for younger adults, it is clear effects such as increased risks of secondary malignancy and that progress has plateaued for children, with survival rates being cardiotoxicity.10,11 Such late effects are of particular concern in substantially inferior to those being achieved for acute paediatrics since treatment occurs during periods of growth and lymphoblastic leukaemia (ALL), where OS exceeds 80%.4,6 development, and the duration of survivorship is much greater Conventional AML therapy is essentially the same for adults and than adults. Less toxic and more effective therapies for AML in children and based on intensive use of or other children, and adults, are therefore urgently needed. The ever- analogs and anthracyclines. Etoposide is frequently expanding array of molecular abnormalities associated with AML used in paediatric induction regimens, although there is no good promises to further refine current risk-stratified treatment and evidence it is of benefit in adult studies and when combined with facilitate individualised therapy with novel therapeutics cytarabine and in children, etoposide provided no specifically targeting these leukaemogenic abnormalities.5 Such advantage compared to thioguanine with cytarabine and an approach should improve outcomes for those children who daunorubicin.2,7 Although chemotherapy will induce complete have historically done poorly and, potentially, permit de- remission (CR) in approximately 90% of children, approximately intensification of therapy for those children with low-risk one third relapse.4,5 Relapsed AML is associated with high disease, as has been achieved with paediatric ALL. morbidity and mortality, with allogeneic haematopoietic stem Several early-phase clinical trials of novel therapies have been cell transplantation (HSCT) generally regarded as the most completed over the past decade and an encouraging number of effective anti-leukaemic therapy available.6 Most re-induction studies are in progress (Table 1). A major limitation for progress in regimens, at least in children, involve high-dose cytarabine in childhood AML, however, is that paediatric studies with novel combination with other agents such as anthracyclines. Addition of agents usually don’t commence until preliminary toxicity and liposomal daunorubicin to the commonly utilized FLAG regimen efficacy profiles have been established in adults. Whilst this may (fludarabine, high-dose cytarabine and G-CSF) has been shown to be justifiable from a patient-safety perspective, it slows the improve the early response rates to chemotherapy from 58 to 68% process of paediatric drug development. Furthermore, it is (P ¼ 0.047).8 Not surprisingly, the likelihood of achieving a second increasingly recognized that AML biology is at least partially CR decreases dramatically with each failed therapeutic attempt, different in children compared to adults, so extrapolating

1Queensland Children’s Medical Research Institute, The University of Queensland, Brisbane, Australia; 2Children’s Health Queensland Hospital and Health Service, Brisbane, Australia; 3Divisions of Clinical Studies and Therapeutics, The Institute of Cancer Research and Royal Marsden, Sutton, UK; 4School of Cancer Sciences, University of Birmingham, Birmingham, UK; 5Innovative Therapies for Children with Cancer (ITCC) European Consortium; 6Department of Haematology, School of Medicine, Cardiff University, Cardiff, UK and 7Pediatric Oncology/Hematology, Erasmus MC/Sophia Children’s Hospital, Rotterdam, The Netherlands. Correspondence: Dr AS Moore, Queensland Children’s Medical Research Institute, Royal Children’s Hospital, Herston Rd, Herston, Qld, 4029, Australia. E-mail: [email protected] Received 6 March 2013; revised 24 March 2013; accepted 4 April 2013; accepted article preview online 8 April 2013; advance online publication, 3 May 2013 Novel therapies for childhood AML AS Moore et al 1452 Table 1. Current clinical trials of new agents in childhood AML

Class Phase Ages (years) Target/Comments Trial number

Nucleoside analogues Clofarabine I 0–28 In combination with , and NCT00462787 MSKCC-07012 Clofarabine II 1–30 In combination with cytarabine NCT00372619 COG-AAML0523 Clofarabine I-II 0–30 In combination with cytarabine and TBI as conditioning NCT00529360 for allo-HSCT Clofarabine I 0–18 In combination with cytarabine and liposomal daunorubicin Dutch Trial Registry 1880

FLT3 inhibitors (CEP-701) III 0–60 FLT3-mutated patients randomised to CEP-701 or ISRCTN55675535 placebo after induction I; small numbers of paediatric patients. (PKC412) I-II 3 mo - 18 FLT3-mutated AML; MLL-rearranged ALL NCT00866281 EudraCT No. 2008-006931-11 Sorafenib III 0–29 For FLT3-ITD þ patients with high-allelic ratio (4 0.4) NCT01371981 COG-AAML1031 (AC220) I 1mo - 21 Combination with cytarabine and etoposide NCT01411267 T2009-004

Aurora kinase inhibitors AT9283 I 6 mo –18 IV, multi-kinase inhibitor (Aurora, ABL, FLT3, JAK2) EudraCT No. 2009-016952-36 NCT01431664 MLN8237 II 1–21 Selective Aurora A inhibitor NCT01154816 COG-ADVL0921

Monoclonal antibodies Gemtuzumab ozogamicin III 0–29 Anti-CD33 mAb conjugated to cytotoxic calicheamicin NCT01407757 COG-AAML0531a Brentuximab vedotin II X6 Anti-CD30 mAb conjugated to anti- NCT01461538 compound MMAE

Proteasome inhibitors III 0–29 Bortezomib in combination with standard chemotherapy NCT01371981 (ADE x 2, AE, A/Mitox) COG-AAML1031

Other novel agents Panabinostat I 8–21 HDAC inhibitor NCT01321346 T2009-012 INCB18424 I 1–21 JAK1/2 inhibitor NCT01164163 COG-ADVL1011 MK2206 I 1–21 Akt inhibitor NCT01231919 COG-ADVL1013 Obatoclax mesylate I 0–21 BCL-2 inhibitor, in combination with , NCT00933985 and dexrazoxane COG-ADVL0816 II 1–16 as epigenetic priming for NCT01177540 induction chemotherapy with ADE Fenretinide I 0–21 Cytotoxic NCT01187810 Abbreviations: TBI, total body irradiation; allo-HSCT, allogeneic haematopoietic stem cell transplantation; AML, acute myeloid leukaemia; ALL, acute lymphoblastic leukaemia; MLL, mixed lineage leukaemia gene; mo, months; mAb, monoclonal antibody; MMAE, monomethyl auristatin E; HDAC, histone deacetylase; ADE, cytarabine, daunorubicin, etoposide; AE, cytarabine etoposide; A/Mitox, cytarabine and . aOngoing, but closed to recruitment.

anticipated efficacy from adult studies to children can be and adults. It is not surprising that novel cytarabine derivatives challenging. For example, ‘epigenetic’ mutations of DNMT3A, have delivered some promising results. and fludarabine IDH1/2, TET2 and NPM1 all occur much less frequently in children were the first two such derivatives developed. Unlike cytarabine, than in adults.12–16 they inhibit both DNA polymerase and ribonucleotide reductase, Novel classes of therapeutics investigated in childhood AML in thereby depleting deoxynucleotide pools.17 Cladribine has shown recent years include nucleoside analogs, small molecule encouraging results in combination with cytarabine,18 topotecan19 kinase inhibitors and immunomodulatory therapies. Here we and .20 Interestingly, the FAB M5 subtype of AML review the results of these trials and highlight some of the more appears more responsive to cladribine than other morphological promising agents emerging from adult trials. subtypes.21 , in combination with cytarabine±an , has become a widely used re-induction regimen for children with relapsed AML.8,9,22,23 The dose-limiting NOVEL PURINE NUCLEOSIDE ANALOGS neurotoxicity of cladribine and fludarabine however, prompted The cytidine analog and DNA polymerase inhibitor cytarabine the development of clofarabine. After efficacy was demonstrated forms the backbone of conventional AML therapy in both children for relapsed/refractory ALL, in December 2004 clofarabine became

Leukemia (2013) 1451 – 1460 & 2013 Macmillan Publishers Limited Novel therapies for childhood AML AS Moore et al 1453 the first anti-leukaemic drug approved for use in childhood prior proceeded to HSCT. The 40 mg/m2 clofarabine cohort is being to adult use approval.17 The efficacy and potential role of extended to accrue more efficacy data.29 clofarabine in paediatric AML is discussed in detail below. There is intense ongoing interest in clofarabine for AML in adults and children, with no fewer than 35 open studies of the drug in AML currently registered on ClinicalTrials.gov. A major Clofarabine challenge, however, remains in defining how best to use Clofarabine is a structural hybrid of cladribine and fludarabine, clofarabine in both upfront and second-line AML treatment designed to have improve efficacy, through reduced deamination regimens. Results of randomised studies in adults are expected by and improved stability, whilst avoiding in the near future, including the NCRI AML16 trial for adults over the formation of toxic metabolites such as 2-F adenine caused by 60 years of age, which randomised newly-diagnosed patients to glycosidic bond cleavage of fludarabine.17 Clofarabine is induction with either daunorubicin and cytarabine or daunorubi- converted intracellularly to clofarabine monophosphate by cin and clofarabine (ISRCTN 11036523). The current NCRI AML17 deoxycytidine kinase (dCK), then to clofarabine triphosphate by trial for younger adults with AML is randomising poor risk patients additional kinases. The triphosphate form of clofarabine impairs (defined by a statistically-derived risk score based on presenting DNA synthesis and repair via inhibition of both ribonucleotide disease characteristics and remission status after course I reductase and DNA polymerase and induces apoptosis via chemotherapy), to either daunorubicin plus clofarabine or FLAG- mitochondrial pathways.24 As a potent ribonucleotide reductase Ida prior to HSCT (ISRCTN55675535). For childhood AML, a inhibitor, clofarabine increases dCK activity, thus potentiating its randomised phase II study sponsored by St Jude Children’s own activation and theoretically that of other drugs such as Research Hospital (SJCRH) is ongoing, comparing end-of-induction cytarabine.24 The maximum tolerated dose (MTD) as a single- MRD for children treated with clofarabine and cytarabine to agent is 52 mg/m2/day for 5 days in children and 40 mg/m2/day conventional ADE therapy (cytarabine, daunorubicin, etoposide; for 5 days in adults with hematologic malignancies, with reversible NCT00703820). hepatotoxicity and skin rash being the main non-haematological dose limiting toxicities (DLTs).25 Although the and skin toxicities are reversible when clofarabine is given as a single FLT3 INHIBITION agent, it should be noted that a potential interaction with Internal tandem duplication (ITD) or tyrosine kinase domain (TKD) intrathecal exists, with at least one case report mutations of the fms-like tyrosine kinase 3 (FLT3) gene occur describing fatal skin and hepatoxicity in a child with a CNS-relapse frequently in AML, resulting in constitutive FLT3 signaling and of pre-B ALL given clofarabine 52 mg/m2/day for 5 days, together stimulation of leukaemic proliferation.30 The incidence of FLT3-ITD with triple intrathecal therapy (hydrocortisone, methotrexate and in AML increases with age, occurring in approximately 23% of cytarabine).26 adults and 12% of children overall with de novo AML, whilst the A phase II study of clofarabine as a single agent in children with incidence of FLT3-TKD is relatively constant across all age groups relapsed/refractory AML had an overall response rate (ORR) of 26%, at approximately 7%.31,32 Although FLT3-TKD mutations do not comprising one CR with incomplete recovery (CRp) and 10 appear to impact on prognosis, the presence of FLT3-ITD has partial responses (PR).25 Although most responses were partial, the consistently been associated with inferior outcome, principally as trial population was heavily pretreated, with patients having a result of increased relapse rate.33 High FLT3-ITD/FLT3-WT allelic received a median of two prior treatment regimens (range one to ratios (AR) carry a particularly poor prognosis, with survival rates of five). Furthermore, six of 28 (21%) patients refractory to prior less than 20% in both adults and children.31,34,35 Furthermore, therapy responded and approximately one third of clofarabine ex vivo studies have demonstrated increased dependence of high treated patients proceeded to HSCT.25 Subsequent interpretation AR FLT3-ITD þ AML blasts to FLT3 signaling, consistent with the of the HSCT data is problematic however, considering that most concept of ‘oncogene addiction’.36 The identification of secondary patients went to HSCT in PR, rather than CR. The toxicity profile was FLT3-TKD mutations in patients treated with FLT3 inhibitors is also reported as being expected for the patient population, with evidence of clonal selection and cellular dependency on frequent (X15%) grade 3 adverse events being febrile constitutive FLT3 signaling.37–39 neutropaenia, catheter-related infection, epistaxis, , Small molecule inhibition of FLT3 kinase has therefore been , , elevated transaminases and hypokalaemia.25 vigorously pursued as a therapeutic strategy over the past decade Clofarabine has also been trialed in combination with conven- and detailed critiques of each individual FLT3 inhibitor developed tional and targeted therapeutic agents. In a phase I study of the to date have recently been published.33,40 Initial ‘first generation’ multi-kinase/FLT3 inhibitor sorafenib in combination with clofar- FLT3 inhibitors, such as CEP-701 (lestaurtinib) and PKC412 abine and cytarabine, responses were seen in both FLT3-wild type (midostaurin), were non-selective compounds already in early (-WT) and FLT3-ITD þ patients.27 This trial is discussed further clinical development and subsequently identified as having below. A phase I trial of clofarabine in combination with etoposide potent anti-FLT3 activity. These compounds have undergone and for children with relapsed and refractory extensive preclinical and clinical evaluation with major phase III acute leukemias resulted in an ORR of 55% (9/20 CR þ 2/20 CRp) trials ongoing. So-called ‘second-generation’ FLT3 inhibitors such for ALL patients and 100% (1/5 CR þ 4/5 CRp) for AML patients.28 as AC220 (quizartinib), designed with increased potency and When given for five consecutive days during induction and four selectivity for FLT3, are currently being evaluated in early-phase consecutive days in consolidation for up to eight cycles (median clinical trials. time between cycles 34 days), the recommended phase II doses Preclinical studies have demonstrated that sustained inhibition (RP2D) of clofarabine, cyclophosphamide and etoposide were 40, of FLT3 phosphorylation to less than 15% of baseline is required to 440 and 100 mg/m2 respectively. The toxicity profile was similar to achieve cytotoxicity.41,42 Furthermore, several pharmacokinetic that seen in the monotherapy phase II study of clofarabine.28 (PK) and pharmacodynamic (PD) studies from clinical trials have A phase I study of clofarabine in combination with liposomal consistently confirmed that clinical responses are unlikely in daunorubicin for children with relapsed/refractory AML was patients who do not achieve adequate FLT3 inhibition.41–45 recently completed. Patients received clofarabine on days 1–5 Finally, although newer more potent and selective FLT3 (30 mg/m2, escalated to 40 mg/m2), and liposomal daunorubicin inhibitors such as AC220 are showing encouraging results in (60 mg/m2) days 1, 3 and 5. Nine patients were enrolled, with the single-agent trials, it appears that FLT3 inhibitors are likely to be of most common severe adverse event being febrile neutropaenia, most benefit when used in combination with conventional but no DLTs observed. One third of patients achieved CR and chemotherapy.33 It is worth noting that myelosuppression from

& 2013 Macmillan Publishers Limited Leukemia (2013) 1451 – 1460 Novel therapies for childhood AML AS Moore et al 1454 conventional chemotherapy itself has been shown to increase the patients. Ten patients had PD studies performed (flow cytometric production of FLT3 ligand (FL) which can decrease the efficacy of a analysis of phosphorylated AKT, 4E-BP1 and S6RP), with decreases number of FLT3 inhibitors, at least in vitro.46 These observations in percentage of positive cells and mean fluorescence intensity highlight the need for effective PD biomarker assays to monitor unrelated to FLT3-mutational status.27 Six patients proceeded to how effectively FLT3 is being targeted. Assays such as the plasma HSCT.27 The most common toxicity associated with sorafenib was inhibitory activity (PIA) assay have been used to demonstrate that skin toxicity, with all 12 patients experiencing some degree of effective FLT3 inhibition can still be achieved clinically however, hand-foot skin reaction and/or rash. Skin toxicity was dose-limiting despite elevations in FL (discussed in more detail below).47 at 200 mg/m2 but not 150 mg/m2, which was the final recommended dose of sorafenib.27 The hand-foot skin (HFS) reaction to sorafenib is classically characterised by tender, scaling Sorafenib skin with erythematous halos on areas of pressure or skin creases The multi-kinase inhibitor, sorafenib, has potent activity against which may blister and can progress to hyperkeratosis and FLT3 and increased activity against FLT3-ITD þ AML cells impaired movement and function.54 Consensus guidelines for compared to FLT3-WT cells.48 Although sorafenib was assessed the management of sorafenib and induced HFS reactions by the Pediatric Preclinical Testing Program (PTPP), only one AML have been developed and include avoidance of skin trauma, cell line was tested.49 In the PTPP screen, the FLT3-WT cell line liberal use of emollients and dose reduction/cessation of 49 54 Kasumi-1 had an in vitro IC50 of 0.02 mM. Pre-clinical studies of sorafenib. The high incidence of skin toxicity in this paediatric sorafenib and another multi-kinase inhibitor, sunitinib, at SJCRH study compared to previous single agent adult trials was examined a broader panel of AML cell lines and demonstrated attributed the concurrent use of cytarabine and clofarabine, as similar in vitro sensitivity of Kasumi-1 (sorafenib IC50 0.04 mM), but well as the higher concentration of the active metabolite sorafenib markedly increased sensitivity of the FLT3-ITD þ paediatric cell line N-oxide observed in the paediatric trial.27 MV4-11 (sorafenib IC50 0.002 mM). In vitro activity of sorafenib The current Children’s Oncology Group (COG) phase III trial against primary paediatric AML samples (4/6 FLT3-WT, 2/6 (AAML1031, NCT01371981) is using a FLT3-ITD/FLT3-WT allelic unknown FLT3 status) was also demonstrated.50 Increased ratio 40.4 (high-AR) to non-randomly stratify children with in vitro sensitivity of FLT3-mutated, primary paediatric AML FLT3-ITD þ AML to receive sorafenib and HSCT in first remission, samples has also been demonstrated for SU11657, a compound if an appropriate donor is available. Although the HSCT data for similar to sunitinib with anti-FLT3 activity51 Unlike sorafenib, adults is inconclusive,2 matched related donor allogeneic HSCT sunitinib is no longer being evaluated as a FLT3 inhibitor in AML has been demonstrated to be of benefit for FLT3-ITD þ children due to poor tolerability at doses required to inhibit FLT3.33 with a high-AR.31 The dismal prognosis for children with high-AR Several clinical trials of sorafenib in adult AML have been FLT3-ITD þ AML (3 year DFS/OSp20%) clearly warrants novel reported,33 with a recent phase I/II study in combination with treatment approaches however, the number of children with high- cytarabine and idarubicin demonstrating overall CR rates of 93% AR disease transplanted on the CCG-2941 and CCG-2961 trials was (14/15, plus 1 with CRp) in FLT3-ITD þ patients and 66% (24/36 small (total FLT3-ITD þ ¼ 11; high-AR ¼ 6), and all high-AR patients plus 3 with CRp) in FLT3-WT patients.52 In the phase II arm of the on the current COG AAML1031 trial will receive both sorafenib and study, sorafenib was given at a dose of 400 mg twice daily for up allogeneic HSCT, so it may be difficult to determine the extent to to 28 days during induction and consolidation (both with which sorafenib further improves survival when compared to this concurrent cytarabine and idarubicin), then continued as historical control. maintenance therapy for up to one year.52 In contrast, a Paediatric trials of other FLT3 inhibitors, including AC220, placebo-controlled, randomized phase II study of sorafenib CEP-701 and PKC412, are currently in progress (Table 1). An combined with standard 7 þ 3 induction chemotherapy and emerging issue with a number of FLT3 inhibitors, including intermediate-dose cytarabine consolidation then continued for 1 midostaurin and the more selective compounds AC220 and year as maintenance in 197 older patients (460 years) failed to sorafenib, is resistance mediated by secondary FLT3-TKD muta- show any difference in CR rates, EFS, OS for FLT3-WT or FLT3-ITD þ tions.37–39,55,56 Given the biologically similar nature of FLT3-ITD þ patients, although the small number of FLT3-ITD þ patients AML in children and adults, similar resistance patterns could be (n ¼ 28; 14.2%) may have limited the power of subset analysis. expected although the relatively small number of children with Despite being reportedly well tolerated, there was a trend towards FLT3-ITD þ AML may make this a rare occurrence. Finally, the slower leucocyte and platelet recovery in the sorafenib arm during benefit of ‘maintenance’ FLT3 inhibition (e.g. post-HSCT) is yet to the induction cycles.53 be proven, however it is plausible that such strategies with Encouraging results from a paediatric phase I study of sorafenib compounds like AC220 or sorafenib may promote acquired in combination with cytarabine and clofarabine for children with resistance, mediated by secondary FLT3-TKD mutations. Despite relapsed/refractory acute leukaemia were recently published. the compelling preclinical data supporting FLT3 inhibition as a Sorafenib was given as a single agent on days 1 to 7 and then therapeutic strategy in FLT3-mutated AML, until therapeutic concurrently with clofarabine (40 mg/m2, or 20 mg/m2 if the benefit is proven from ongoing clinical trials in adults and patient had HSCT within 6 months or fungal infection within 1 children, up-front use of FLT3 inhibitors for childhood AML should month) and cytarabine (1 g/m2) on days 8 to 12. If tolerated, be limited to clinical trials. However, for children with relapsed or sorafenib alone was continued until day 28. Repeated courses of refractory FLT3-ITD þ AML, limited therapeutic options (e.g. from sorafenib, clofarabine and cytarabine, maintenance therapy with anthracycline cardiotoxicity) and no access to a clinical trial, FLT3 single-agent sorafenib, or transplantation were administered inhibition could be considered to help achieve disease control, for according to clinical judgment.27 Eleven of the 12 patients (aged example prior to HSCT or possibly even as a palliative strategy. 6–17 years) had AML, including 3 who had previously undergone allogeneic HSCT (2 FLT3-ITD þ ,1FLT3-WT). Of these 11 AML patients, 5 were FLT3-ITD þ and all achieved morphological CR on day 22 (3 CR þ 2 CRp) in combination with cytarabine and IMMUNOTHERAPY clofarabine, including both patients who had previous allogeneic Gemtuzumab ozogamicin HSCT.27 Of the 6 FLT3-WT AML patients, 3 achieved morphological Gemtuzumab ozogamicin (GO), a humanized anti-CD33 mono- CR and one a PR. Of note, the initial seven-day course of clonal antibody conjugated to the cytotoxic compound N-acetyl- single-agent sorafenib reduced bone marrow blast percentages in g-calicheamicin dimethylhydrazine, has been extensively investi- 10 patients (median 66%, range 9–95%), including all FLT3-ITD þ gated in adults and children over the past decade. Therapeutic

Leukemia (2013) 1451 – 1460 & 2013 Macmillan Publishers Limited Novel therapies for childhood AML AS Moore et al 1455 targeting of CD33 has a sound rationale in AML, since fractionated dosing, further evaluation of GO in children is approximately 80% of AML cases express CD33.57 High CD33 planned. expression in childhood AML is associated with adverse disease characteristics such as FLT3-ITD and independ- 58 ently predicts poor outcome. Initial experience with GO STEM CELL TARGETING monotherapy in children with relapsed/refractory AML Bortezomib demonstrated response rates of 53% (8/15 patients), with 6 The , bortezomib, was initially approved for patients proceeding to HSCT.59 Subsequent trials, including GO use in multiple myeloma. Pre-clinical studies of proteasomal combined with chemotherapy and HSCT have demonstrated its inhibition in combination with idarubicin in AML demonstrated safety.60–63 Monotherapy studies and case reports of GO in down-regulation of NF-kB and preferential cell death in leukaemic relapsed/refractory childhood AML have resulted in a number stem cells but not normal haemopoietic stem cells (HSCs).72 A clinically meaningful responses, whilst phase II studies in single-agent phase I study in children demonstrated acceptable combination with chemotherapy have shown similar results to toxicity, with a RP2D of 1.3 mg/m2 per dose when administered historical controls. The recently reported multi-centre AML02 twice weekly for 2 weeks.73 Although NF-kB inhibition was phase III trial included a randomization of GO (3 mg/m2) during observed in 2 of 5 evaluated patients, there were no objective induction II chemotherapy with cytarabine, daunorubicin and clinical responses. Phase I studies of bortezomib (twice weekly for etoposide.64 Although the specific effects of GO versus no GO two weeks) in combination with chemotherapy in adult AML74 were not reported, GO in combination with ADE resulted in and paediatric ALL75 were also well tolerated, with predictable, reductions in minimal residual disease (MRD) in 93% (27/29) of chemotherapy-related toxicity. In the adult phase I combination patients who had responded poorly to induction I, including all 8 study, nine patients were less than 60 years of age with relapsed patients with MRD425% after induction I. Of the 8 patients with AML, with the remaining 22 patients being over MRD425%, half became MRD-negative with ADE þ GO. Of 60 years with untreated AML.74 Overall, 7/9 (78%) of relapsed the remaining 21 patients, who all had MRD41% after patients and 15/22 (68%) untreated older patients achieved CR induction I, the ADE þ GO combination reduced MRD in 19 74 (including 3 CRp). In addition to administering Sorafenib to (90%), with 9 patients becoming MRD-negative (43%).64 Paediatric children with high-AR FLT3-ITD þ AML, the current COG phase III phase III studies of GO in combination with conventional trial (AAML1031, NCT01371981) is randomising patients with de chemotherapy are ongoing. novo AML (including those with low-AR FLT3-ITD) to receive The potential for veno-occlusive disease (VOD) of the liver conventional chemotherapy with or without Bortezomib for all following GO has been of concern in adult patients, particularly in courses. those patients proceeding to HSCT within 3.5 months of receiving 6 or 9 mg/m2 GO.65 The recently reported UK NCRI AML15 trial (1,113 patients) administered a lower GO dose (3 mg/m2) in adults and failed to show an increased risk of hepatic toxicity, even when NOVEL AGENTS WITH PROMISING DATA IN ADULTS administered within 120 days of HSCT.66 The successor trial, UK Aurora kinase inhibition NCRI AML17 is randomizing adult patients to 3 vs 6 mg/m2.In Aurora kinases are a family of serine/threonine kinases with critical pediatric studies liver toxicity has been less of an issue, and GO roles in mitosis.76 Aurora A kinase plays a key role in centrosome seems better tolerated in children.67 maturation and mitotic spindle assembly whilst Aurora B kinase Other dosing strategies of GO have also been examined, regulates the mitotic checkpoint, forms part of the chromosomal including fractionated therapy, facilitating cumulative doses passenger complex (with inner centromere protein, Borealin and of up to 27 mg/m2 (refs 68–70). A phase I study in combination Survivin) and is required for final cytokinesis.76 Although with and cyclophosphamide HSCT conditioning for 12 therapeutic success in solid tumours has been limited, Aurora children with CD33 þ AML (median age 3 years, range 1–17) failed kinase inhibitors have shown encouraging responses in leukaemia, to identify any GO-associated DLTs at 3.0, 4.5, 6.0 or particularly AML and Philadelphia-positive leukaemias.77 A recent 7.5 mg/m2 and had no 100-day transplant-related mortality.61 phase I/II trial of the selective Aurora B inhibitor, AZD1152 In June 2010 the United States Food and Drug Administration (barasertib) had an overall response rate of 25%.78 The toxicity (FDA) withdrew marketing approval for GO, based on lack of profile, including mucositis and myelosuppression, was reported benefit in relapsed/refractory AML and increased induction as being manageable in an older population. FLT3 kinase is a mortality in adults. It has been highlighted, however, that the secondary target of AZD115279 and although the FLT3 status of increased induction mortality associated with GO was relative to patients on the trial was not reported, it is of interest that a an unusually low mortality in the control arm of one of the studies number of responses occurred below the MTD and 55% of the FDA based their decision on.66 Furthermore, subsequent patients on the trial had cytogenetically normal AML (CN-AML). data from 1,113 newly diagnosed patients treated on the UK Given the high incidence of FLT3 mutations in CN-AML, it is NCRI AML15 trial have demonstrated a significant benefit of possible that the anti-FLT3 activity of AZD1152 may have GO for patients with core binding factor (CBF) AML when given contributed to efficacy in patients with FLT3 mutations. A phase as a single dose of 3 g/m2 during courses one and three.66 II/III study of AZD1152, alone and in combination with low-dose The biological explanation for this is somewhat unclear, since cytarabine in older adults (460 years), is currently ongoing responses to GO were not related to blast CD33 expression.66 (NCT00952588). A recent preclinical study has demonstrated that Interestingly, children with CBF AML on the NOPHO-AML 2004 paediatric AML cell lines are sensitive to Aurora B knockdown by trial accounted for 35% (N ¼ 17) of relapses, independent of shRNA and small molecule inhibition with AZD1152.80 One whether or not GO (5 mg/m2 Â 2 doses) was given as post- potential limitation of the AZD1152 compound is its delivery via consolidation therapy.71 Definitive results from larger paediatric a 7 day continuous IV infusion. trials of GO are awaited. The recently reported French trial ALFA- The selective Aurora A inhibitor MLN8237 (alisertib), has shown 0701 confirmed the survival advantage for adults with de novo response rates of 17% in an adult phase II trial81 and preclinical AML and favourable cytogenetics, but also for those with activity in paediatric leukaemia.82 Of note, a recent preclinical intermediate-risk cytogenetics. A notable feature of the ALFA- study in acute megakaryoblastic leukaemia (AMKL) demonstrated 0701 study was the delivery of fractionated, higher cumulative that Aurora A inhibition with MLN8237 could induce doses of GO (3 g/m2 days 1, 4 and 7 during induction and in two polyploidization in both a Down Syndrome (DS) AMKL cell line consolidation cycles).70 On the basis of these adult studies with (CMK) and primary blasts from children with non-DS-AMKL.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1451 – 1460 Novel therapies for childhood AML AS Moore et al 1456 MLN8237 also had in vivo activity against a non-DS-AMKL model.83 combining with cytotoxic chemotherapy in adults with A paediatric phase II trial of MLN8237 in children with relapsed/ relapsed/refractory AML. When used in combination with refractory solid tumours and leukaemia is currently ongoing mitoxantrone, etoposide and cytarabine, plerixafor increased (NCT01154816, COG-ADVL0921). Similarly, the Aurora kinase mobilization of AML blasts into the peripheral circulation.103 inhibitor AT9283, which also has potent activity against ABL, Overall CR rates of 46% were achieved and no dose-limiting JAK2 and FLT3 kinases, has shown encouraging responses toxicities were observed. in adult leukaemia trials and is currently being assessed in a paediatric phase I study (EudraCT No. 2009-016952-36; Epigenetic therapies NCT01431664). Genes regulating DNA methylation and demethylation such as DNMT3A, IDH1/2 and TET2 are frequently mutated in adult AML MEK inhibition and have prognostic significance.104–107 Consequently, targeting Mutations of N- and K-RAS in AML are examples of class I epigenetic processes has become an attractive therapeutic 84 mutations, conferring a proliferative/survival stimulus. A recent strategy in adult AML. The DNA methyltransferase inhibitors comprehensive pre-clinical predictive biomarker analysis of 218 decitabine and are currently being evaluated in clinical solid tumor and 81 haematological cancer cell lines revealed AML trials of adults with AML and paediatric evaluation programs are and CML cell lines as being particularly sensitive to the MEK being designed. A recent analysis of 46 patients treated on two trials 85 inhibitor, GSK1120212. Amongst solid tumour cell lines, RAF/RAS of decitabine has shown an improved response rate in patients with 85 mutations were predictive of sensitivity. In a panel of 12 AML cell DNMT3A mutations compared to those with wild-type DNMT3A (CR 108 lines, single-nanomolar IC50’s were reported for all 6 RAS-mutant rate 75% (6/8) vs 34% (13/38) respectively, P ¼ 0.05). Studies of (N- or K-RAS) cell lines, as well as 2 RAS-wt cell lines. The paediatric azacitidine alone and in sequential combination with lenalidomide cell line Kasumi-1 (RAS-wt) was resistant, although other paediatric have shown responses of approximately 50% in older adults with þ cell lines, THP-1 (N-RAS mutant) and MV4-11 (FLT3-ITD , RAS-wt), newly-diagnosed AML.109,110 85 were sensitive. A phase I trial of GSK1120212 was reported at the Another focus of epigenetic therapy has been histone 2010 ASH meeting, with 12 of 14 patients treated having AML deacetylase (HDAC) inhibition with compounds such as valproic 86 (including 2 transformed from MDS). Preliminary anti-leukaemic acid, and panabinostat. Widely used as an anti- activity was reported, with one patient achieving CR. A phase I/II convulsant, valproic acid was first demonstrated to inhibit HDAC study of GSK1120212 in AML is ongoing (NCT00920140). and cause partial differentiation of the paediatric AML cell line Kasumi-1 over a decade ago.111 Preclinical studies, including Aminopeptidase inhibition paediatric models, have also demonstrated single cytotoxic 112,113 Aminopeptidases play a key role in removing amino acids from synergy with cytarabine and clofarabine. Clinical responses to valproic acid alone or in combination with cytarabine in adults cellular peptides, a process required for protein regulation and 114–116 recycling. Aminopeptidase inhibition depletes intracellular amino with AML however, have been variable. It should be noted 87 that when combined with decitabine, valproic acid has been acids required for new protein synthesis. AML cells have been 117 shown to be sensitive to aminopeptidase inhibition, undergoing associated with encephalopathy. apoptosis, whilst normal bone marrow cells are less susceptible.88 Although a single-agent randomised phase II trial of vorinostat in adults with relapsed or high-risk untreated AML showed only The aminopeptidase inhibitor tosedostat has shown promising 118 activity in phase I/II trials of older adults with predominantly minimal responses (1 CR from 37 patients) a recent phase II trial relapsed/refractory AML, with single-agent response rates of up to of vorinostat in combination with idarubicin and cytarabine (IA) in 27%.89,90 Tosedostat appears well tolerated with thrombocytopaenia patients with AML and no CBF abnormalities resulted in an EFS of being the most common adverse event reported. 47 weeks (range, 3 to 134 weeks) and a CR rate of 76%. Although not randomised, these responses compared favourably to historical data, the combination was reported as not excessively c-KIT inhibition toxic compared to IA alone and all 11 FLT3-ITD þ patients Activating c-KIT mutations are common in paediatric and adult responded (10 CR, 1 CRp).119 As discussed above, the relative AML, particularly in CBF AML (incidence 21–54.5% with inv(16) rarity of mutations in genes regulating DNA methylation in 91–95 and 17–46.8% with t(8;21)). Although c-KIT mutations confer childhood AML may limit the broader applicability of these agents 93 an increased risk of relapse in adults with CBF AML, this does not for children, particularly if responses are confirmed to be 94,95 appear to be the case in children. Murine models have associated with mutational status. demonstrated c-KIT mutations cooperate with AML1-ETO to cause Although adult clinical trials have only recently been initiated, it aggressive AML in vivo, but are responsive to c-KIT inhibition with is worth noting one potential epigenetic therapy that may be of 96 dasatinib. Dasatanib has also been shown to have single-agent significant importance to childhood leukemias with MLL gene activity against the t(8;21) paediatric cell line, Kasumi-1, which also rearrangements, given their relatively high incidence (AML 18%, harbours an N822K point mutation in the activation loop of ALL 8.3%).4 The histone H3 methyltransferase, DOT1L, plays a key 97,98 c-KIT. Results of several ongoing clinical trials in AML, role in MLL-fusion mediated leukemogenesis (reviewed in Ref 120). particularly those enriched with CBF patients, are eagerly awaited. A small molecule inhibitor of DOT1L, EPZ004777, was recently shown to have in vivo preclinical efficacy against MLL-rearranged CXCR4 antagonism leukemia.121,122 A phase I trial of an optimized compound, EPZ- The critical role of the bone marrow micro-environment, or ‘niche’ 5676, opened in September 2012 for adults patients with advanced in regulating normal and malignant haematopoiesis is becoming hematological malignancies, including MLL-rearranged AML and increasingly recognised. The CXCR4 antagonist, plerixafor, has ALL (NCT01684150). demonstrated efficacy in the mobilization of normal HSCs from the marrow for use in autologous transplantation in non-Hodgkin lymphoma and multiple myeloma.99,100 Plerixafor has also been FINDING AND HITTING THE RIGHT TARGET IN AML: demonstrated to be a feasible and effective second-line strategy PREDICTIVE AND PHARMACODYNAMIC BIOMARKERS in mobilizing HSCs in children.101,102 Based on pre-clinical data Of critical importance for the rational and efficient development of demonstrating AML blasts could be mobilized into the peripheral targeted agents in cancer and leukaemia is the use of validated blood, a recent phase I/II study demonstrated the feasibility of predictive and PD biomarkers. Predictive biomarkers are those

Leukemia (2013) 1451 – 1460 & 2013 Macmillan Publishers Limited Novel therapies for childhood AML AS Moore et al 1457 that identify which patients are most likely to derive benefit from of subsequent HSCT and the time required to screen, enroll and a given targeted agent, whilst PD biomarkers are needed to verify treat patients with the novel agent prior to HSCT. whether or not the desired molecular target is being modulated, Accelerating drug development for childhood AML will rely and establish how target modulation relates to dose, toxicity and heavily on effective international collaboration, particularly if response. This information, together with careful examination of enrolment on targeted therapeutic trials is to be restricted to the any resistance mechanisms or treatment failures, not only relatively small number of patients with predictive biomarkers of decreases the attrition rate of targeted therapeutics, but also response. Groups such as the Innovative Therapies for Children improves the ongoing pre-clinical development of new agents. with Cancer (ITCC) European Consortium and the Therapeutic FLT3 inhibitors provide arguably the best example of how Advances in Childhood Leukaemia and Lymphoma (TACL) robust predictive and PD biomarkers have been successfully consortium have an existing focus on early-phase trials of novel applied in AML. It is well established that patients without agents for childhood leukaemia. Although legislative, regulatory activating mutations of FLT3 are less likely to benefit from FLT3 and pharmaceutical supply barriers prevent the enrolment of inhibition.33 Even with potent, second generation FLT3 inhibitors children from outside Europe or North America on certain trials, such as AC220, response rates in FLT3-WT patients are lower than attempts to facilitate truly international early-phase studies are in FLT3-ITD þ patients (34 vs 44%).123 FLT3 mutational status ongoing. Novel approaches to early-phase trial design are also therefore serves as a robust predictive biomarker for personalized critical in accelerating drug development, particularly in children. AML therapy. Furthermore, results from trials of CEP-701 clearly Important strategies include phase I trials with expansion cohorts demonstrate the importance of combining a predictive biomarker at the RP2D enrolling patients with predictive biomarkers, and with a robust PD assay, since adequate FLT3 inhibition correlates adaptive randomised trials incorporating a number of new agents with outcome for FLT3-mutated patients treated with CEP-701.45,47 with the aim of ‘picking-the-winner’ or ‘dropping-the-loser’.125 An important PD assay developed for FLT3 inhibitors is the PIA Such approaches hold promise of reducing the number of large assay.42 This assay involves incubation of a FLT3-ITD þ cell line in phase III trials needed and improving the likelihood of beneficial the plasma of patients treated with FLT3 inhibitors. The degree of treatments being identified for malignancies like AML with FLT3 inhibition seen ex vivo for a patient at a given time-point increasingly heterogeneous biology. The important question of during or after therapy is normalized to the amount of FLT3 how much can be extrapolated from adult biology and trial data phosphorylation observed when cells are incubated with that remains difficult to answer, hence defining the minimum dataset patient’s pre-treatment plasma. FLT3 ligand (FL) levels and protein in children is an ongoing challenge. binding can impact on the efficacy of FLT3 inhibition.46 The PIA Although significant challenges remain for novel drug devel- assay is therefore superior to simply calculating the free drug opment in paediatric oncology, particularly AML, the encouraging concentration, since the plasma sample used to treat the cell line results of initial paediatric trials of therapies such as clofarabine ex vivo contains not only free drug, but also the patient’s actual FL and sorafenib, together with the large number of new agents in and plasma proteins at that time-point. Using the PIA assay, it has the pipeline is encouraging. Although survival rates in childhood recently been demonstrated that when adequate FLT3 inhibition AML are significantly better than adults with the disease, is achieved in combination with chemotherapy, relapse rates are outcomes for children with AML are dramatically inferior lower and overall survival can be improved.47 compared with paediatric ALL.4 Improving outcomes in AML The relatively small circulating blood volume and need for with cytotoxic chemotherapy alone is highly unlikely, given the general anaesthetic when performing bone marrow aspiration can current intensity of conventional therapy. Continued efforts to find limit the number and nature of PD assays undertaken in paediatric effective novel therapies are therefore of critical importance if leukaemia trials. Assays such as the PIA assay have been survival is to be improved and long-term morbidity reduced. successfully adapted for use in children, such that smaller blood volumes are required, permitting use in phase I paediatric trials.124 Furthermore, the PIA assay has been successfully adapted and CONFLICT OF INTEREST validated for therapeutic targets other than FLT3, with inhibition ASM and ADJP are past and present employees respectively of The Institute of Cancer of phosphorylated histone H3 being used as a PD biomarker of Research, which has a commercial interest in drug development programmes (see Aurora kinase inhibition in a paediatric trial of AT9283 (EudraCT www.icr.ac.uk), and are subject to a ‘Rewards to Inventors Scheme’ which may reward No. 2009-016952-36, NCT01431664).124 contributors to a programme that is subsequently licensed. ASM has received competitive research funding from Pfizer Inc., by way of a Pfizer Australia Cancer Research Grant.

FUTURE DIRECTIONS AND TRIAL DESIGN ACKNOWLEDGEMENTS The prognosis for children with AML has improved over recent ASM is supported by the National Health and Medical Research Council (Australia) decades, but such improvements are largely attributable to more and the Children’s Health Foundation Queensland. ADJP is supported by Cancer intensive use of cytotoxic chemotherapy and better supportive Research UK (programme grant C1178/A10294). ASM and ADJP acknowledge NHS 6 care. Although a number of novel therapeutic agents have been funding to the NIHR Biomedical Research Centre at The Institute of Cancer Research recently developed and show promising efficacy in adult AML, and the Royal Marsden NHS Foundation Trust. significant issues still exist for executing early phase clinical trials of such drugs in children. Often, patients may progress rapidly between screening and commencing a novel therapy, by which REFERENCES time they may become ineligible with poor performance status or 1 Dores GM, Devesa SS, Curtis RE, Linet MS, Morton LM. Acute leukemia incidence complicating conditions such as uncontrolled, invasive infection. and patient survival among children and adults in the United States, 2001–2007. Furthermore, patients relapsing soon after allogeneic HSCT may Blood 2012; 119: 34–43. have poor organ function deeming them ineligible. One possible 2 Burnett A, Wetzler M, Lowenberg B. Therapeutic advances in acute myeloid solution is to treat patients with primary refractory or first-relapsed leukemia. J Clin Oncol 2011; 29: 487–494. AML with targeted novel agents in a ‘window’ period prior to 3 Gibson BE, Webb DK, Howman AJ, De Graaf SS, Harrison CJ, Wheatley K. Results of a randomized trial in children with Acute Myeloid Leukaemia: Medical allogeneic HSCT. Several factors need to be considered for this Research Council AML12 trial. Br J Haematol 2011; 155: 366–376. approach though, including the likelihood of achieving CR with 4 Pui CH, Carroll WL, Meshinchi S, Arceci RJ. Biology, Risk Stratification, and second or third line chemotherapy, the potential of the novel Therapy of Pediatric Acute Leukemias: An Update. J Clin Oncol 2011; 29: agent causing organ toxicity which could compromise the success 551–565.

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