Novel agents for the treatment of childhood acute leukemia

Abstract

Together, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) make up approximately one-third of all pediatric cancer diagnoses. Despite remarkable improvement in the treatment outcomes of these diseases over the past several decades, the prognosis for certain high-risk groups of leukemia and for relapsed disease remains poor. However, recent insights into different types of ‘driver’ lesions of leukemogenesis, such as the aberrant activation of signaling pathways and various epigenetic modifications, have led to the discovery of novel agents that specifically target the mechanism of transformation. In parallel, emerging approaches in cancer immunotherapy have led to newer therapies that can exploit and harness cytotoxic immunity directed against malignant cells. This review details the rationale and implementation of recent and specifically targeted therapies in acute pediatric leukemia. Topics covered include the inhibition of critical cell signaling pathways [BCR-ABL, FMS-like tyrosine kinase 3 (FLT3), mammalian target of rapamycin (mTOR), and Janus-associated kinase (JAK)], proteasome inhibition, inhibition of epigenetic regulators of gene expression [DNA methyltransferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, and disruptor of telomeric signaling-1 (DOT1L) inhibitors], monoclonal antibodies and immunoconjugated toxins, bispecific T-cell engaging (BiTE) antibodies, and chimeric antigen receptor-modified (CAR) T cells.

Introduction

Remarkable progress has been made in the past several decades in the treatment of childhood acute lymphoblastic leukemia (ALL), with 5-year survival rates now approaching 90% [Hunger et al. 2012]. This success has been achieved with a risk stratification design incorporating minimal residual disease (MRD) status, and the intensification of cytotoxic chemotherapy for those at highest risk [Pui et al. 2011a]. However, up to 20% of children with ALL will be refractory to treatment or relapse following treatment, and the survival rate for relapsed ALL remains poor. In addition, although the majority of children with acute myeloid leukemia (AML) will achieve remission with conventional chemotherapy, less than 60% will be long-term survivors. Cytotoxic therapy intensification has been maximized in the treatment of AML, highlighting the need for additional, targeted novel therapies in this disease [Pui et al. 2011b]. Targeted therapies in pediatric leukemia are largely unproven to date, with the clear exceptions of tyrosine kinase inhibitors (TKIs) in BCR-ABL (Philadelphia chromosome) positive leukemia [Schultz et al. 2009], and all-trans retinoic acid (ATRA) in acute promyelocytic leukemia (APML) with PML-RARa fusions [Tallman, 2004]. Challenges have included the effective incorporation of novel agents into current chemotherapy regimens, the development of resistance to targeted therapies, and failure to eradicate the leukemia stem cell (LSC) population in an individual disease. In this article, we summarize recent and developing novel drugs for pediatric acute leukemia, review open and recently completed clinical trials using these agents (listed in Table 1), and discuss plans to implement these therapies into pediatric ALL or AML treatment regimens.

Table 1.

Active and recently completed clinical trials with novel agents in pediatric acute leukemia.

Drug	Target	Disease	Phase	Clinical trial identifier(s)
Dasatinib	BCR-ABL	ALL (Ph+)	II	NCT01460160	*completed
				COG AALL1122
Nilotonib	BCR-ABL	ALL, CML (Ph+)	I	NCT01077544
				CAMN107A2120
Lestaurtinib	FLT3	Infant ALL	III	NCT00557193	*completed
		AML	I	COG AALL0631
				NCT00469859	*completed
				COG AAML06P1
Midostaurin	FLT3	Infant ALL, AML	I/II	NCT00866281
Quizartinib	FLT3	ALL, AML	I	NCT01411267	*completed
				TACL 2009-004
Sorafenib	FLT3, other RTKs	AML	III	NCT01371981
		Infant ALL, AML, MDS	I	COG AAML1031
		ALL, AML	I	NCT00908167
				RELHEM
				NCT00665990
				ANGIO1
Rapamycin	mTOR	ALL, NHL	I	NCT01658007
		ALL	III	2012-0361
				NCT00382109	*completed
				COG ASCT0431
Temsirolimus	mTOR	ALL, NHL	I	NCT01403415
				COG ADVL1114
Everolimus	mTOR	ALL	I	NCT01523977
		ALL	I/II	11-237
				NCT00968253
				2009-0100
Ruxolitinib	JAK 1/2	ALL, AML	I	NCT01164163	*completed
				COG ADVL1011
Bortezomib	Proteasome	ALL, AML	I	NCT00077467	*completed
		AML	II	COG ADVL0317
		ALL	II	NCT00666588	*completed
		AML	III	COG AAML07P1
				NCT00873093	*completed
				COG AALL07P1
				NCT01371981
				COG AAML1031
Azacitidine	DNMT	ALL, AML	I	NCT01861002	*completed
				TACL 2011-002
Vorinostat	HDAC	ALL	I/II	NCT01483690
				TACL 2009-003
Panobinostat	HDAC	ALL, AML, HL, NHL	I	NCT01321346
				TACL 2009-012
EPZ-5676	DOT1L	ALL, AML (MLL-r)	IIb	NCT02141828
Gemtuzomab	CD33	AML	III	NCT00372593	*completed
				COG AAML0531
Epratuzumab	CD22	CD22+ ALL	I/II	NCT00098839	*completed
		ALL	II	ADVL04P2
				NCT01802814
				IntReALL SR 2010
Inotuzumab	CD22	ALL	I/II	NCT01134575	*completed
				2009-0872
Moxetumomab	CD22	ALL or NHL	I	NCT00659425
			II	CAT-8015-1004
		ALL		NCT02227108
				CAT-8015-1036
Blinatumomab	CD3, CD19	ALL	I/II	NCT01471782
		ALL	III	MT103-205
				NCT02101853
				COG AALL1331
CAR T-cells	CD19	CD19+ ALL /lymphoma	I/IIa	NCT01626495
		CD19+ ALL /lymphoma	II	10-007706
		CD19+ ALL	I	NCT01593696
		CD19+ ALL	I	120112, 12-C-0112
		CD19+ ALL	I/II	NCT01860937
				13-052
				NCT01683279
				PLAT-01
				NCT02028455
				PLAT-02

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; Ph+, Philadelphia chromosome positive; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; NHL, non-Hodgkin lymphoma; HL, Hodgkin lymphoma; MLL-r, mixed lineage leukemia gene rearranged; DNMT, DNA methyltransferase; HDAC, histone deacetylase; DOT1L, Disruptor of telomerase-1; CAR T-cells, chimeric antigen receptor.

Tyrosine kinases (TKIs)

BCR-ABL

Among the successful stories of targeted therapies in pediatric acute leukemia is the introduction of the TKI imatinib into upfront therapy for Philadelphia chromosome positive (Ph+) ALL patients. The Children’s Oncology Group (COG) trial AALL0031 (2002–2006) incorporated imatinib into an upfront, intensive chemotherapy backbone for Ph+ ALL pediatric patients. Initial results from this trial demonstrated a 3-year event-free survival (EFS) of 88%, doubling that of historical controls [Schultz et al. 2009]. In addition, longer follow-up revealed that Ph+ ALL patients receiving chemotherapy with continuous imatinib had similar 5-year EFS rates to patients undergoing either related or unrelated donor hematopoietic stem-cell transplant (HSCT; 71%, 64% and 63%, respectively; p = 0.77) [Schultz et al. 2014]. Retrospective analysis of patients who relapsed after treatment demonstrated remission reinduction rates similar to other high-risk non-Ph+ ALL patients treated on contemporaneous trials, allowing these patients to proceed to HSCT as salvage therapy [Schultz et al. 2014]. Imatinib was FDA-approved for the treatment of Ph+ ALL in children in 2013. However, a well-known mechanism of resistance to TKI therapy is the outgrowth of resistant clones, often mediated through the development of point mutations in the kinase domain of ABL. In a recent review of 272 adult patients with relapsed Ph+ ALL, 70% harbored a kinase domain point mutation, including T315I, E255K and Y253H [Soverini et al. 2014]. Interestingly, kinase point mutations seem to be less common in pediatric Ph+ ALL at relapse, although they have been demonstrated to occur [Chang et al. 2012].

Dasatinib, a second-generation TKI, replaced imatinib in the most recent COG trial AALL1122 for Ph+ ALL patients, after COG phase I/II trial AALL0622 demonstrated good tolerability and rapid efficacy of dasatinib in combination with chemotherapy. There is in vitro evidence that dasatinib has superior central nervous system (CNS) penetration compared with imatinib [Porkka et al. 2008]. Dasatinib is effective against many resistant mutations, with the exception of point mutation T315I [Talpaz et al. 2006]. Nilotinib is another second-generation TKI that has been less studied in both adults and pediatrics, but some reports show efficacy against certain dasatinib-resistant mutations, although not T315I [Jabbour et al. 2008; Sekimizu et al. 2013]. A phase II COG study is investigating the efficacy of nilotinib in pediatric chronic myeloid leukemia (CML), and a multi-institutional phase I study of nilotinib is open for pediatric patients with CML or relapsed/refractory Ph+ ALL (Table 1). The third-generation TKI, ponatinib, is active against the increasingly clinically significant mutation T315I, but toxicities of arterial thrombosis risk were documented [Cortes et al. 2013], temporarily halting its development in clinical trials. As of January 2014, ponatinib is FDA-approved for adults with Ph+ leukemia resistant to other TKIs, now carrying the additional warning of thrombosis. Other classes of kinase inhibitors are being explored in adult Ph+ leukemia in an attempt to prevent the development of resistance, such as the Janus-associated kinase (JAK) inhibitor, ruxolitinib, in combination with nilotinib [ClinicalTrials.gov identifiers: NCT01702064 and NCT01914484].

FMS-like tyrosine kinase 3 (FLT3)

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase expressed on human CD34⁺ hematopoietic stem and early progenitor cells, and FLT3 signaling is central to cell proliferation and differentiation [Small et al. 1994]. FLT3 is aberrantly expressed on the majority of leukemic blasts regardless of CD34 expression [Carow et al. 1996]. Of note, the most consistently overexpressed gene in mixed lineage leukemia-rearranged (MLL-r) infant ALL is wild-type FLT3 [Armstrong et al. 2002]. Also, mutations of FLT3 occur in 20–25% of pediatric AML patients, and result in ligand-independent constitutive activation of the receptor [Kondo et al. 1999; Meshinchi et al. 2001]. Roughly two-thirds of these mutations are internal tandem duplications (ITD) of the juxtamembrane domain of the gene, and the remaining one-third are point mutations of the tyrosine kinase domain (TKD) [Meshinchi et al. 2001; Yamamoto et al. 2001]. Multiple studies have documented decreased overall survival and increased rate of relapse in FLT3-ITD mutant AML [Iwai et al. 1999; Kondo et al. 1999; Meshinchi et al. 2001]. In one study, children with ITD mutations had 8-year overall survival (OS) and EFS rates of 13% and 7%, respectively, compared with an OS of 50% and EFS of 44% for children without ITD mutations [Meshinchi et al. 2001]. A large retrospective review determined that an ITD allelic ratio of 0.4 or higher identified the highest risk group with the worst prognosis, whereas children with allelic ratios <0.4 had similar outcomes to those with wild type FLT3 [Meshinchi et al. 2006]. These data provide strong rationale for the use of FLT3 inhibitors in pediatric acute leukemia.

The FLT3 inhibitor lestaurtinib (CEP-701) has shown modest efficacy in adult trials as monotherapy [Smith et al. 2004; Knapper et al. 2006] or in combination with chemotherapy [Levis et al. 2011]. In pediatrics, a phase I trial of lestaurtinib with chemotherapy for relapsed/refractory AML has been completed, though clinical data are not yet published. However, at each dose level, five of six patients tested had >80% inhibition of FLT3 phosphorylation at the majority of lestaurtinib trough time points. Recently completed COG phase III trial AALL0631 investigated lestaurtinib in combination with chemotherapy for newly diagnosed infant ALL. Intermediate-risk (MLL-r and >90 days old) and high-risk (MLL-r and <90 days old) infants were randomized to receive lestaurtinib after induction chemotherapy. Trial efficacy results are pending.

Midostaurin (PKC412) is a multi-TKI that has activity against FLT3, and early phase adult clinical trials showed hematological responses in patients with mutant FLT3 [Stone et al. 2005; Fischer et al. 2010]. In pediatrics, a phase I/II clinical trial open in Europe and some US centers is currently recruiting MLL-r infant ALL and FLT3-mutant AML patients to receive midostaurin as a single agent. Laboratory correlatives include evaluation of FLT3 phosphorylation before and after receiving drug.

Quizartinib, or AC220, is a far more potent and selective FLT3 inhibitor than lestaurtinib and midostaurin. In a phase II study of quizartinib as monotherapy in relapsed/refractory adults with FLT3-ITD mutant AML, a complete response (CR) or a complete response with incomplete blood count recovery (CRi) was induced in 9 of 17 patients (53%) [Levis et al. 2012]. Current adult studies are investigating quizartinib in combination with chemotherapy. In pediatrics, a recently completed Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) pilot study investigated quizartinib in combination with cytarabine and etoposide in patients with MLL-r ALL or relapsed/refractory AML. Laboratory correlatives showed near-complete inhibition of FLT3 phosphorylation; importantly, four of six patients with FLT3-ITD mutant AML achieved a CR or CRi, and the other two ITD patients had stable disease [Cooper et al. 2013].

Sorafenib, a multi-TKI with activity against FLT3, induced clinical responses specifically in ITD-mutant cases of relapsed/refractory AML in three independent phase I adult trials [Zhang et al. 2008; Crump et al. 2010; Pratz et al. 2010]. A phase I/II trial in newly diagnosed adult AML combined sorafenib with cytarabine and idarubicin and impressively, 38 of 51 (75%) patients achieved a CR, including 14 of 15 FLT3-ITD patients [Ravandi et al. 2010]. In 12 pediatric patients with relapsed/refractory AML, 5 of 5 FLT3-ITD and 3 of 7 wild type FLT3 patients achieved a CR or CRi with sorafenib in combination with clofarabine and cytarabine [Inaba et al. 2011]. Given these encouraging reports, the current COG phase III trial for newly diagnosed pediatric AML nonrandomly assigns ITD-mutant patients with high allelic ratios (>0.4) to receive sorafenib in combination with chemotherapy, and includes a maintenance phase with single agent sorafenib for 1 year. There are also two phase I studies open at St. Jude Children’s Research Hospital, combining sorafenib with chemotherapy in patients with refractory hematological malignancies.

Serine/threonine kinases

mTOR

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase, and is centrally integrated in several key signal transduction pathways critical to cell growth and proliferation. Aberrant activation of the mTOR pathway has been demonstrated in multiple tumor types, and inhibition of mTOR by the macrolide rapamycin (sirolimus) or one of its analogs (temsirolimus, everolimus) has shown antitumor activity in preclinical models and in early phase clinical trials.

Constitutive activation of the mTOR pathway has been demonstrated in the majority of cases of AML [Min et al. 2003; Xu et al. 2003; Recher et al. 2005b]. Rapamycin treatment causes G0/G1 cell cycle arrest in AML cell lines, and inhibits the clonogenic properties of AML patient samples without significantly affecting healthy donor CD34⁺ bone marrow (BM) cells [Recher et al. 2005a]. Rapamycin induced clinical responses in 4 of 9 relapsed/refractory adult AML patients when given as a single agent for a 28-day course [Recher et al. 2005b]. A phase I/II study of single-agent everolimus in adults with relapsed/refractory leukemia demonstrated good tolerability and a modest clinical response [Yee et al. 2006]. Currently, there are no ongoing trials investigating mTOR inhibitors in childhood AML.

Rapamycin has also been shown to have activity in pre-B cell ALL cell lines, primary patient samples, and a xenograft model [Brown et al. 2003; Maude et al. 2012]. mTOR inhibitors also synergize with methotrexate in pre-B ALL cells [Teachey et al. 2008]. In addition, rapamycin was shown to sensitize steroid-resistant malignant lymphoid cells to glucocorticoid-induced apoptosis via modulation of anti-apoptotic MCL1 [Wei et al. 2006], and could therefore have a role in overcoming glucocorticoid resistance, a known poor prognostic factor in ALL. These studies provide strong rationale to pursue mTOR inhibition in combination with chemotherapy in pediatric ALL, and there are multiple ongoing early phase clinical trials investigating these agents. A single-institution pilot trial at Cincinnati Children’s hospital combines rapamycin with the mitoxantrone arm of UKALLR3 reinduction chemotherapy [Parker et al. 2010] in patients with relapsed ALL up to 30 years of age. The phase I COG study ADVL1114 is investigating temsirolimus in combination with a similar reinduction chemotherapy backbone in relapsed pediatric ALL patients. A multi-institutional phase I trial combines everolimus with four-drug reinduction for pediatric ALL patients in first bone marrow relapse. Finally, a single-institution phase I/II trial investigating everolimus in combination with hyper-CVAD chemotherapy is underway for patients aged 10 years and older with relapsed/refractory ALL.

mTOR inhibitors such as rapamycin (sirolimus) are also effective in graft versus host disease (GVHD). The recently completed phase III COG trial, ASCT0431, investigated whether the addition of sirolimus to GVHD prophylaxis in children with ALL would decrease relapse rates as well as acute GVHD (aGVHD). Results showed that although sirolimus decreased aGVHD, survival was not improved [Pulsipher et al. 2014].

JAK/STAT

Hyperactive signaling of the Janus-associated kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been documented in several types of high-risk leukemia. Activating mutations of JAK2 are well documented in AML [Daver and Cortes, 2012]. Aberrant activation of JAK signaling has also been described in Philadelphia chromosome-like (Ph-like) ALL, a recently defined subtype of ALL with gene expression patterns similar to Ph+ cases but lacking the BCR-ABL fusion [Mullighan et al. 2009]. Xenograft models of eight cases of Ph-like ALL demonstrated decreased leukemic burden when treated with a selective JAK1/2 inhibitor, ruxolitinib, and six of these xenograft models harbored either a JAK2 mutation or CRLF2 rearrangement [Maude et al. 2012]. The remaining two Ph-like ALL patient samples contained some other activating signature of hyperactive JAK/STAT signaling, despite lacking a point mutation. This suggests that a JAK2 activation footprint may be more significant than the presence of a mutation, in terms of predicting response to JAK2 inhibition. Interestingly, the mTOR pathway is also often aberrantly activated in Ph-like pre-B ALL patients, and single agent rapamycin demonstrated activity in all eight of these xenograft models [Maude et al. 2012]. Combining mTOR inhibitors with JAK2 inhibition, or combining JAK2 inhibition with cytotoxic chemotherapy, has not yet been studied in pediatric leukemia. The phase I COG study ADVL1011 recently investigated the safety and dosing of single-agent ruxolitinib in children with relapsed/refractory hematologic malignancies and solid tumors, and results are pending.

Furthermore, a detailed genomic analysis of 154 patients identified as Ph-like ALL by gene expression profiling was recently reported [Roberts et al. 2014]. Greater than 90% of patients in this cohort were found to have a kinase-activating lesion. Rearrangements involving ABL1, ABL2, and JAK2 were among the most common alterations, and fusion protein expression caused cell proliferation and activated STAT5 signaling. Importantly, Ph-like leukemia cells with fusions involving ABL 1/2 were sensitive to dasatinib in vitro, and cells with JAK2 rearrangements were sensitive to ruxolitinib. Trials are needed to determine whether identifying Ph-like patients and incorporating targeted TKIs into therapy will improve outcomes in this patient population.

JAK2 inhibitors were also identified in a high-throughput screen of kinase inhibitors as potential therapeutic agents in resistant FLT3-mutant AML [Weisberg et al. 2012]. The combination of JAK inhibitors with the FLT3 inhibitor PKC412 in AML cell lines demonstrated synergistic cytotoxicity and inhibition of downstream pathway signaling, specifically overcoming resistance to PKC412 in the presence of stroma [Weisberg et al. 2012]. An adult phase III study is investigating pacritinib, a dual JAK2/FLT3 inhibitor, in myelofibrosis [ClinicalTrials.gov identifier: NCT02055781].

Proteasome inhibitors

Proteasome inhibitors impair tumor growth through a variety of mechanisms [Rajkumar et al. 2005] and inactivate nuclear factor kB (NF-kB) by blocking the degradation of IkBa, a negative regulator of NF-kB. NF-kB is a transcriptional activator with anti-apoptotic properties, and is thought to be a key survival factor in various malignancies [Wang et al. 1996]. NF-kB is aberrantly constitutively activated in primary AML samples, specifically in the quiescent CD34⁺/CD38⁻ LSC compartment [Guzman et al. 2001]. NF-kB activity is also increased in leukemia cells after treatment with chemotherapy [Maestre et al. 2001]. Treatment of AML samples with a proteasome inhibitor led to down-regulation of NF-kB and its downstream targets, and induced apoptosis in CD34⁺ leukemia cells, but not in normal CD34⁺ bone marrow [Guzman et al. 2001]. Combining a proteasome inhibitor with idarubicin induced a rapid apoptotic response in primary AML samples in vitro, and effectively ablated the ability of the LSC population to engraft in a xenograft model, though normal hematopoietic cells were viable and able to engraft [Guzman et al. 2002]. This data led to the clinical investigation of a proteasome inhibitor, bortezomib, hypothesizing that selective targeting of the LSC population would result in more effective and durable responses in combination with standard treatment.

Bortezomib

Although bortezomib treatment inhibits NF-kB activity, it has little antileukemia activity as a single agent [Horton et al. 2007]. Adult trials have demonstrated that bortezomib may be safely combined with AML chemotherapy, although there have been rare cases of acute respiratory distress syndrome (ARDS) in combination with high-dose cytarabine [Attar et al. 2008]. The phase II COG protocol AAML07P1 combined bortezomib with standard AML chemotherapy and though results are not yet published, no children developed ARDS. Thus, while the available data on bortezomib in pediatric AML are limited, in vitro and adult data demonstrate that bortezomib may be combined safely with AML chemotherapy and may augment the efficacy of standard AML therapy. Phase III COG trial AAML1031 is evaluating bortezomib in a randomized fashion in newly diagnosed pediatric AML, in combination with standard chemotherapy.

A phase I TACL trial determined that bortezomib is well tolerated in combination with four-drug reinduction chemotherapy in relapsed/refractory pediatric ALL [Messinger et al. 2010]. The expanded phase II portion of this trial evaluated 22 relapsed ALL pediatric patients and demonstrated a 73% overall response rate, including 14 CRs and 2 CRis [Messinger et al. 2012]. The recently completed phase II COG study AALL07P1 combined bortezomib with reinduction chemotherapy for relapsed ALL. End of induction CR was achieved in 42 of 61 (68%) of pre-B ALL patients, as well as 11 of 17 (65%) T-cell ALL patients (unpublished data, Horton, COG open meeting, 2013), prompting the planned incorporation of bortezomib in a randomized fashion in the next phase III COG study for newly diagnosed T-cell ALL, AALL1231.

Carfilzomib

Carfilzomib is another proteasome inhibitor that is structurally and mechanistically distinct from bortezomib, demonstrating less reactivity against non-proteosomal proteases [Arastu-Kapur et al. 2011] and achieving higher levels of proteasome inhibition than bortezomib in preclinical models [Yang et al. 2011]. In early phase trials of adults with multiple myeloma, carfilzomib showed good tolerability both alone and in combination with chemotherapy, and clinical responses were achieved in both bortezomib-naïve and bortezomib pretreated patients [Siegel et al. 2012; Wang et al. 2013]. Carfilzomib induced apoptosis in a variety of pediatric tumor cell lines, and synergized with etoposide and cyclophosphamide [Jayanthan et al. 2013; Ruan et al. 2013]. Based on this encouraging preclinical data, a phase I Pediatric Oncology Experimental Therapeutics Investigator’s Consortium (POETIC) study is planned, evaluating the tolerability of carfilzomib with cyclophosphamide and etoposide in relapsed/refractory pediatric leukemia and solid tumors. A TACL phase I/II study is also planned for children with relapsed/refractory leukemia, investigating carfilzomib in combination with UKALLR3 reinduction chemotherapy.

Epigenetic targeting

Epigenetic modifications, including methylation of CpG islands in gene promoter regions and chromatin modifications by histone acetylation, play a critical role in gene expression. Transcriptional silencing of tumor suppressor genes can lead to malignant transformation in many cancers, including leukemia [Herman et al. 1997; Seedhouse et al. 2003]. These modifications can be targeted by DNA methyltransferase (DNMT) inhibition or histone deacetylase (HDAC) inhibition in an attempt to reverse the epigenetic silencing of crucial regulatory genes and modify the malignant phenotype [Gore, 2005].

DNMT

The two azanucleosides, 5-azacytidine (azacitidine) and 5-aza-2′-deoxycytidine (decitabine), are cytosine analogs that incorporate into nucleic acids, thereby affecting multiple molecular pathways. After incorporation into DNA, both drugs covalently ‘trap’ DNMTs, which are then degraded [Santi et al. 1984]. There is in vitro evidence that these drugs are effective in inducing DNA demethylation and epigenetic gene reactivation [Stresemann et al. 2006], although differential demethylating effects on various human AML cell lines is seen [Stresemann et al. 2008].

Both azacitidine and decitabine are effective in the treatment of adult patients with MDS [Silverman et al. 2002; Kantarjian et al. 2006], and have been FDA-approved for this disease. Decitabine has also been approved in Europe for the treatment of adult AML. Combining DNMT inhibitors with HDAC inhibitors is being studied extensively in adult MDS and AML, with promising initial results [Garcia-Manero et al. 2006; Gore et al. 2006; Soriano et al. 2007]. Some studies suggest that despite good clinical responses, no correlation is seen between response and methylation or gene expression patterns [Fandy et al. 2009]. However, another study compared gene expression and methylation patterns of paired diagnostic-relapsed ALL samples, and demonstrated a relapse-specific signature associated with chemoresistance [Bhatla et al. 2012]. Treating primary relapsed samples or leukemia cell lines in vitro with the HDAC inhibitor vorinostat alone or in combination with decitabine restored expression of genes that were preferentially silenced at relapse, and seemed to ‘reprogram’ the abnormal relapsed gene expression profile. Furthermore, these drugs demonstrate synergistic cytotoxicity when given prior to chemotherapy [Bhatla et al. 2012]. A recently completed phase I TACL trial combined azacitidine with chemotherapy for relapsed/refractory pediatric ALL or AML, and an ongoing phase I/II TACL trial is evaluating the efficacy of epigenetic therapy with decitabine and vorinostat followed by reinduction chemotherapy in relapsed/refractory pediatric ALL.

HDAC

HDACs remove acetyl groups from lysine residues on histones, causing decreased accessibility of chromatin and thus, transcriptional repression and epigenetic silencing [Lucas et al. 2010]. In vitro experiments demonstrate efficacy of HDAC inhibitors in both AML and ALL. One investigation demonstrated synergy of vorinostat and methotrexate in ALL cell lines by increasing the expression of the synthetase that metabolizes methotrexate to its active form, leading to increased cytotoxicity [Leclerc et al. 2010]. Another study demonstrated restoration of BH3-only BCL2 family member (BIM) expression after vorinostat treatment in glucocorticoid-resistant ALL xenografts, resensitizing this disease to glucocorticoids [Bachmann et al. 2010]. In an AML cell line, treatment with vorinostat resulted in increased expression of p21, leading to p53-independent cell cycle arrest [Vrana et al. 1999]. HDAC inhibitors have also been shown to abolish the fusion proteins AML1-ETO and PML-RARa in AML [Kramer et al. 2008]. Stumpel and colleagues demonstrated that treatment with HDAC inhibitors induced cytotoxicity in t(4;11) MLL-r primary infant ALL cells, and HDAC inhibitor treatment was shown to neutralize the MLL-AF4 fusion protein [Stumpel et al. 2012]. These data and others led to the investigation of HDAC inhibitors in acute leukemia.

A phase I adult trial with vorinostat monotherapy demonstrated some antileukemia effect and good tolerability [Garcia-Manero et al. 2008]. Subsequently, a phase I pediatric trial with vorinostat in solid tumors and leukemia also demonstrated tolerability, although patients with refractory leukemia required a lower dose [Fouladi et al. 2010]. As stated above, vorinostat is currently being investigated in a phase II pediatric clinical trial in combination with decitabine in relapsed ALL, in an attempt to ‘reprogram’ the gene expression signature and restore sensitivity to chemotherapy. In addition, a phase I TACL trial recently opened and is investigating the tolerability of the newer HDAC inhibitor, panobinostat, in refractory pediatric hematological malignancies.

DOT1L

Approximately 60–80% of infant leukemia harbor rearrangements of the MLL gene (MLL-r) [Ayton and Cleary, 2001] and have a poor overall prognosis with standard chemotherapy approaches. Similar gene expression patterns have been described in MLL-r leukemia despite the variety of possible fusion proteins [Tsutsumi et al. 2003], including upregulation of homeobox (Hox) gene expression [Krivtsov et al. 2006]. It has been demonstrated that various MLL-fusion proteins form a complex with Disruptor of telomeric signaling-1 (DOT1L) [Zhang et al. 2006; Buttner et al. 2010]. DOT1L is the sole methyltransferase that methylates lysine 79 of histone 3 (H3K79), causing transcriptional activation [Lacoste et al. 2002]. The interaction of MLL-fusion proteins with DOT1L can lead to the mistargeting of DOT1L and subsequent methylation of H3K79 at inappropriate gene promoter sites, likely playing an important role in leukemogenic transformation [Okada et al. 2005; Mueller et al. 2007; Krivtsov et al. 2008]. For example, hypermethylation of H3K79 is seen at the promoter of HoxA9 in MLL-r leukemia [Krivtsov et al. 2008; Yokoyama et al. 2010].

In vivo and in vitro experiments have demonstrated that H3K79 methylation by DOT1L is in fact required for both initiation and maintenance of MLL-AF9 induced leukemogenesis [Chang et al. 2010; Bernt et al. 2011; Nguyen et al. 2011]. A potent small molecule inhibitor of DOT1L, EPZ004777, selectively killed MLL-r cells while having little effect on non-MLL translocated cells, and led to increased survival in an MLL-AF9 xenograft model [Daigle et al. 2011]. Similar results were then demonstrated in MLL-AF10 and MLL-AF6 cells [Chen et al. 2013; Deshpande et al. 2013]. Furthermore, treatment with EPZ004777 led to decreased expression of HoxA9 and Meis1 in all MLL-rearranged leukemia cells. These data provide strong rationale for targeting DOT1L in MLL-r leukemia. A phase I/II trial of DOT1L inhibitor EPZ-5676 is currently underway for adults with advanced MLL-r hematological malignancies [ClinicalTrials.gov identifier: NCT01684150], and a phase I multi-institutional trial of EPZ-5676 recently opened for pediatric patients with relapsed/refractory MLL-r leukemia.

Antibody-based immunotherapy

CD33

CD33 cell surface antigen is expressed in 88% of childhood AML blasts [Creutzig et al. 1995]. CD33 is also present on normal maturing hematopoietic progenitor cells, but is not present on hematopoietic stem cells or other normal tissue, making it an attractive target [Sievers et al. 2001]. Gemtuzumab (CMA-676 or GO) is an anti-CD33 antibody conjugated to calicheamicin, a potent antitumor antibiotic that cleaves double-stranded DNA at specific sequences, leading to apoptosis [Zein et al. 1988; Hinman et al. 1993; Sievers et al. 1999]. COG phase III study AAML0531 incorporated GO in a randomized fashion in combination with standard chemotherapy in de novo AML. Results showed a modest improvement in 3-year EFS in the GO arm (53% versus 47%, p = 0.05) and a trend toward improved OS [Aplenc et al. 2013]. However, GO was withdrawn from the US market in 2010 after adults receiving GO in induction therapy experienced increased mortality, despite recently published studies of GO in children [Burnett et al. 2011; Cooper et al. 2012; Hasle et al. 2012] and adults [Castaigne et al. 2012] showing that a dose of 3 mg/m² has been well tolerated. Furthermore, use of GO as consolidation therapy after HSCT in AML patients [Roman et al. 2005], or as part of a conditioning regimen for HSCT [Satwani et al. 2012] in early phase studies demonstrated safety in both settings. Reports also demonstrate reduction of MRD status with GO in otherwise refractory pediatric AML patients, thereby optimizing disease status prior to HSCT [Rubnitz et al. 2010; O’Hear et al. 2013]. These reports provide good rationale to consider ‘resurrecting’ GO [Ravandi et al. 2012].

CD22

CD22 is expressed in 96% of pre-B ALL patients on at least 90% of blasts [Gudowius et al. 2006]. Epratuzumab is a humanized monoclonal anti-CD22 antibody, and cells demonstrate rapid intracellular localization of the antibody complex after binding [Carnahan et al. 2003]. COG phase I/II study ADVL04P2 combined epratuzumab with reinduction chemotherapy in children with CD22⁺ relapsed ALL, and response was assessed after block one. In the phase II portion of the study, 65% of 98 evaluable patients achieved a CR, and 46% of these were MRD negative [Raetz et al. 2011]. This was significantly higher than the 25% of CR patients achieving negative MRD with chemotherapy alone on predecessor study AALL01P2 (p = 0.001). The upcoming European phase III IntReALL study will randomize pediatric ALL patients in standard risk first relapse to receive epratuzumab.

Since CD22 is internalized upon binding of monoclonal antibody, it is attractive for targeted delivery of immunotoxin [Press et al. 1989]. Inotuzumab ozogamicin (CMC-544 or IO) is an anti-CD22 humanized monoclonal antibody conjugated to calicheamicin, similar to anti-CD33 GO [DiJoseph et al. 2004]. IO was originally developed for use in CD22-expressing non-Hodgkin lymphoma (NHL), and two early phase trials of IO in adults with refractory NHL showed promising clinical responses as a single agent [Advani et al. 2010] and in combination with rituximab [Fayad et al. 2013]. Preclinical evidence with pre-B ALL cell lines and mouse models demonstrated that IO has good cytotoxic effect against CD22+ lymphoblasts in vitro, and abrogates xenograft tumors and disseminated leukemia in vivo [Dijoseph et al. 2007]. IO demonstrated clinical activity in a phase II study of adults and children with relapsed/refractory CD22+ pre-B ALL at dosing of 1.3–1.8 mg/m² every 3–4 weeks [Kantarjian et al. 2012]. Of 49 patients, 28 (57%) achieved CR, CRi or complete response with incomplete platelet recovery (CRp; platelets <100 × 10⁹/L). An additional 41 patients were enrolled at 1.8 mg/m² divided into a weekly dosing regimen (0.8, 0.5, and 0.5 mg/m²/week) and had a similar overall response rate of 59% [Kantarjian et al. 2013]. The most common toxicities were grade 1–2 fever, hypotension, elevated transaminases and hyperbilirubinemia. A retrospective review of the five pediatric patients on this trial demonstrated good tolerability and activity at both dosing schedules [Rytting et al. 2014]. Three of five patients achieved a CR or CRp and went to HSCT, including one patient who had achieved an MRD negative status after two cycles of the weekly dosing schedule. Current trials are investigating IO in adults with ALL in relapsed disease (phase III trial [ClinicalTrials.gov identifier: NCT01564784]), in the elderly population (phase I/II trial [ClinicalTrials.gov identifier: NCT01371630]), and prior to allogeneic transplant (phase I/II trial [ClinicalTrials.gov identifier: NCT01664910]). There are no open pediatric trials investigating IO at the time of this review.

Moxetumomab pasudotox (MP, previously CA-8015 or HA22) is a second-generation anti-CD22 immunotoxin, composed of the variable fragment of the antibody (Fv) conjugated to a protein derivative (PE38) of Pseudomonas exotoxin A [Pastan et al. 2006]. BL22, the first-generation predecessor of moxetumomab, demonstrated cytotoxicity against malignant CD22⁺ cells in vitro [Kreitman et al. 2000] and clinical activity in a phase I trial of pediatric ALL patients [Wayne et al. 2010]. Likewise, moxetumomab demonstrated good in vitro activity against relapsed, steroid-resistant, and de novo pediatric pre-B ALL samples [Mussai et al. 2010]. Immunotoxin-resistant ALL cells were found to be heavily hypermethylated at the DPH4 promoter, rendering these cells refractory to moxetumomab, and suggesting a role for concurrent demethylating agents [Wei et al. 2012]. A phase I study of moxetumomab in CD22⁺ pediatric malignancies is ongoing, and preliminary results report 4 CRs in 17 evaluable patients (24%), 1 partial response (6%) and 7 patients (41%) with >50% reduction in peripheral blood blasts or improvement in blood counts [Wayne et al. 2011]. Toxicities were generally mild and reversible, although capillary leak syndrome was dose limiting in several patients. Fourteen percent of patients developed neutralizing antibodies, necessitating further study of the potential challenges of this drug. A phase II multicenter study of moxetumomab recently opened in August 2014 for relapsed/refractory pre-B ALL or B-cell lymphoblastic lymphoma.

CD19

Blinatumomab is a CD19/CD3 bispecific T-cell engaging (BiTE) antibody that binds to CD3⁺ T-cells and colocalizes them with CD19⁺ B-cells, thereby activating the T-cells and inducing perforin-mediated death of the targeted B-cells [Loffler et al. 2000]. After promising results in a phase I trial of adults with NHL [Bargou et al. 2008], blinatumomab was moved in a phase II study in adult pre-B ALL. This study yielded striking results; of 20 evaluable patients with detectable MRD by polymerase chain reaction (PCR), 16 (12 primary refractory and 4 relapsed) achieved an MRD negative remission. Eight of these 16 patients received a HSCT and remained in remission post-transplant. Common adverse events were pyrexia, chills and lymphopenia with hypogammaglobulinemia [Topp et al. 2011]. At a median follow-up of 33 months, relapse-free survival in these patients was 61% overall, with 6 relapses reported [Topp et al. 2012]. Of 4 earlier relapses (at 3–7 months), two had CD19-negative marrow disease, one had an isolated CNS relapse, and one had a testicular relapse. Interestingly, the CNS and testes are relatively T-cell deplete compartments. The two later relapses (at 19 and 31 months) were both CD19⁺ [Topp et al. 2012]. Importantly, four responders received no subsequent therapy yet remain in remission, suggesting that as a single agent, blinatumomab can induce a durable remission in MRD-positive disease. Remarkably, the effector memory T-cell subset was amplified in all patients, which could partially explain the duration of response in these patients, though amplification occurred in non-responders as well [Klinger et al. 2012]. The US Intergroup ECOG is currently investigating blinatumomab in upfront adult pre-B ALL in a phase III clinical trial [ClinicalTrials.gov identifier: NCT02003222].

In children, Handgretinger and colleagues evaluated blinatumomab monotherapy in a small case series of heavily pretreated, post-HSCT pediatric ALL patients. All three of three children achieved negative MRD with tolerable and similar toxicities. Two of three ultimately relapsed, one after a second transplant, and the other after four courses of blinatumomab. The third patient received a second transplant and remained in a CR at 23 months [Handgretinger et al. 2011]. A multicenter phase I trial in 34 children with relapsed/refractory pre-B ALL administered blinatumomab as a continuous 4-week infusion, and established the maximum tolerated dose (MTD) as 15 µg/m²/day [Zugmaier et al. 2013]. The dose-limiting toxicity was cytokine-release syndrome. Across all dose levels, the overall response was 41% [Zugmaier et al. 2013]. A randomized COG phase III study for pre-B ALL children in first relapse is scheduled to open in late 2014, combining blinatumomab with UKALLR3 reinduction chemotherapy.

Cellular-based immunotherapy

Chimeric antigen receptor-modified T-cells (CAR T-cells) with CD19 specificity are generating excitement as a novel therapy for B-cell malignancies. CAR T cells are patient-derived T-cells, transduced to express a chimeric antigen receptor, which includes an anti-CD19 antibody fragment fused to a T-cell intracellular signaling domain [Barrett et al. 2014]. Second-generation CAR T cells also encode for a costimulatory domain, such as CD28 or members of the tumor necrosis factor receptor family such as CD27, CD137 (4-1BB) and CD134 (OX40). The costimulatory domains activate the CAR T-cells, allowing for targeting and lysis of CD19⁺ cells. Clinical trials with second-generation CD19 CAR T-cells in adult chronic lymphocytic leukemia (CLL) demonstrated massive T-cell expansion in vivo, lysis of CD19⁺ tumor cells, and aplasia of normal CD19⁺ B cells [Kalos et al. 2011]. Importantly, these relapsed and refractory CLL patients demonstrated durable remissions after infusions of CD19 CAR T-cells.

Several institutions in the United States have open pediatric trials of CD19 CAR T-cells in pre-B cell ALL. The first two pediatric patients treated at Children’s Hospital of Philadelphia (CHOP) with refractory/relapsed pre-B ALL achieved a CR within a month of CAR T-cell infusion [Grupp et al. 2013]. In both patients, dramatic expansion of CAR T-cells was documented in the peripheral blood, peaking around day 10. Surprisingly, CAR T-cells were detected in the CNS in both patients. Toxicity related to a cytokine-release syndrome after CAR T-cell infusions is significant, and in severe cases can be reminiscent of macrophage-activation syndrome. However, anticytokine therapy with the anti-IL6 monoclonal antibody, tocilizumab, can rapidly induce clinical improvement in this setting [Grupp et al. 2013]. As expected, complete B-cell aplasia occurs, and the duration of this effect is undefined and must be supported with intravenous immunoglobulin (IVIG) administration. Of the original two pediatric patients, one remains in remission without any subsequent disease-directed therapy. The other patient relapsed in 2 months with a CD19-negative clone, signifying the strong selective pressure of this therapy.

At the American Society of Hematology (ASH) 2013 annual meeting, the CHOP group presented their updated outcomes on this trial, having now enrolled 16 pediatric and 4 adult patients with CD19⁺ leukemia. A total of 14 of 17 evaluable patients (82%) achieved a CR at 1 month. Three of these 14 CR patients subsequently relapsed. Aside from the patient mentioned above, the other two relapsed patients did not have a CD19 negative clone. All patients have demonstrated some sort of cytokine release syndrome corresponding with the peak T-cell expansion in vivo [Frey et al. 2013]. Seven of 20 required treatment for respiratory or hemodynamic instability, and all improved rapidly after tocilizumab given with or without steroids. In all patients with durable responses, CAR T-cells were detectable by flow cytometry for a range of 1–15 months. Given these encouraging results, multicenter pediatric trials are now being planned to investigate CD19 CAR T-cells in a phase II setting.

Finally, a phase I clinical trial of CD22 CAR T-cells for pediatric patients with relapsed/refractory ALL is currently in development, based on promising preclinical data [Haso et al. 2013].

Conclusion

There is compelling rationale for the ongoing discovery and implementation of novel and targeted agents in pediatric leukemia. Future goals of treatment for pediatric ALL and AML, as well as other forms of cancer, are to personalize treatment to the specific molecular aberrancies driving each individual’s disease, while decreasing the intensity and toxicities of conventional chemotherapy regimens. Pediatric leukemia is a disease prototype for this area of translational and clinical research, given the relatively smaller number of cumulative ‘hits’ necessary for malignant transformation. However, there are significant challenges to the development of novel agents. The process of drug development, including enrolling adequate numbers of patients to complete clinical trials, will be particularly challenging when only specific subgroups of leukemia patients are appropriate to study for each new agent. As mentioned previously, incorporating a new agent in the safest and most effective manner into existing chemotherapy regimens is complex, and this complexity will undoubtedly increase as combinations of targeted agents are proposed; particularly since another challenge is the development of resistance that occurs upon applying strong selective pressure with targeted agents. Finally, newer targeted agents must be effective in eradicating not only the bulk leukemia population, but also the elusive and often quiescent leukemic stem cell (LSC) population, in order to effectively prevent relapse and improve long-term survival. Despite these challenges, the ongoing discovery of oncogenic lesions in pediatric leukemia and the exciting parallel development of diverse novel therapeutic agents provide true promise for improving outcomes in high-risk and relapsed pediatric acute leukemia.