Novel Therapies for Pediatric Cancers

Abstract

The current high cure rates for children diagnosed with cancer can in part be attributed to emphasis on large cooperative group clinical trials. The significant improvement in pediatric cancer survival over the last few decades is the result of optimized chemotherapy drug dosing, timing, and intensity; however, further alterations in traditional chemotherapy agents are unlikely to produce substantially better outcomes. Furthermore, there remains a subset of patients who have a very poor prognosis due to tumor type or stage at presentation, or who have a dismal prognosis with relapse or recurrence. As such, innovative approaches to therapy and new drugs are clearly needed for introduction into the current pediatric oncology arsenal. A variety of biologically targeted therapies which have shown promise in preclinical studies and early phase adult clinical trials are now being explored in pediatric clinical trials. These novel agents hold the promise for continuing to drive forward improvements in patient survival with potentially less toxicity than exists with traditional chemotherapy drugs.

INTRODUCTION

Pediatric cancer is relatively uncommon, however, it does remain the leading cause of death from disease in persons under the age of 19.[1] Despite dramatic improvements in outcome, some patients will either not achieve remission or relapse with refractory disease. Once a patient relapses, conventional chemotherapy or radiation and/or bone marrow transplant are much less effective.[2-7] Because further increases in dose intensity are neither likely tolerable nor effective, new approaches to therapy are needed, particularly for refractory or high risk patients.

Current multi-agent therapies are intensive and carry with them a significant risk for acute toxicity and late effects. It is doubtful that significant gains will be achieved by continued intensification of conventional chemotherapy regimens without paying a significant price of morbidity to patients. This makes the development of targeted therapies with greater efficacy and less toxicity of paramount importance.

One major focus of current investigation in pediatric oncology is to differentiate patients with a high likelihood of relapse from those with a favorable prognosis. Yet even within subsets of patients with favorable prognostic factors, relapse remains problematic. New technologies have improved our understanding of cancer biology, and expanding knowledge of underlying molecular pathways and genomic aberrations is facilitating the development of new therapies for pediatric cancers.[8, 9] We present a brief review of selected new agents under investigation for pediatric malignancies, both solid tumors and hematologic malignancies (Figure 1). The majority of agents are in early phase clinical trials and some remain in pre-clinical exploration. The “novelty” of such agents comes from their more selective nature in capitalizing on a feature or features unique or more predominant in malignant cells, compared to their normal counterparts. Table 1 summarizes the drugs discussed and the current stage of clinical development. It should be noted that the current rate of drug development far outpaces the realistic ability for any review to be comprehensive in nature. However, an attempt is made to focus on a number of promising molecularly and/or mechanistically targeted agents, with the hope of characterizing pathways which may be utilized to more effectively treat patients while minimizing toxicities. Traditionally, the therapies for different pediatric tumor types have been fundamentally distinct, as our understanding of the diseases takes on a more molecular basis, analysis of the targets for therapy is increasingly likely to take on a more “cross-platform” nature.

Figure 1.

Summary of mechanisms of action of novel therapies directed against pediatric tumors and leukemia. Drugs are listed in italics demonstrating their effect on targeted proteins, selected signaling pathways, and gene transcription. RTK = receptor tyrosine kinase; VEGF = vascular endothelial growth factor; HDAC = histone deacetylase; SAHA = suberanilohydroxamic acid

Table 1.

Selected therapies under investigation in pediatric malignancies.

Target/Class	Patient Population	Drug Name: generic (brand)	Route of Administration	Pediatric Status
BCR-ABL TKIs	Ph +ALL, Solid tumors	imatinib (Gleevec™)	PO	•Pediatric phase I complete •Pediatric phase III in Ph+ ALL closed, early analysis – improved early EFS (COG) •Pediatric phase II single agent trial in solid tumors – no activity
	Ph +ALL, Solid tumors	dasatinib (Sprycel®)	PO	•Adult phase I complete; phase II and III on-going •Pediatric phase I in imatinib resistant Ph+ALL and solid tumors ongoing •Pediatric phase II in Ph+ ALL with imatinib in development
	Ph +ALL, Solid tumors	nilotinib (Tasigna®)	PO	•Adult phase I complete; phase II and III on-going •Pediatric phase I in development
VEGF pathway inhibitors	Leukemias, Solid tumors	bevacizumab (Avastin®)	IV	•Pediatric phase I study in solid tumors complete •Pediatric phase I study with combination chemotherapy opening •Pediatric phase II study in combination with chemotherapy for relapsed Ewing Sarcoma ongoing
	Solid tumors	VEGF trap	IV	• Pediatric phase I study in refractory solid tumors ongoing
	Solid tumors	Sunitinib (Sutent®)	PO	• Pediatric phase I study in relapsed/refractory solid tumors ongoing
IGF1R inhibitors	Solid tumors	R1507 IMC A12	IV	•Multiple adult Phase 1 complete; phase II, III ongoing •2 Pediatric Phase I studies on-going •2 Pediatric Phase II studies approved
Multi-target TKIs	Leukemias, Solid tumors	Sorafenib (Nexavar®)		• Adult phase I complete, phase II/ III ongoing • Pediatric phase I on-going
FLT3 inhibitors	MLL rearranged infant ALL, AML	lestaurtinib (CEP-701)	PO	•Pediatric phase I/II complete •Phase III in high risk infants with intensive chemotherapy ongoing •Pediatric pilot study in relapsed/refractory AML with cytarabine and idarubicin in development
Purine nucleoside analogues	Acute Leukemias	clofarabine (Clolar®)	IV	•Pediatric Phase I, II study complete •Pediatric phase I/II study in relapsed/refractory acute leukemias combined with cyclophosphamide and etoposide ongoing •Pediatric phase I/II study in relapsed/refractory leukemia with cytarabine ongoing •Pediatric phase I/II study in relapsed/refractory ALL, AML, MDS combined with cyclophosphamide ongoing
	T-cell ALL, T-cell Lymphomas	nelarabine (Arranon®)	IV	•Pediatric phase II study complete •Pediatric phase III study in newly diagnosed T-cell ALL in combination with chemotherapy ongoing •Pediatric phase I study in relapsed/refractory T-cell ALL in combination with cyclophosphamide and etoposide in development
	ALL	forodesine (Fodosine™)	IV	•Pediatric phase II study complete •Pediatric phase II and phase I/II combination study in development
Proteasome inhibitors	Leukemias, Lymphoma	bortezomib (Velcade®)	IV	•Adult Phase I study complete •Pediatric phase Ib/II study in relapsed/refractory AML with chemotherapy in development •Pediatric phase I/II combination study in ALL in development •Pediatric phase II combination in Hodgkin's Disease ongoing
mTOR inhibitors	Leukemias, Solid tumors	rapamycin (sirolimus, Rapamune®)	PO	•Pediatric phase III study in ALL patients post-bone marrow transplant ongoing •Phase II combination study in ALL in development •Phase I combination study with temozolamide and irinotecan in refractory sarcomas in development
	Leukemias, Solid tumors	temsirolimus	PO	• Adult Phase I, II studies complete, analyses pending
	Leukemias, Solid tumors	everolimus	PO	• Pediatric phase I study in solid tumors complete
	Leukemias, Solid tumors	deforolimus	PO	•Adult phase I study complete •Pediatric phase I study in solid tumors ongoing
Anti CD22 MoABs	B-cell ALL	epratuzumab (IMMU-103)	IV	•Pediatric phase I/II study complete •Pediatric phase II combination study in relapsed CD22 positive B-cell ALL ongoing
	B-cell ALL, Lymphoma	tositumomab (Bexxar™)	IV	• Adult Phase I studies in lymphoma complete, phase II/III ongoing
	B-cell ALL, Lymphoma	Ibritumomab	IV	• Pediatric phase I study in lymphoma complete, analysis pending
γ-Secretase inhibitors	T-cell ALL	LY-411575 and LY450139	PO	•Adult Phase I complete •Pediatric phase I studies in development
Anti CD52 MoAB	T-cell ALL	alemtuzumab (CamPath®)	IV	• Pediatric phase II in relapsed/refractory ALL with combination chemotherapy closed due to poor accrual
angiogenesis inhibitors	AML, Solid tumors	thalidomide, lenalidomide	PO	• Pediatric phase I studies in solid tumors completed or ongoing
Farnesyl transferase inhibitors	Leukemias, MDS	tipifarnib (Zarnestra™)	PO	•Pediatric phase I study in relapsed/refractory leukemia completed •Pediatric phase I trial in refractory solid tumors completed •Phase II window study in JMML with combination chemotherapy completed • Phase II study in pediatric brain tumors completed
MAPK pathway inhibitors	Solid tumors	AZD6244	PO	• Adult phase I complete, phase II ongoing
	Solid tumors	CI-1040	PO	• Adult phase I complete, phase II ongoing
HDAC inhibitors	Leukemias	valproic acid	PO	•Adult combination studies complete and on-going •Studies in pediatric solid tumors ongoing •Pediatric phase I combination study with 5-azacytadine approved
	Leukemias, Solid tumors	Suberanilohydroxamic acid (SAHA)/vorinostat (Zolinza™)	PO	•Pediatric phase I study in solid tumors and refractory leukemias ongoing •Pediatric phase I study in relapsed/refractory leukemias and solid tumors with cyclophosphamide and topotecan opening •Pediatric phase I combination with cis-retinoic acid complete •Pediatric phase I combination with sorafenib in development
		depsipeptide	IV	• Pediatric phase I study in relapsed/refractory leukemia complete
DNA methylation inhibitors	Leukemias	decitabine (Dacogen®)	IV	•Adult Phase I/II studies complete, phase III on-going •Pediatric phase I study completed, analyses pending •Pediatric phase I combination with MGCD0103 (HDAC inhibitor) in development
	Leukemias, MDS, Solid Tumors	5-azacytadine	IV	• Pediatric phase I trial in relapsed/refractory leukemias and solid tumors in combination with valproic acid approved
Anti CD33 MoABs	AML	gemtuzumab ozogamicin (Mylotarg®)	IV	•Pediatric Phase I complete •Pediatric phase III study in newly diagnosed AML using combination therapy ongoing
HSP90 inhibitor	Leukemias, Solid tumors	17-AAG	IV	• Pediatric Phase I complete

ALL = acute lymphoblastic leukemia; AML = acute myeloid leukemia; PO = oral; IV = intravenous; Ph+ = Philadelphia chromosome positive; TKIs = tyrosine kinase inhibitors; VEGF = vascular endothelial growth factor; JMML = juvenile myelomonocytic leukemia; MAPK = mitogen-activated protein kinase; MoABs = monoclonal antibodies; HDAC = histone deacetylase; HSP90 = heat shock protein 90

Tyrosine Kinase Inhibitors

Tyrosine kinase receptors are implicated in a multitude of malignancies, and are currently an active approach in many targeted therapies. Therapeutics directed against selected pathways of interest are all in active development and discussed below.

BCR-ABL and Src-ABL tyrosine kinase inhibitors (TKIs)

The t(9;22)(q34;q11.2) translocation, also known as the Philadelphia chromosome, is found in fewer than 5% of pediatric ALL patients and confers a high risk of relapse and poor outcome.[10, 11] The resulting BCR-ABL fusion transcript encodes an 8.5 kB chimeric mRNA that translates to a constitutively active ABL tyrosine kinase resulting in increased cell proliferation and survival, and altered cell adhesion.[12, 13] Imatinib mesylate (STI-571, Gleevec™) is an orally available multi-targeted small molecule inhibitor that binds the ATP-binding pocket of the BCR-ABL complex, stabilizing it in the inactive form[14] and is often cited as the first molecularly targeted agent to “make it” to mass market. Its use has been applied to chronic myelogenous leukemia (CML), Philadelphia chromosome positive (Ph+) ALL, and gastrointestinal stromal tumors (GIST), with limited sustained effect as a single agent in the latter two. The most recent Children's Oncology Group clinical trial for Ph+ ALL which incorporated imatinib into a backbone of conventional chemotherapy is demonstrating substantial improvement in early EFS.[15] Despite good initial response, resistance to imatinib is an increasing concern. To circumvent this, two other orally available ABL TKIs, dasatinib (BMS-354825, Sprycel®) and nilotinib (AMN107, Tasigna®), were developed and are in advanced development. Both dasatinib and nilotinib have documented activity against many imatinib-resistant mutations, although neither has significant activity against the most refractory T315I mutation. Third generation ABL inhibitors are in early development which demonstrate activity against T315I.

Src family kinases have an important role in initiation and progression of many malignancies through regulation of cellular survival, proliferation, cellular adhesion, invasion, migration and angiogenesis.[16, 17] Dasatinib, which is much more potent than imatinib against Abl, also inhibits Src, a non receptor tyrosine kinase, with a low nM IC50, compared to the μM IC50 of imatinib for Src. The Src kinases Lyn, Hck, and Fgr are thought to be required for transformation of Ph+ALL (but not CML), therefore, given its inhibition profile, dasatinib may be an important addition to the arsenal of Ph+ALL therapy. Src overexpression has been studied in a variety of tumor types, including neuroblastoma[18], osteosarcoma[19], lymphoid [20] and myeloid leukemia,[21] making it a desirable therapeutic target for a variety of cancers. Anti-tumor activity is noted preclinically with Src kinase inhibitors against pediatric osteosarcoma [22], Ewing Sarcoma[23] and rhabdoid tumors.[24]

FLT-3 inhibitors

FLT3, a receptor tyrosine kinase (RTK) involved in hematopoiesis, is expressed in a variety of malignancies, including MLL gene rearranged infant and childhood ALL. It is the most frequently mutated gene in AML, and is aberrantly expressed in up to 90% of AML patient samples.[25-27] Dysregulation of FLT3 may occur by one of three different mechanisms: protein overexpression, internal tandem duplication (FLT3/ITD), and activating loop mutations (FLT3/ALM). Mutations leading to constitutive activation of FLT3 are seen in 20-25% of children with AML, with 10-15% having FLT3/ITD and the remainder having FLT3/ALM.[28] FLT3/ITD mutations are associated with a poorer outcome, with lower rates of remission induction and survival. Additionally, a high ratio of mutant to wild type FLT3 confers a poorer prognosis.[29] Several FLT3 TKIs, including lestaurtinib (CEP-701), the most selective of the FLT3 targeted agents, as well as midostaurin (PKC412), and sunitinib (SU11248, Sutent®), are in various stages of clinical testing for hematologic malignancies.[30-33] Lestaurtinib, an oral FLT3 TKI has selective in vitro activity against MLL-rearranged cells as well as synergistic activity with other chemotherapy agents in a sequence dependent manner.[34, 35] The addition of lestaurtinib to an intensive chemotherapy regimen is currently being evaluated in the most recent COG infant ALL trial. Several other multi-targeted kinase inhibitors, including several Aurora kinase inhibitors also demonstrate clinically achievable FLT-3 inhibition, but these agents are only now entering trials, primarily in adults, to a significant degree.

FLT3 expression in cancer is not unique to leukemia; FLT3 and its ligand are expressed in solid tumors, including neuroblastoma.[36] FLT3 inhibition decreases cell growth and proliferation.[37] Lestaurtinib also has been evaluated in phase I study of relapsed neuroblastoma, however the target in this disease is thought to be primarily through the TrkB pathway.[38]

c-Kit inhibitors

c-Kit, a RTK important for tumor growth and progression, is normally expressed in hematopoietic progenitor cells, and is expressed in 50-80% of pediatric AML, with 11% having activating mutations.[39, 40] Three types of activating mutations are known: internal tandem duplication (ITD) of the juxtamembrane domain, activation loop mutations at D816, and exon 8 mutations. All confer a poor prognosis.[41-43] Imatinib has activity against c-Kit and platelet derived growth factor receptor. Targeting c-Kit has shown some promise pre-clinically in a spectrum of pediatric cancers including osteosarcoma, Ewing sarcoma, neuroblastoma and Wilm's tumor.[44-47] Unfortunately, a phase II trial of imatinib in pediatric solid tumors demonstrated little to no activity as a single agent.[48] However, it may have some utility in combination with chemotherapy in vitro[49] or in patients who are less heavily pretreated. Imatinib does not appear to be active against the D816 mutations seen in AML.[50] However, other more potent agents including dasatinib and midostaurin as well as other new compounds on the horizon may be effective in this setting.[51, 52]

Vascular endothelial growth factor (VEGF) inhibitors

The role of angiogenesis, particularly in solid tumors, remains a very active area of research. The effect of tumor vasculature on proliferation and migration is crucial for tumor expansion and growth. Anti-angiogenesis therapy, through vascular endothelial growth factor (VEGF) blockade has been demonstrated to be effective in many adult tumor types, including renal cell carcinoma, colorectal, breast and lung cancer.[53-57] Subsequently, targeting angiogenesis, particularly via the VEGF pathway, has become an area of increasing interest in pediatric malignancies. VEGF and its RTKs are overexpressed in acute leukemias[58, 59] and microvessel density is increased in the bone marrow of ALL, AML, and MDS patients.[60, 61] As a result, anti-angiogenic therapies (thalidomide; thalidomide analogs like lenalidomide; bevacizumab, an anti-VEGF humanized monoclonal antibody; and small molecule inhibitors such as sunitinib, vatalanib, and telatinib) are being pursued as treatment strategies in the acute leukemias, MDS, and solid tumors.[62-65]

VEGF inhibition in pediatric solid tumors, specifically neuroblastoma, glioblastoma, Wilm's tumor, hepatoblastoma, Ewing Sarcoma and rhabdomyosarcoma results in anti-tumor activity in vitro.[66-70] Pediatric preclinical testing of two small molecule tyrosine kinase inhibitors with anti-VEGF receptor activity, sunitinib and cediranib (AZD2171), showed promising anti-angiogenic effects in many of the solid tumor xenographs treated.[71, 72] Many new anti-VEGF agents are small molecule inhibitors that target more than one receptor. As a result, these “dirty” or “promiscuous” inhibitors may provide improved anti-tumor efficacy than antibodies alone through multi-target inhibition.[73] They may also have increased toxicities that are important to delineate and address.

Insulin-like Growth Factor Receptor -1 Inhibitors

Targeting of the insulin-like growth factor 1 receptor (IGF1R) pathway is a very active area of therapeutics currently. The binding of IGF1 and IGF2 to the IGF1R results in autophosphorylation and subsequent activation of multiple signaling pathways including the Ras-Raf-MAPK, PI3K/Akt pathway which both activate the mammalian target of rapamycin (mTOR) pathway, stimulating cell growth.[74] Not only is IGF1R over-expressed in many malignancies, there is also evidence to suggest that anti-cancer treatments including chemotherapy and radiation also result in increased IGF1R signaling activity.[75, 76] Many pediatric tumors have dysregulated IGF1R signaling, including rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, Wilm's tumor, desmoplastic small round cell tumor, astrocytoma, and medulloblastoma.[77-82] Several in vitro studies have shown synergistic interactions with chemotherapeutic agents and IGF1R inhibitors in Ewing sarcoma.[83, 84] The development of anti-IGF1R therapies is active in adult oncology with more than a dozen antibodies and small molecule inhibitors in development.[85, 86] The role of IFG1R in leukemia is less well defined, although both ALL and AML cells have been shown to express the receptor.[87, 88]

Inhibitors of Ras Activity/ Mitogen-activated protein kinase (MAPK) pathway

The Raf-MAPK-ERK pathway including its upstream activators is often constitutively activated in tumors and is important in cell proliferation and survival; it has emerged as another attractive target for inhibition. Raf, a serine/threonine kinase is the principal effector of Ras and is required for phosphorylation of the mitogen associated/extracellular regulated kinase-1 (MEK). Inhibitors of B-Raf, sorafenib (BAY 43-9006, Nexavar®) and MEK (CI-1040/PD184352, AZD6244, PD325901) are being studied in AML and MDS, as well as a variety of solid tumors.[89, 90] Of note, sorafenib has inhibitory activity of several other protein kinases, include VEGFR2, PDGFRβ, Flt3, c-kit.[91] Specific targeting of the epidermal growth factor (EGF) signaling cascade is another means of inhibiting the downstream pathway of MAPK-ERK, although there are several other pathways, including PI3K-Akt, protein kinase C and phospholipase pathways, associated with EGF receptor signaling (reviewed in [92]). There has been success in this area, with antibodies (including cetuximab, a chimeric monoclonal IgG1 antibody) and TKIs (gefitinib and erlotinib) which are FDA approved for adult malignancies including metastatic colon carcinoma, non-small cell lung cancer, and pancreatic cancer. Cetuximab is also approved for use in combination with radiation for head and neck cancer. EGFR antibodies including cetuximab, panitumomab, and nimotuzumab are moving through pediatric cancer clinical trial development.

Ras proteins are small molecular weight GTP-binding proteins that act downstream of RTKs and upstream of the Raf-MAPK-ERK pathway. They are involved in multiple RTK signaling cascades. Activating mutations of RAS have been shown to occur in approximately 20% of pediatric AML, although the clinical significance of these mutations is unclear.[28, 39, 93] Mutations leading to RAS activation may play an important role in the pathogenesis of juvenile myelomonocytic leukemia (JMML) and MDS.[94] They have also been found in rhabdomyosarcoma.[95] The gene for neurofibromatosis encodes a protein, neurofibromin which negatively regulates Ras. Tumors in NF-1 patients, JMML and malignant peripheral nerve sheath tumors, have documented hyperactive Ras.[96]

In order to function properly, Ras must be plasma membrane bound via a series of post-translational modifications leading to the attachment of a farnesyl group to the protein. The enzyme farnesyl transferase is required for this process. The farnesyl protein transferase inhibitors (FTIs) tipifarnib (Zarnestra™, R115777) and lonafarnib (Sarasar®, SCH 66336) were developed to target Ras, although their anti-tumor effect is likely also due to inhibition of other farnesylated proteins, such as the Rho family GTPases. FTIs showed early promise in the treatment of JMML, AML, and MDS,[97-100] although their more recent development has lagged.

Purine nucleoside analogues

Nucleoside analogues, Cladribine (2-CDA, Leustatin®), fludarabine (Fludara®), and clofarabine (Clolar®) are used commonly in patients who have relapsed. Purine nucleoside analogues are similar in structure to adenosine or guanosine, but their mechanisms of action differ. Cladribine, fludarabine, and clofarabine (a second-generation purine nucleoside analog designed as a “hybrid” molecule) require intracellular phosphorylation for cytotoxicity, through the inhibition DNA polymerases and/or ribonucleotide reductase, leading to apoptosis.[101, 102]

Purine nucleoside phosphorylase (PNP) inhibitors such as nelarabine (compound 506U78, Arranon®) and forodesine (BCX-1777, Fodosine™) are directed therapies for T-lineage disease. PNP phosphorylates 2’-deoxyguanosine (dGuo) to the guanine nucleobase and 2’-deoxyribose-1-phosphate. The rare genetic deficiency of PNP results in lymphopenia and altered T-cell immunity prompting the rational development of drug-induced PNP inhibition for the treatment of T-cell malignancies.[103, 104]

Nelarabine, a 6-prodrug of ara-G, is rapidly demethylated to the active form of ara-G. Ara-G is then intracellularly phosphorylated to ara-G triphosphate, where its accumulation leads ultimately to apoptosis. [102] Nelarabine is 10-fold more soluble, thus is more attractive clinically than ara-G.[105, 106]

Forodesine, another PNP inhibitor that has shown promise in refractory ALL, blocks intracellular deoxyguanine cleavage to guanine resulting in deoxyguanosine triphosphate accumulation leading to apoptosis.[102, 103] While both agents were initially developed for T-cell disease, forodesine also has demonstrated activity against B-lineage ALL.[107, 108]

Proteasome inhibitors

The proteasome-ubiquitin pathway controls critical cell functions including transcription, apoptosis, and cell cycle progression by degrading important regulatory proteins. Malignant cells are more sensitive to proteasome blockade, a result of altering the recycling of regulatory proteins.[109] Proteasome inhibition also reduces chemoresistance and increases apoptosis by blocking chemotherapy-induced NF-κB pathway activation.[110] Bortezomib (PS-341, Velcade®) is a highly selective, reversible proteasome inhibitor with activity in B-cell leukemia.[111] It also has been shown to effect neuroblastoma cell growth both in vitro and in xenograft models.[112, 113] Ewing sarcoma and osteosarcoma cell lines have shown sensitivity to proteosome inhibitors as demonstrated by increased apoptosis.[114, 115] Preclinical evaluation of bortezomib against a panel of pediatric tumors in vitro showed uniform sensitivity. However, when applied to a selection of tumors in vivo, several ALL lines showed the greatest response.[116] It is FDA approved for use in multiple myeloma and mantle cell lymphoma, and is being investigated in combination with a variety of conventional chemotherapy and targeted agents in both adults and children presently. A phase 1 COG study of bortezomib in pediatric leukemia and solid tumors has been completed.[117, 118] Second generation proteasome inhibitors salinosporamide[110, 119, 120], CEP18770[121, 122], carfilzomib[123] are being actively developed. Salinosporamide demonstrates a synergistic interaction with bortezomib suggesting a role for different mechanisms of action.[124-126]

mTOR inhibitors

The mammalian target of rapamycin (mTOR) is important in cell proliferation and cell cycle progression.[127] Rapamycin (sirolimus, Rapamune®), a macrolide antibiotic, is commonly used as an immunosuppressant following allograft and stem cell transplantation. Rapamycin, everolimus (RAD001), and temsirolimus (CC-I779, a water soluble ester of rapamycin) also decrease cellular proliferation through inhibition of mRNA translation to proteins required for cell cycle progression from G₁ to S phase, as well as decreasing angiogenesis.[128-130] Rapamycin and temsirolimus affect cellular growth in B-cell leukemia, both in vitro and in xenograft models.[131, 132] As mTOR inhibitors block activation of many signaling molecules involved in oncogenesis, they have also shown activity in solid malignancies, including rhabdomyosarcoma, neuroblastoma, osteosarcoma, and medulloblastoma.[133-136] In xenograft models of pediatric tumors, responses have been seen in ALL as well as osteosarcoma, rhabdoid tumor and rhabdomyosarcoma.[137] suggesting that these agents may have potential in the clinical setting.

Histone Deacetylase (HDAC) inhibitors

Histone deacetylase inhibitors regulate transcription and protein function of genes through the control of histone acetylation. The acetylation of histones results in a relaxed chromatin structure, promoting transcriptional activation of genes (reviewed in [138]). HDAC inhibition likely leads to alteration in transcription regulation of genes important in cell cycle regulation and regulation of apoptosis, including p21^WAF1/CIP1, p53, RB, bcl2, bcl6, bcl-xl, and mcl-1[138]. Several HDAC inhibitors, including valproic acid, suberanilohydroxamic acid (SAHA, vorinostat, Zolinza™), entinostat (formerly MS-275, now SNDX-275), and depsipeptide (Romidepsin), are under broad investigation in malignancies particularly hematologic, both alone and in combination[139, 140], although their use has been limited in pediatric malignancies.

DNA methyltransferase inhibitors

DNA methylation in promoter regions of genes is another control mechanism for gene transcription. When cytosine methylation occurs, promoters and gene transcription are suppressed. DNA hypermethylation in promoters regions and consequent inactivation of tumor suppressor genes, including p15^INK4B, p16^INK4, p14^ARF, and p21^WAF1/CIP1, is thought to play a role in the pathogenesis of many tumors, including AML, Ewing sarcoma, osteosarcoma, neuroblastoma and rhabdomyosarcoma.[141-144] DNA methylation inhibitors bind cytosine to prevent DNA methylation resulting in increased or restoration of normal gene transcription. Several methyltransferase inhibitors, including 5-azacytidine (Vidaza®), decitabine (5-aza-2-deoxycitadine, Dacogen®), and zebularine, are being studied in the adult acute leukemias, particularly in combination with HDAC inhibitors.[145-147]

Antibody therapies

Another strategy employed in current drug development is the targeting of proteins expressed in cancer cells but with limited expression in normal cells. Antibodies targeted to these proteins may be used alone, or can then be conjugated to radioactive or cytotoxic agents.

Gemtuzumab ozogamicin

CD33 is a transmembrane receptor expressed on the surface of myeloid and monocytic lineage cells, as well as AML cells. Gemtuzumab ozogamicin (Mylotarg®) is a humanized anti-CD33 antibody conjugated to a derivative of the cytotoxic antibiotic calicheamicin. Upon binding to CD33 on the surface of an AML cell, the antibody is internalized and the calicheamicin molecule is released through hydrolysis, leading to DNA damage and apoptosis. [148] This antibody is actively being evaluated in pediatric AML trials.

Anti-CD-22 targeted therapies

CD22 is normally expressed on the surface of mature B-cells and serves as a negative modulator of B-cell activation via B-cell antigen receptors, though its function is not fully understood. It is also expressed in more than 90% of B-precursor ALL cases[149], making it an attractive and specific target against CD22 positive malignancies. Epratuzumab (IMMU-103) and BL22 are monoclonal antibodies directed against CD22. BL22 is a monoclonal antibody fused to a portion of Pseudomonas exotoxin A.[150] Once bound, these antibodies are rapidly internalized and in vitro studies show increased cell death via antibody-dependent cellular cytotoxicity.[151, 152] Epratuzumab is currently being studied in the COG relapsed pediatric ALL trial. Because of the rapid internalization, these agents are also an attractive carrier molecule for cytotoxic drugs, such as inotuzumab ozogamicin, a humanized anti-CD-22(CMC-544) conjugated with calicheamin[153], or with radiation, such as the anti-CD20 antibodies linked to ⁹⁰yittrium ibritumomab tiuxitan (Zevalin™) or I¹³¹ linked tositumomab (Bexxar™). A COG Phase I trial has been completed with ibritumomab tiuxitan.[154]

Alemtuzumab

Alemtuzumab (CamPath®) is a humanized monoclonal antibody against CD52. CD52 is normally expressed on all lymphocytes as well as lymphoblasts. As with many monoclonal antibodies, the mechanism of action of alemtuzumab is poorly understood. Apoptosis may be induced by either antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. The degree of apoptosis is dependent on CD52 antigen density on the cell surface. Alemtuzumab is used in stem cell transplantation and chronic lymphocytic leukemia and its use is being extrapolated to acute leukemias.[155, 156]

Other Targeted Therapies

Heat Shock Protein Inhibitors

17-AAG, a geldanamycin that disrupts the chaperone function of hsp90, has shown initial promise in the treatment of both AML and ALL, particularly those leukemias with defined fusion proteins.[157] Hsp90 has an essential role in promoting cell survival by stabilizing and enhancing the activity of many signaling proteins involved in oncogenesis, including tyrosine kinases, raf and steroid hormone receptors.[158-161] It is thought that inhibition of function of heat shock protein 90 by 17-AAG, and a newer water-soluble analog, 17-DMAG, leads to depletion of many intracellular proteins, including Raf-1, AKT, and SRC kinases, as well as regulators of angiogenesis, possibly explaining sensitivity of some AML cell lines to the drug.[162, 163] 17-AAG has also shown promise preclinically against malignant glioma as well as neuroblastoma and osteosarcoma.[164-166] Phase I testing of 17-AAG has been completed in children[167, 168] and newer generation HSP90 inhibitors are being evaluated now, particularly in combination with proteasome inhibitors and a range of multi-targeted tyrosine kinase inhibitors.[169]

γ-Secretase inhibitors

The NOTCH1 gene encodes a regulatory transmembrane receptor essential for normal T-cell development. After post-translational modification of Notch, γ-secretase, a membrane bound protease, cleaves Notch at the transmembrane domain, generating intracellular Notch which enters the nucleus as a transcriptional activator that upregulates NFκB. NFκB promotes proliferation and activates the anti-apoptotic PI3 Kinase/AKT pathways.[170] Aberrant NOTCH1 activation has been implicated in tumorigenesis of many cancers, including T-ALL [170], via the PI3/AKT or c-MYC pathways. Notch has also been implicated in neural development, playing a role in the determination of fate for multipotent neural stem cells.[171] This role for Notch is thought to explain why its expression is upregulated in neuroblastoma and the more neural phenotype of Ewing sarcoma.[172, 173] Hes-1, a notch pathway gene, has been shown to be important in osteosarcoma invasion and metastasis.[174] In the hematologic malignancies, approximately 50% of T-cell ALL patients express activating mutations in NOTCH1 in the heterodimerization domain and/or the PEST domain.[175, 176] Interestingly, NOTCH1 mutations have not been observed in B-ALL patients.[176]

Targeting γ-secretase prevents intracellular Notch formation. Patients treated with γ-secretase inhibitors (LY-411575 and LY450139) for Alzheimer's disease, the drug's initial intended application, experienced altered lymphopoiesis and thymocyte development, supporting interest in use for T-cell malignancies.[177] One γ-secretase inhibitor, MK-0752 entered clinical trials for patients with refractory acute leukemias after passing initial safety and tolerability studies in normal volunteers. However, in phase I trial for patients with refractory acute leukemias, neurologic toxicities limited progression of these studies, and further work is ongoing. The mechanism for such neurotoxicity in leukemia patients has been unclear, although Notch has been implicated in neural development.[178, 179] While evidence for the role of Notch in solid tumors is increasing, trials in solid malignancies are just proceeding in adults with several newer generation γ-secretase inhibitors.

CONCLUSIONS

A wide variety of targeted agents are being studied for the treatment of a variety of malignancies in both adult and pediatric settings, and early results are promising. The molecular characterization of each patient's cancer will likely be important in the development of tailored therapy. Despite the overall cure rate in pediatric cancer, many patients cured of their cancer with current treatment protocols will suffer some late effects. The development of targeted agents may not only improve cure rates, but also help decrease the usage and thereby, the side effects of standard cytotoxic chemotherapy. A significant challenge is to determine the optimal combinations of one or several agents in conjunction with more traditional chemotherapy that will improve cure rates and decrease short- and long-term morbidity associated with treatment for these diseases. Because there are a host of new agents available, it is imperative to design clinical trials that will optimize patient utilization and resources, and increase the “signal to noise” ratio in order to develop a more comprehensive understanding of who is likely to benefit from which targeted agents. The ability to identify targeted agents with efficacy in specific patient populations will hopefully lead to continued improvements in overall patient survival in pediatric cancers and diminished toxicities from therapy.