Abstract
Improved understanding of the molecular changes involved in oncogenesis has generated considerable interest in developing therapeutic agents that target dysregulated pathways and malfunctioning or overexpressed molecules in cancer. This process has been challenging in the case of neuroblastoma because of its clinical heterogeneity. This chapter highlights promising new targeted therapies for this disease, such as new pathway inhibitors, norepinephrine-targeted agents, interventions to improve T cell-mediated cytotoxicity, and immune modulators that enhance the function of natural killer cells in the context of monoclonal antibody therapy. We review the biologic rationale for these treatments and discuss future clinical implications.
Keywords
Immunotherapy, Neuroblastoma, Pediatric oncology, Small molecular targets
Introduction
Major strides have been made in targeted cancer therapy over the past decades due to better understanding of underlying cancer biology, identification of cancer vulnerabilities, and integration of multiple therapy approaches . These advances have led to the development of small-molecule inhibitors and monoclonal antibodies that target dysregulated pathways and malfunctioning or overexpressed molecules. For example, imatinib mesylate, the ABL tyrosine kinase inhibitor, has been successfully used to treat chronic myeloid leukemia patients with the BCR – ABL translocation and is associated with very high cytogenetic and hematologic response rates . The monoclonal antibody dinutuximab against the disialoganglioside GD2 has been approved by the Food and Drug Administration (FDA) for patients with high-risk neuroblastoma. Dinutuximab facilitates the recognition and elimination of GD2 expressing neuroblastoma cells by the immune system, which results in improved event-free survival (EFS) of patients receiving this immunotherapy . Therapies have also been directed toward downstream signaling molecules, epigenetic mechanisms, and metabolic pathways within tumor cells or inhibition of angiogenesis and modulation of interactions with immune cells in the tumor microenvironment ( Fig. 3.1 ).
Although these new therapies have very specific mechanisms of action, therapy escape, and immune evasion, driven by the gain of new mutations in the tumor or change in epitope expression, respectively, pose a major challenge for molecularly targeted treatments. For example, approximately one-third of patients with neuroblastoma, while receiving monoclonal antibody therapy, experience treatment failure , and little is known about immune escape mechanisms such as a change in human leukocyte antigen or GD2 expression during the therapy. Real-time genomic and biologic testing during treatment can help unravel the mechanisms of clonal selection and aid the development of more effective therapies in the future.
Despite the highly effective infrastructure of clinical trials, conducting clinical studies in pediatric oncology has been inherently challenging because of low disease incidence, insufficient preclinical models and testing programs, and different perspectives on therapy success (i.e., striving for cure vs. prolonged survival). The heterogeneous clinical behavior of neuroblastoma adds yet another layer of complexity. Neuroblastoma is a rare childhood malignancy with an incidence of 11 per million children under the age of 15 years . It is the most common cancer in infants and toddlers , affecting patients at a median age of 17 months . Neuroblastoma is a developmental tumor that originates from primitive cells of the autonomic system . The heterogeneous biology of this tumor is reflected by disproportionate survival rates among patients. For example, in children with disseminated disease (stage 4), infants with stage 4S (special) disease may have spontaneous tumor regression and cure rates greater than 90% with very limited medical intervention . However, infants with MYCN amplification and those aged 18 months or older with stage 4 disease, irrespective of MYCN status, have dismal survival rates and account for 15% of childhood cancer deaths . Given this disparity in survival, several clinical features and molecular markers that predict biological behavior and outcome in neuroblastoma have been considered for developing a more accurate risk stratification system. Risk group assignment dictates the intensity of treatment and depends on clinical features such as age and initial tumor stage as well as biological factors, most importantly MYCN status . As such, it is important to note that despite sharing the same diagnosis, patients may respond differently to molecularly targeted therapy. Thus, further refinement in grouping might be necessary to identify the subset of patients in whom the effect of these agents can be maximized.
In past decades, some promising treatments for neuroblastoma have been studied and advanced in clinical trials. Treatments include tyrosine kinase inhibitors (e.g., anaplastic lymphoma kinase [ALK] inhibitors) and norepinephrine-targeted agents (e.g., 131 I-metaiodobenzylguanidine [MIBG]). Other strategies seek to improve immunotherapy by adding immune modulators (e.g., lenalidomide) or by specifically enhancing T cell-mediated cytotoxicity (e.g., cancer vaccination or chimeric antigen receptor [CAR] T cells). This chapter reviews the biological rationale of promising treatments that are currently under different stages of development and discusses their potential clinical implications.
Growth Factor Signaling and Oncogenes
Small-molecule ALK Kinase Inhibitors
The classic tyrosine kinase ALK belongs to the superfamily of insulin receptors and is important for neurogenesis and learning processes across multiple species . Its role in oncogenesis first emerged in the 1980s, when several research groups independently reported the t(2; 5) (p23; q35) translocation in anaplastic large cell lymphoma (ALCL) , which was later characterized as the pathognomonic nucleophosmin-ALK (NPM-ALK) fusion . Subsequently, other oncogenic ALK rearrangements were identified in B-cell non-Hodgkin lymphomas and nonlymphoid cancers, including non-small-cell lung cancer (NSCLC) and inflammatory myofibroblastic tumor (IMT) . In neuroblastoma, activating mutations in ALK have been identified in 8% of all patients with familial and sporadic disease combined , which by risk group affects 14% of children with high-risk, 6% with intermediate-risk, and 8% with low-risk disease . These patients have significantly poorer survival (5-year overall survival [OS] 59% ± 3.6%) than those without ALK aberrations (5-year OS 78% ± 1.8%), reinforcing the need for a better understanding of the role of ALK in neuroblastoma for developing new therapeutic strategies for this subgroup .
In most cancers, alterations in ALK involve chromosomal rearrangements that engage the intracellular tyrosine kinase domain of ALK and lead to constitutive activation of downstream signaling ( Fig. 3.2 ). However, patients with neuroblastoma have point mutations in ALK at three hotspot residues R1275 (43%), F1174 (30%), and F1245 (12%), which account for the vast majority of ALK mutations in this group . These alterations result in autophosphorylation of ALK and activation of the full-length ALK receptor. Mutated ALK has been shown to be the primary driver of neuroblastoma growth in preclinical studies, wherein targeted expression of mutated ALK induced tumor development in transgenic mice and knockdown of ALK resulted in proliferation arrest of neuroblastoma cells in vitro . Given these findings and the poor prognosis associated with ALK-mutant neuroblastoma, research efforts over the past years have concentrated on testing ALK kinase inhibitors that have made their way into the clinic for many adult cancers with ALK aberrations.
Oral crizotinib (PF-02341066) is a first-generation adenosine triphosphate (ATP)–competitive ALK inhibitor that is the ultimate in clinical testing and use. The compound was originally developed as an inhibitor of the mesenchymal-epithelial transition growth factor but was also found to inhibit ALK and ROS1 receptor tyrosine kinases . It acts by competitively blocking the ATP-binding pocket of ALK and prevents autophosphorylation and ALK activation. Crizotinib has been tested against various ALK rearranged and mutated tumors in vitro and in vivo and successfully translated into the clinic as first-line and subsequent-line therapy for NSCLC .
Crizotinib was first tested in the Children’s Oncology Group (COG) Phase I dose-escalation trial ADVL0912 . In addition to accruing patients with relapsed solid tumors by using a rolling-six design, children with ALK -mutated tumors were eligible to participate and receive crizotinib at any point during the enrollment period. Crizotinib was tolerated up to a recommended Phase II dose of 280 mg/m 2 , which is nearly twice that of the dose recommended for adults. Hematologic and hepatic toxicities were the most common grade four adverse effects. This trial also included a Phase II expansion cohort for ALK -driven ALCL, neuroblastoma, and other cancers such as IMT. Dose-dependent response rates in this subcohort were 90% for ALCL at the recommended Phase II dose and 86% for IMT across all dose levels tested . In contrast, objective responses of the 11 patients with neuroblastoma and known ALK mutation were much lower; one patient achieved a complete response, and two patients had stable disease. Interestingly, two of the patients with responses carried an R1275Q germline mutation, which has been reported to confer sensitivity to crizotinib in preclinical studies . Of seven patients with progressive disease, three carried a sporadic F1174L mutation that has been reported in tumors with intrinsic resistance to crizotinib . Although initial biochemical studies concluded that changes in ATP-binding affinity of F1174-mutant ALK were responsible for crizotinib resistance , structural analysis later revealed that the F1174 residue was located outside of the ATP-binding pocket and therefore unlikely to induce resistance by this mechanism . It was not surprising that second-generation ALK inhibitors with a 20-fold higher ATP-binding affinity and potency than crizotinib could still not fully overcome the problem of drug resistance in F1174-mutant tumors . As discovered later, the substitution of phenylalanine by a smaller leucine residue causes a very subtle change in the ALK protein structure, primarily affecting the geometry of the αC helix and resulting in a catalytically active conformation change of ALK that will not respond to inhibition by crizotinib . This example highlights the need for more differential therapeutic targeting of ALK and raises the question of whether evaluating the conformation change of ALK rather than drug–protein interactions is more effective to assess preclinical drug sensitivity to ALK inhibitors in this subset and those with other genetically related tumors.
Current early phase clinical trials for neuroblastoma are summarized in Table 3.1 and include testing of second-generation ALK inhibitors such as entrectinib (RXDX-101; ClinicalTrials.gov identifier NCT02650401 ) and lorlatinib ( NCT03107988 ). Results from these studies are pending. In the next Phase III COG trial for newly diagnosed high-risk neuroblastoma, one treatment arm will be dedicated to ALK mutated tumors and evaluate 3-year EFS in patients receiving crizotinib in addition to standardized multimodal therapy. The trial plans to accrue patients with confirmed activating mutations or ALK copy number changes in the tumor as detected by ALK tyrosine kinase domain sequencing and fluorescence in situ hybridization, respectively. Correlating the genetic aberrations in these patients with therapy response may identify mutations associated with therapy failure and provide opportunities to understand and ultimately overcome therapeutic challenges related to resistance.
| Agent | NCT Number | Phase | Status | Sponsor |
|---|---|---|---|---|
| Ceritinib | NCT02780128 | I/Ib | Recruiting | CHOP |
| Crizotinib | NCT00939770 | I/II | Closed | COG |
| Ensartinib | NCT03155620 | II | Recruiting | NCI |
| Ensartinib | NCT03213652 | II | Recruiting | NCI |
| Entrectinib | NCT02650401 | I/Ib | Recruiting | Ignyta, Inc. |
| LDK378 | NCT01742286 | I | Recruiting | Novartis Pharmaceuticals |
| Lorlatinib | NCT03107988 | I | Recruiting | NANT |
Targeting MYCN
The human v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) (MYCN), is a member of the MYC family of proto-oncogenes and encodes the MYCN transcription factor . Similar to the other two MYC proteins c-MYC and MYCL, MYCN contains a transcriptional activation domain in the N-terminus and a basic helix-loop-helix leucine zipper in the C-terminus that engages in heterodimerization with the Max protein, sequence-specific DNA binding, and subsequent transcriptional regulation . MYC proteins play important roles in diverse physiological processes during growth and development, such as stemness, cell metabolism, and cell cycle progression. However, deregulated MYC expression, for example, due to chromosomal translocation, insertional mutagenesis, or gene amplification, can lead to increased cellular proliferation and evasion of apoptosis, which are two critical aspects in carcinogenesis. Other mechanisms of deregulation, such as growth factor stimulation, alterations in downstream messengers of related pathways, and changes in MYC metabolism can have a similar effect, which highlights the complex cellular and molecular network through which MYC exerts its pleiotropic effects .
Amplification of MYCN occurs in approximately 30% of all patients with neuroblastoma and is associated with a significantly worse prognosis, independent of risk group assignment . Two animal models have revealed the specific role of MYCN in the development and progression of neuroblastoma. In the TH-MYCN transgenic mouse model that harbors human MYCN under the rat tyrosine hydrolase (Th) promoter in cells of the peripheral sympathetic nervous system, MYCN was expressed in migrating neural crest cells during early phases of embryonic development and drove the generation of spontaneous neuroblastoma-like tumors in the sympathetic ganglia . When treated with MYCN anti-sense RNA, mice showed reduced numbers of tumors and smaller tumor size . In the Cre-conditional human MYCN mouse model, MYCN was conditionally expressed in dopamine β-hydroxylase–expressing cells in the neural crest, thereby giving rise to neuroblastoma that anatomically, histologically, and molecularly resembled human neuroblastoma .
Investigators have tried to pharmacologically drug the “undruggable” transcription factors for cancer therapy. Although some attempts have been successful, such as retinoic acid and arsenic trioxide that target the oncogenic fusion transcription factor promyelocytic leukemia–retinoic acid receptor alpha (PML–RARα) in acute promyelocytic leukemias , no MYC inhibitor has yet advanced to the clinic. It was earlier speculated that MYC inhibition would cause devastating side effects in healthy tissues, given its crucial and pleiotropic role in development and growth. In addition, MYC was deemed “undruggable” because of the lack of a deep binding pocket that has traditionally served as the target for drug discovery initiatives that use small-molecule inhibitors. However, the recent progress made has led to the development of various strategies to potentially, directly and indirectly, target MYC.
Proteasomal degradation of MYCN is mediated by the Fbxw7 ubiquitin ligase but is halted when MYCN and the Aurora kinase form a stable protein–protein complex . By using the Aurora-A kinase inhibitors MLN8054 and alisertib (MLN8237), investigators demonstrated in preclinical experiments that formation of the Aurora–MYCN complex could be disrupted, leading to degradation of MYCN . As a consequence, the expression of MYCN – dependent genes was broadly downregulated; analogously, in MYCN-driven neuroblastoma mouse models, treatment with MLN8054 prolonged the survival of tumor-bearing animals . Clinical trials are currently underway to test alisertib for neuroblastoma. The COG has completed Phase I and II studies ( NCT02444884 and NCT01154816 ) on children with refractory and relapsed solid tumors, and results are pending. In combination with irinotecan and temozolomide, alisertib is being tested by the New Approaches to Neuroblastoma Therapy (NANT) consortium in patients with neuroblastoma in a Phase I study ( NCT01601535 ) that includes a Phase II expansion cohort. This study is still accruing patients and results are pending.
The role of functional chromatin states in cancer is a subject of ongoing research. A growing number of molecules that set (“writers”) or remove (“erasers”) modifications to DNA or histones or that bind (“readers”) to epigenetically modified chromatin sites have been implicated as drivers of oncologic processes. Epigenetic modifications that lead to side-chain acetylation of lysine residues on histone tails enforce an open chromatin state and transcriptional activation . Bromodomains, which are present in several human proteins, exclusively recognize acetylation motifs and thereby engage respective proteins with acetylated histones. The bromodomain and extraterminal domain (BET) family of proteins, comprising BRD2, BRD3, and BRD4, plays an important role in transcriptional regulation, epigenetic memory, and cell growth. Therefore, targeting the bromodomain in BET proteins is being pursued as a pharmacologic strategy in cancer-directed therapy.
In a large screen of more than 600 well-characterized cancer cell lines, many cell lines showed significant sensitivity to the BET bromodomain inhibitor JQ1, and cytotoxicity correlated with MYCN amplification status . For neuroblastoma cell lines, sensitivity was highly correlated with depletion of MYCN protein. JQ1 induced displacement of BRD4 at the MYCN promoter site, which was the likely cause for MYCN depletion. JQ1 also affected cell proliferation, apoptosis, stemness, and neural differentiation, thus showing that it exhibited a broader therapeutic effect than previously thought. Consistent with these findings, neuroblastoma-bearing mice had decreased tumor growth after being treated with JQ1 . A Phase I trial ( NCT01587703 ) on the BET bromodomain inhibitor I-BET 762 is currently underway and enrolling adults with cancer and children older than 16 years with neuroblastoma.
Despite MYCN being the most common genetic aberration in neuroblastoma, efforts to pharmacologically target MYCN have been challenging. Owing to a better understanding of interactions and mechanisms that control MYCN metabolism, promising new therapeutic strategies have emerged that have resulted in the development of small-molecule inhibitors that are being currently tested in clinical trials ( Table 3.2 ). As we await the results of these clinical studies, expanding our knowledge of the basic mechanisms of MYCN function in normal and pathologic states will be essential to advance in this area of translational research.
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