Recent Advances of Cell-Cycle Inhibitor Therapies for Pediatric Cancer Christopher C

Published OnlineFirst November 2, 2017; DOI: 10.1158/0008-5472.CAN-17-2066 Cancer Review Research

Recent Advances of Cell-Cycle Inhibitor Therapies for Pediatric Cancer Christopher C. Mills1, EA. Kolb2, and Valerie B. Sampson2

Abstract

This review describes the pivotal roles of cell-cycle and check- MLN8237), Wee1 kinase (MK-1775), KSP (ispinesib), and tubu- point regulators and discusses development of speciﬁc cell-cycle lin (taxanes, vinca alkaloids), are presented. The design of mech- inhibitors for therapeutic use for pediatric cancer. The mechanism anism-based combinations that exploit the cross-talk of signals of action as well as the safety and tolerability of drugs in pediatric activated by cell-cycle arrest, as well as pediatric-focused drug patients, including compounds that target CDK4/CDK6 (palbo- development, are critical for the advancement of drugs for rare ciclib, ribociclib, and abemaciclib), aurora kinases (AT9283 and childhood diseases. Cancer Res; 1–10. 2017 AACR.

Introduction of DNA damage, epigenetics, metabolism, proteolytic degra- dation, stem cell self-renewal, neuronal functions, and sper- Recent preclinical and clinical studies of highly selective matogenesis (2). agents that target various regulators of the mammalian cell Selective members of the CDK family of protein kinases act as cycle demonstrate cell-cycle arrest, inhibition of transcription, oncogenic stimuli in several types of cancer (e.g., CDK1 in breast and apoptotic cell death in models of human cancer. The cell- cancer and colon cancer (3, 4), CDK4 in familial melanoma (5), cycle drives proliferation of cells by the duplication of chromo- and CDK6 in MLL-rearranged leukemia (6). Abnormal activity somes and their distribution to a pair of genetically identical is associated with the malignant transformation of cells, inhi- daughter cells. The fidelity of these processes relies on a series of bition of DNA transcription and low response to standard drug ordered and highly regulated steps that control the transition of treatment (7). Pharmacological inhibition of CDKs typically cells through DNA synthesis (S-phase) and cell division (M- results in cell cycle arrest, apoptosis, and transcriptional repres- phase), which are separated by gap phases G and G . Activa- 1 2 sion to provide the rationale for therapeutically targeting tion of cyclin-dependent kinases (CDK) and association with CDKs in cancer. This review focuses on the cell-cycle inhibitors regulatory cyclins enable successful progression through the that have entered clinical trials for development against child- cell cycle. Nine CDKs regulate different processes in the cell- hood cancer. cycle machinery, through formation of specialized and tissue- specific cyclin/CDK complexes (1). Association of cyclin D1 Cyclin-dependent kinase 4/6 inhibition with CDK4 and CDK6 drives progression through G1 stage, Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have cyclin E and cyclin A bind CDK2 to regulate centrosome emerged as promising cell-cycle therapeutics. The CDK4 and duplication and also to target helicases and polymerases during CDK6 genes encode the CDK4 and CDK6 cyclin-dependent – G1 S and S-phase, and cyclin A/cyclin B/CDK1 complexes serine-threonine kinases (CDK 4/6), respectively. Mitogenic – regulate the G2 M checkpoint (1). stimuli (e.g., estrogen and human epidermal growth factor Inhibitory proteins further regulate activity of cyclin/CDK receptor) and pro-proliferative factors (e.g., oncogenic MYC complexes. p27 (CDKN1B gene), p21 (CDKN1A gene), and and RAS) trigger quiescent cells to express D-type cyclins and CDKN1C p57 ( gene) block the interaction of G1-phase CDKs enter the cell cycle (8). These stimulate formation of CDK4/6 (CDK4 and CDK6) with their respective targets. In addition, complexes with cyclins D1-D3, leading to phosphorylation and the family of Ink4 proteins (inhibitors of kinase 4), p15 activation of the retinoblastoma tumor suppressor gene prod- (CDKN2B gene), and p16 (CDKN2A gene) bind CDK4 and uct (Rb). Rb protein phosphorylation releases E2F transcription CDK6 and control mid-G1 stage by decreasing the phosphor- factors that regulate genes that are required for G0 or G1 phase ylation of target proteins. Over 30 cyclin/CDK/Inhibitors are transition (pre-DNA synthesis) to S phase, in which DNA implicated in cellular functions regulating transcription, repair synthesis occurs (9). CDK4/6 signaling also has roles independent of cell-cycle regulation. These include senescence suppression via regulation of the FOXM1 transcription factor (10) and 1University of Delaware, Newark, Delaware. 2Nemours Center for Cancer and transcriptioninhematopoieticcells(1).HighexpressionofD- Blood Disorders, Nemours/Alfred I. duPont Hospital for Children, Wilmington, type cyclins, genetic mutations or amplification of the CDK4 Delaware. and CDK6 loci, or loss of the p16Ink4A inhibitory protein that Corresponding Author: Valerie B. Sampson, Nemours/A.I. duPont Hospital for regulates cyclin D/CDK4/6 complexes, are associated with Children, 1701 Rockland Road, Wilmington, DE 19803. Phone: 302-651-4832; Fax: unrestricted cancer cell growth. In addition, deletion of RB1 301-651-4827; E-mail: [email protected] occurs in many tumors and accelerates proliferation indepen- doi: 10.1158/0008-5472.CAN-17-2066 dently of cyclin D–CDK4/6 activity. This suggests that activa- 2017 American Association for Cancer Research. tion of the cyclin D/CDK4/CDK6/Rb axis is a molecular

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hallmark of cancer (11). Currently, three selective CDK4/6 ALL mouse models in combination therapy with corticosteroids inhibitors are approved: palbociclib (PD-0332991), ribociclib and mTOR inhibitors (18). Dual inhibition of MEK1/2 (binime- (LEE011), and abemaciclib (LY2835219). tinib) and CDK4/6 (ribociclib) achieved preclinical synergy (19), as well as ALK (ceritinib) and CDK4/6 (ribociclib; ref. 20) in Palbociclib (PD-0332991). Palbociclib (Pfizer) is developed for neuroblastoma. treatment of ER-positive and HER2-negative breast cancer. Estro- Ribociclib was assessed in one pediatric phase I study for 32 gen receptor (ER) pathway activation induces cyclin D1 and patients with malignant rhabdoid tumors (MRT) and neuroblas- combining aromatase inhibition with CDK4/6 inhibition signif- toma. Results demonstrated acceptable safety and pharmacoki- icantly reduces cyclin D–CDK4/6 activity (11). Palbociclib shows netics (21). Patients received escalating once-daily oral doses 2 selective potency against CDK4 and CDK 6 (IC50 9–11 nmol/L (3-weeks-on/1-week-off). The MTD was 470 mg/m and RP2D and 15 nmol/L, respectively) in comparison with a range of other was 350 mg/m2. These values were similar to those in adults. The kinases (12). Preclinical studies indicate palbociclib shows anti- most common grade 3 DLTs were fatigue (280 mg/m2; n ¼ 1) tumor activity in pediatric malignancies. Barton and colleagues and thrombocytopenia (470 mg/m2; n ¼ 2). There were several (13) demonstrated that a single dose of gamma radiotherapy adverse grade-3/4 hematologic events, including neutropenia followed by daily treatment with palbociclib increased survival in (63%), leukopenia (38%), anemia (3%), thrombocytopenia genetically engineered brainstem glioma mouse models by 19% (28%), and lymphopenia (19%) and fatigue (3%). Observations in comparison with radiotherapy alone. In another investigation, of prolonged stable disease in a subset of patients with neuro- palbociclib plus PLX4720, an inhibitor against v-raf murine blastoma (n ¼ 7) and primary CNS MRT (n ¼ 2) support further sarcoma viral oncogene homolog B1 (BRAF) extended survival clinical evaluation of combination therapies to optimize CDK4/6 in pediatric malignant astrocytoma, relative to either monother- targeting in neuroblastoma and MRT. An integrated analysis of apy (14). Response was specific for a subset of pediatric astrocy- ribociclib-induced cellular senescence could help to identify tomas with BRAF (V600E) mutation and CDKN2A (encoding unresponsive tumors, and may have potentially important ther- cyclin-dependent kinase inhibitor 2A) deficiency. apeutic implications. Two phase-I clinical trials are ongoing to test palcociclib in Rb-positive solid tumors and leukemia. Investigation of the Abemaciclib (LY2835219). Abemaciclib (Eli Lilly and Company) maximum tolerated dose (MTD)/phase II recommended dose is an ATP-competitive, reversible kinase inhibitor with break- as well as toxicities of palcociclib in children with Rb-positive through therapy designation for patients with refractory hor- þ recurrent, progressive or refractory primary central nervous mone-receptor–positive (HR ) advanced or metastatic breast system (CNS) tumors is planned (NCT02255461). One clinical cancer. Abemaciclib shows potency against CDK4, CDK6 and study for adults with various advanced solid tumors, reported CDK9 at 2, 10, and 57 nmol/L, respectively (22). One area of dose-limiting toxicities (DLT) in patients were grade 3 neutro- clinical study is the ability of abemaciclib to penetrate the blood– penia (12%), anemia (7%), and leukopenia (2%). Other com- brain barrier (23). This agent has entered clinical evaluation for mon adverse events included fatigue, nausea, and diarrhea newly diagnosed diffuse intrinsic pontine glioma (DIPG) and (15). For the planned pediatric clinical development, patients advanced malignant brain (Grade III/IV, including DIPG; MBT) will receive 26 courses of palbociclib, given on days 1 to 21, and solid tumors (neuroblastoma, Ewing sarcoma, rhabdomyo- over 4 weeks. Correlates of drug activity with the expression of sarcoma, osteosarcoma and rhabdoid tumor; NCT02644460). Rb protein will determine whether this provides clinical ben- The primary objective of the phase I study is to identify the MTD efit. In another planned study (NCT03132454), the safety and for abemaciclib, to evaluate safety and tolerability using 2 arms, effects of palbociclib, when given alone and in combination either 30–33 fractions over 6 weeks or twice daily for 28 days with standard drugs (sorafenib, decitabine, and dexametha- (which defines one cycle), each for the duration of 2 years. The sone) will be investigated in patients with hematopoietic/ secondary objectives are to assess DLTs, preliminary activity and lymphoid cancer, acute myelogenous leukemia (AML), and investigate the pharmacokinetics profile starting at a dose level acute lymphoblastic leukemia (ALL). Side effects and best of that is 80% of the adult dose. different dose schedules of the standard drugs will be studied for consideration for further development. Flavopiridol (alvocidib)/other CDK inhibitors. Although CDK4/6 Ribociclib (Novartis and Astex Pharmaceuticals) received has emerged as highly relevant cell-cycle targets, agents that target recent approval for use as a frontline agent in combination with other CDKs or a broad spectrum of CDKs are also under devel- aromatase inhibition for metastatic breast cancer. Novel applica- opment. Flavopiridol (alvocidib) was the first pan-CDK inhibitor tions in adult patient populations are being studied for melano- in human clinical trials. This drug demonstrates potent in vitro ma, neuroblastoma, and liposarcoma that are Rb-positive. Pre- inhibition of CDK1 (40 nmol/L), CDK2 (40 nmol/L), CDK4(40 clinical characterization in human neuroblastoma indicated ribo- nmol/L), CDK6(40 nmol/L), and CDK7 (300 nmol/L; ref. 24). ciclib-induced cell-cycle arrest and reduced proliferation in 12 of The Children's Oncology Group conducted a Phase I study to 17 cell lines (mean IC50 ¼ 307 68 nmol/L in sensitive lines) assess the safety of flavopiridol in 25 children with refractory solid through selective CDK4/6 inhibition (16). Growth delay of neu- tumors (25). Patients received doses of flavopiridol at 37.5 to 80 roblastoma xenograft tumors and a correlation between ribociclib mg/m2/d over 3 consecutive days. The MTD was 62.5 mg/m2/d activity and sensitivity against MYC-amplified neuroblastoma and the main DLTs experienced were neutropenia and diarrhea. were demonstrated. Importantly, observations of dose-depen- No antitumor activity was observed in this population. The dent decreases in phosphorylated Rb and FOXM1, highlight activity of flavopiridol in pediatric patients with relapsed or cellular senescence could be an important mechanism associated refractory solid tumors or lymphoma was also recently evaluated with the clinical activity of this agent. Ribociclib was active in vitro (NCT00012181) using flavopiridol IV over 1 hour on days 1 to 3 in leukemia cells (17) and in vivo in mutant NOTCH1-driven T- on a 21-day schedule. Results for these studies are pending.

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The CDK inhibitor SCH 727965 (dinaciclib) inhibits CDKs 1, Selective inhibition of Aurora-A blocks Thr228 phosphoryla- 2, 5, and 9. Preclinical evaluation of SCH 727965 by the tion, promotes formation of monopolar spindles, cell-cycle Pediatric Preclinical Testing Program against a panel of human arrest at G2–M phase and apoptosis (35). Preclinical evidence cell lines and pediatric-derived xenografts and ALL, induced of activity of AT9283 was demonstrated by growth suppression significant delays in event free survival distribution compared in imatinib-resistant BCR–ABL-positive leukemic cells and in with control in 64% of solid tumor xenografts and in 43% of mice engrafted with BaF3/BCR-ABL, human BCR-ABL(þ)cell ALL xenografts (26). The highest efficacy observed was a com- lines, or primary patient samples expressing BCR-ABL/T315I or plete response in one leukemia model and stable disease for a glutamic acid 255 to lysine imatinib-resistant mutation. Pre- single osteosarcoma xenograft. Although the use of dinaciclib clinical reports also indicate that AT9283 synergizes with dasa- in preclinical models has generated important data about tar- tinib and enhances the repression of medulloblastoma survival geting CDKs, the clinical development of this agent for pediatric and migration (36). cancer is not planned. SNS-032, an inhibitor of CDK2/9 activity, The first phase I/II trial with AT9283 in 33 pediatric patients demonstrated potent single-agent toxicity in primary AML blasts with solid tumors (NCT00985868) led to a partial response in and synergistic effects when combined with cytarabine (27), but one patient diagnosed with CNS-primitive neuroectodermal showed limited clinical activity in adult patients with CLL and tumor (PNET) and disease stabilization in 38% of patients, with multiple myeloma (28). manageable hematological toxicity (37). The most commonly reported drug-related toxicities were hematologic events, includ- Aurora kinase inhibition ing neutropenia (36.4%), anemia (18.2%), and thrombocytope- The Aurora A, Aurora B, and Aurora C kinases are a family of nia (21.2%). These were grade 3 in 30.3%, 6.1%, and 3% of highly conserved serine/threonine kinases with essential roles in patients. Other toxicities were fatigue, infections, febrile neutro- mitosis and cytokinesis (29). Aurora-A localizes to chromosomes penia and increased alanine transaminase. The phase I/II trial of in S-phase, and expression and activation are upregulated in early AT9283 for relapsed/refractory acute leukemia (NCT01431664) M-phase by autophosphorylation at threonine 288 (Thr288). This did not accomplish the primary endpoint of identifying a dose for triggers the maturation of centrosomes, formation of the mitotic phase II assessment (38). This study was terminated because there spindle assembly, meiotic maturation, and cytokinesis (29, 30) to was no evidence of clinical efficacy for the doses examined. It is govern the correct passage of genetic material to daughter cells. likely that the evaluation of this agent in a cohort of pediatric Aurora A also directly phosphorylates and activates Polo-like patients with leukemia harboring the JAK, ABL, or FLT3 mutations kinase-1 (Plk-1) to promote checkpoint recovery in G2 (31). might increase the potential of clinical responses. Aurora-B is a catalytic component of the chromosomal passenger complex (CPC) that also contains the inner centromere protein Alisertib (MLN8237). Alisertib (MLN8237) inhibits Aurora-A and (INCENP), survivin, and borealin (32). This complex governs Aurora-B kinase with an IC50 of 1.2 and 396.5 nmol/L, respec- chromosome condensation, proper chromosome–microtubule tively (39, 40). Alisertib displays activity in preclinical models of attachment at the mitotic spindle, the spindle-assembly check- lung, prostate, ovarian, and lymphoma cells (41). In the PPTP in point (SAC), and the final stages of cytokinesis (32). Aurora-C is vitro and in vivo models of childhood cancer, MLN8237 treatment highly expressed in testis, thyroid, and placenta and during led to growth repression in neuroblastoma and Ewing sarcoma meiosis of gametes (33). Recent studies show that increased cell lines as well as maintained complete responses and complete Aurora-C levels are associated with abnormal cell division result- responses in neuroblastoma and ALL xenografts, respectively ing in centrosome amplification and multinucleation in cells (39). Preclinical studies established a correlation between aliser- (33). NIH3T3 mouse fibroblasts overexpressing Aurora-C tib sensitivity and decreased AURKA copy number, in addition induced tumor formation in nude mice, to demonstrate the to a steep dose–response relationship (1 mmol/L). In AML oncogenic potential of this isoform (34). cell lines and primary AML cells, exposure to alisertib induced The observation that several tumors harbor genomic amplifi- antitumor activity, mediated by cell-cycle arrest and apoptotic cell cation of AURKA (Aurora-A kinase gene) and AURKB (Aurora-B death (42). Alisertib synergizes with vorinostat in vitro in pediatric kinase gene) supports the importance of aurora kinase signaling leukemia, medulloblastoma, and neuroblastoma cell lines (43). in cancer. This often correlates with high protein levels, and The Phase I trial to test the safety of alisertib by the Children's cytogenetic abnormalities including chromosome instability and Oncology Group, enrolled 37 patients with advanced solid aneuploidy (32, 33). Alterations in Aurora A and Aurora B vary by tumors. This study demonstrated that once-daily dose regimens, tumor type and reflect different subtypes. Small molecule inhi- every 21 days were more tolerable for children than twice-daily bitors designed against aurora kinases target a highly conserved regimens used for adults (44). DLTs were myelosuppression (3/ ATP-binding pocket, to abrogate kinase activity. These agents 4 patients at 100 mg/m2), mood alteration (1/6 patients at 80 mg/ interrupt the mitotic checkpoint, and lead to the onset of aberrant m2) and mucositis (1/6 patients at 45 mg/m2) on the once-daily mitosis without cytokinesis, accumulation of cells demonstrating dosing. Toxicities on the twice-daily schedule were mucositis and polyploidy and cell death. First-generation agents reported off- myelosuppression (80 mg/m2), alkaline phosphatase (1/5 target effects against other kinases, leading to high toxicities. patients at 60 mg/m2) and hand-foot-skin syndrome (5/11 Current second-generation aurora kinase inhibitors are highly patients). Among the 33 evaluable patients receiving alisertib in specific and more potent to rapidly dividing cancer cells. 2.1 this study, a partial response (n ¼ 1) and prolonged stable disease AT9283 (n ¼ 6) were observed. In another trial (45), single-agent alisertib AT9283 is a multikinase inhibitor with high potency against once daily for 7 days of a 21-day treatment cycle administered to Aurora A and Aurora B kinase activity. AT9283 also inhibits Janus four pediatric patients with recurrent atypical teratoid rhabdoid kinase 2 (JAK2) and JAK3, FMS-like tyrosine kinase 3 (FLT3), and tumors (ATRT), resulted in stable disease and/or durable regres- Abelson tyrosine kinase (ABLT315I; ref. 35) with lower efficacy. sion for all patients. Further clinical development of alisertib

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focused on incorporation with mechanism-based combinations treating newly diagnosed DIPG (NCT01922076); and with iri- could achieve additive beneﬁts in this patient population. notecan hydrochloride in treating advanced solid tumors (NCT02095132) is ongoing. Wee1 kinase inhibition

Wee1 kinase regulates the G2–M cell-cycle checkpoint by mod- Kinesin spindle protein inhibition ulating the activities of CDK1 and CDK2, to halt cell-cycle pro- The kinesin spindle protein (KSP), also known as Hs Eg5, gression in response to DNA damage (46). In comparison belongs to a family of at least 12 kinesins involved in the assembly with normal cells that repair DNA damage during G1 arrest, and maintenance of the spindle during mitotic and meiotic cell deficiencies in the G1 checkpoint in cancer cells result in DNA division. KSP forms a homotetrameric complex that binds micro- repair at the G2–M checkpoint. Ataxia-telangiectasia–mutated tubules and mediates separation of spindle poles and assembly of (ATM) kinase and/or ataxia-telangiectasia-related (ATR) kinase the bipolar mitotic spindle (58). Inhibition of KSP leads to cell mediate the repair of DNA double-strand breaks and DNA single- cycle arrest in mitosis with the formation of characteristic monoa- strand breaks (47), respectively. ATR also phosphorylates the ster spindles (59). Monostrol, was the first small-molecule inhib- checkpoint kinase CHK1, which phosphorylates Wee1. Phos- itor against KSP that was discovered nearly 2 decades ago (60). Its phorylation of tyrosine 15 on CDK1 by Wee1 inhibits CDK1/ mechanism of action was unique because all previously known cyclin B function, resulting in cell-cycle arrest at G2, entry into small molecules that specifically affect microtubule dynamics, mitosis and DNA-damage repair (46). Wee1 is also involved in typically target tubulin. KSP expression is high in proliferating stabilizing DNA replication forks and homologous recombina- human tissues as well as breast, colon, lung, ovary, and uterine tion (HR) repair (48). Because Wee1 functionally interacts with tumors (61). critical regulators of mitosis, inhibition interrupts the DNA repair machinery, leading to mitotic catastrophe and apoptotic cell death Ispinesib. Ispinesib (SB-715992; Cytokinetics and GlaxoSmith- (49). Wee1 is expressed at elevated levels in many adult and Kline) was the first potent, highly specific small-molecule inhib- pediatric malignancies, including osteosarcoma (50), high-grade itor of KSP tested in clinical trials. Ispinesib treatment against the glioma (HGG; ref. 51), DIPG (52), and leukemia (53). Inhibition PPTP in vivo tumor panels led to maintained complete response in of Wee1 could sensitize cancer cells to radiation therapy (52) and one rhabdoid tumor, one Wilms tumor and one Ewing sarcoma chemotherapy (49), by disruption of the G2–Mcheckpoint. xenograft (62). Ispinesib achieved 2 complete and 2 partial responses among 6 evaluable tumors in the ALL xenografts panel. AZD1775 (formerly MK-1775). AZD1775 is the most frequently Reports from one phase I trial of ispinesib in 24 pediatric tested Wee1 inhibitor in preclinical and clinical studies. Com- patients with recurrent or refractory solid tumors (NCT00363272; bining gamma radiation and AZD1775 in HGG cell lines and ref. 63) demonstrate that infusion of ispinesib for 1-hour at either orthotopic HGG and DIPG xenograft tumors led to enhanced 5, 7, 9, or 12 mg/m2/dose once every 3 weeks, did not achieve sensitivity than achieved by radiation treatment alone (51). objective responses as monotherapy. Positive therapeutic Kreahling and colleagues (50) demonstrated single-agent treat- response (n ¼ 3), was stable disease for one patient with ana- ment of several sarcoma cell lines with AZD1775 resulted in plastic astrocytoma, one with alveolar soft part sarcoma, and one unscheduled entry into mitosis and initiation of apoptosis. In with ependymoblastoma, for 4–7 courses of drug treatment. addition, AZD1775 caused activation of cell division cycle protein In combination strategies in clinical trials for adults with 2 (CDC2; also known as CDK1) in an osteosarcoma xenograft and advanced solid tumors, the best response achieved was stable reduced tumor growth by 50% as monotherapy, and by approx- disease for the evaluation of ispinesib with docetaxel (64) and imately 70% when combined with gemcitabine (50). In hema- with capecitabine (65). Neutropenia was the most common DLT. tological malignancies, AZD1775 plus panobinostat demonstrat- Second-generation inhibitors against KSP were also tested in adult ed synergistic antitumor effects in preclinical models of AML (54) trials (SB-743921, a derivative of ispinesib, MK-0731/Merck and and when combined with CDK inhibitor (roscovitine; ref. 53), ARRY-520/Array Biofarma). Further assessment of KSP inhibitors and cytarabine (55). In the latter study, AZD1775 inhibited in prospective clinical trials for pediatric cancer are not planned. cytabarine-mediated S-phase arrest and prevented DNA repair. Furthermore, MYC-driven tumors or CDKN2A mutation com- Microtubule inhibitors bined with TP53 mutation could show aberrations in the G1 Microtubule-targeting agents (MTA) are classed as antimitotics checkpoint and become more reliant on the S- and G2-phase for cancer therapy. This class of agents has received much atten- checkpoints. S- and G2-checkpoint abrogation caused by inhibition among cytotoxic agents due to their broad spectrum of tion of Wee1 may selectively sensitize p53-deficient tumors (49). clinical activity against a number of different types of cancer. The Other studies also show that AZD1775 sensitizes AML cells to microtubule cytoskeleton governs the cell structure and motility cytarabine (56) and HGG cells to radiation (51) irrespective of and is involved in the intracellular transport of organelles and p53 mutation status. These observations suggest that the require- proteins and cell division (66). Microtubule dynamics involve the ment of p53 mutations for sensitivity with combination therapies systematic reorganization of tubulin polymerization (rescue) and using AZD1775 is context-dependent and may not be a critical depolymerization (catastrophe). Stabilizing factors include consideration during development of novel therapies. microtubule-associated proteins, MAPs (MAP4, XMAP215, A clinical study for 25 adults with refractory solid tumors, XMAP230/XMAP4, and XMAP310) and destabilizing factors reported DLTs in patients were supraventricular tachyarrhythmia include (Stathmin1 and XKCM1; ref. 67). Drugs that bind b-tubu- and other common toxicities were myelosuppression and diar- lin have inhibitory effects on microtubule dynamics. Consistent rhea (57). Patient recruitment for two pediatric phase-I trials to with the disruption of microtubules and the mitotic spindle, study side effects and best dose of Wee1 kinase inhibitor MTAs can arrest cell-cycle progression in mitosis, resulting in cell AZD1775 when given together with local radiation therapy in death (66, 68).

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Clinical studies indicate activity in pediatric leukemia (69) and another study, for efficacy against refractory or relapsed leukemia many solid tumors, including rhabdomyosarcoma (70). Howev- (ALL), AML (n ¼ 63), the objective response rate across all varying er, other tumors such as osteosarcoma are generally refractory to dose levels and schedules was <10% (69). The use of nanopar- these drugs, either as first-line therapy or through the develop- ticles [e.g., nab-paclitaxel (Abraxane); nab, nanoparticle albumin ment of mechanisms that allow cells to undergo mitotic slippage, bound] and antibody–drug conjugates are being explored to escape cell-cycle arrest and proliferate. These studies also reveal achieve greater specificity and therapeutic efficacy. A clinical study several limitations, such as neurological and bone marrow tox- evaluating nab-paclitaxel combined with gemcitabine in relapsed icity (69). or refractory osteosarcoma, Ewing sarcoma, rhabdomyosarcoma and other soft tissue sarcoma is underway (NCT02945800). Taxanes. The taxanes (e.g., paclitaxel, docetaxel, cabazetaxel) are Assessments will be response rate and progression-free survival microtubule-stabilizing agents. These agents bind b-tubulin in a as primary outcome measures and adverse effects as secondary hydrophobic pocket between adjacent protofilaments, within the outcome measures. lumen of the microtubule (71). Synthetic compounds, for exam- Pediatric phase I clinical trials of docetaxel (Taxotere) as a ple, docetaxel, as well as analogues of existing compounds, for single-agent completed in children with refractory solid example, taxol-like agents, epothilones have advanced the clinical tumors (74), demonstrated positive therapeutic responses in development of taxanes as a widely applied class of chemother- patients who received a 1-hour infusion of docetaxel. One apeutics. Taxanes are a standard first-line treatment for metastatic patient with rhabdomyosarcoma had a complete response, breast cancer and are also used for ovarian, breast, and non–small one with peripheral primitive neuroectodermal tumor cell lung cancer (72). (PPNET) had a partial response in, and there were minimal A phase I study in 31 children with refractory solid tumors responses in two patients with PPNET. Major toxicities were showed a low overall response rate of 13% (73). Taxol was neutropenia, malaise, myalgias, and anorexia. The clinical administered in 62 courses and the DLT was neurotoxicity. evaluation of the combination of docetaxel and gemcitabine Positive responses were complete response for one patient, partial in 28 children and adolescents with recurrent or refractory response for 2 patients, and a minimal response for one patient. In osteosarcoma, achieved 17.6% complete responses (75). Of

Aurora Cyclin D • AT9283 A/B 1/2/3 • Alisertib (MLN8237) • Palbociclib (PD-0332991) CDK • Ribociclib (LEE011) • Taxanes Microtubules/ 2/4/6 • Abemaciclib (LY2835219) • Vinca alkaloids Mitotic spindle • Flavopiridol (alvocidib) SAC checkpoint Kinesin • Ispinesib spindle M G1 (SB–715992) protein Gap G0

Cell division • MK-1775 Wee1 Quiescence

G2/M checkpoint Gap G1/S checkpoint

G2 S DNA synthesis

Figure 1. Cell-cycle inhibitors evaluated in clinical trials against pediatric cancer. A schematic representation of mammalian cell division and inhibitors that target regulatory proteins at various phases of the cell cycle. Mitogenic signals trigger increases in the expression of D-type cyclins and the formation of complexes with

CDK4 and CDK6 to initiate the cell cycle (G1) in which the cell prepares for DNA synthesis. Cells transition through S-phase where DNA synthesis occurs, the G2-gap phase and M-phase, where cell division occurs. Yellow stars depict three cell-cycle checkpoints: G1–S (the DNA replication checkpoint), G2–M (the DNA damage checkpoint), and SAC (the spindle assembly checkpoint). Regulatory proteins, blue. Blunt red arrows, direct inhibition strategies with targeted drugs. Dotted red lines, indirect inhibition strategies.

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note, the gemcitabine dose of either 675 or 900 mg/m2 did not superfamily. Evaluation of brentuximab vedotin as frontline influence this response rate. treatment in 16 pediatric patients with Hodgkin lymphoma (82)whoreceivedweeklydosingof1.2mg/kgofbrentuximab Vinca alkaloids. The vinca alkaloids are microtubule-destabilizing vedotin, led to toxicities comparable to that of the standard-of- agents that bind the interface of a- and b-tubulin heterodimers care backbone using vincristine. Ongoing trials for the same and inhibit tubulin polymerization (76). Vinblastine and vincris- patient population are evaluating effects of combining bren- tine were the first vinca alkaloids approved for clinical use as tuximab vedotin with doxorubicin (NCT01920932), dacarba- anticancer agents. Vincristine is approved and used for induction zine (NCT02979522), and gemcitabine (NCT01780662). therapy in childhood ALL (77). Several studies in pediatric patients show that vincristine has a sensitizing effect with cyto- Conclusions toxic chemotherapy (78–80). The liposomal formulation (sphin- gomyelin/cholesterol) vincristine sulfate liposome (VSL; Mar- Targeting cell regulation demonstrates efficacy in various pre- qibo) in relapsed ALL was tolerated at approximately 100-fold clinical studies in pediatric malignancies. Validation of effect in lower doses (2.25 mg/m2)/dose of weekly VSL in 21 children with clinical studies is ongoing (Fig. 1, Table 1). However, a single- refractory solid tumors or leukemia, in comparison to standard agent efficacy signal is not likely in studies focused on small vincristine (81). Observed clinical activity included minimal patient subgroups (e.g., metastatic and recurrent disease or residual disease negative complete remission (n ¼ 1) with ALL patients with specific mutations; ref. 36). The biologic rationale and stable disease (n ¼ 9). Brentuximab vedotin (Adcetris), is an for the development of agents targeting cell-cycle regulation is antibody–drug conjugate containing MMAE (monomethyl aur- sound and early efficacy signals reinforce the need for combina- istatin E) and an antibody targeting CD30 [tumor necrosis factor tion studies in patients at highest risk for adverse outcomes. (TNF) receptor superfamily, member 8; TNFRSF8], which is Early phase studies of cell-cycle regulators for pharmacological expressed in classical Hodgkin lymphoma (HL) and systemic inhibition in the treatment of cancer in children demonstrate anaplastic large cell lymphoma (sALCL). CD30 is a type I appreciable tolerability, and support the incorporation into com- transmembrane receptor and shares sequence homology in the bination approaches. These studies also demonstrate differences extracellular domain with other members of the TNF receptor in DLTs in targeting different cell-cycle components. One primary

Table 1. Potential biomarkers for clinical assessment of cell-cycle inhibitors in pediatric cancer Inhibitors Target Pediatric disease Potential biomarker Trials/references Palbociclib CDK4/6 CNS tumors Rb/pRb NCT02255461 MYC Lymphoma/AML/ALL CDK4/6 NCT03132454 Cyclin D1-3 CDKN2A Ribociclib CDK4/6 Rhabdoid tumor (21) Abemaciclib CDK4/6/9 DIPG NCT02644460 Neuroblastoma Ewing sarcoma Rhabdomyosarcoma Osteosarcoma rhabdoid tumor Flavopiridol CDK1/2/4/6/7 Solid tumors/lymphoma NCT00012181 AT9283 Aurora A/B kinase CNS/PNET AURKA (37) JAK2 AURKB JAK3 FLT3 Leukemia JAK2 (38) JAK3 FLT3 Alisertib Aurora A/B kinase Solid tumors (44) Aurora A/B kinase Rhabdoid tumor (45) MK-1775 Wee1 kinase DIPG TP53 NCT01922076 MYC Solid tumors NCT02095132 Ispinesib KSP Solid tumors (63) Paclitaxel Microtubules SAC Solid tumors p27 (73) Stathmin1 blll-tubulin Survivin ALL/AML (69) Nab-paclitaxel NCT02945800 Docetaxel Solid tumors (74) Osteosarcoma (75) Vincristine sulfate liposome ALL (81) Brentuximab vedotin Hodgkin's lymphoma (82)

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concern with the development of therapeutic interventions of the Several other common factors may influence clinical trial out- cell-cycle pathway is that strategies will not distinguish between come for pediatric patients. Some studies are withdrawn when trial normal dividing cells and cancer cells. At present, HR-positive doses are ineffective. Pediatric tumors are rare and biologically status in breast cancer tumors is the only clinical indicator for distinct from tumors in adults, and developmental changes in these agents (83). Because mitogens regulate D-type cyclins, infancy and adolescence associated with growth and maturation of understanding the connection between mitogenic signals and the organs, as well as alterations in metabolism may alter drug effects cell cycle will help identify potential predictive biomarkers. Val- in pediatric patients. It is likely the extrapolation of efficacy from idation and integration of functional assays will also be required adult and other data to the pediatric population by using relatively to successfully translate these inhibitors for pediatric use. low starting doses in children, in comparison with the adult MTD, In addition to agents discussed in this review (Fig. 1, Table 1), could diminish clinical efficacy for eligible patients. The extrapo- the development of inhibitors against other components of the lation of preclinical results to the clinical setting requires more cell-cycle machinery, have not yet been explored in pediatric focused studies on doses and dose schedules in relevant preclinical patients. For example, investigations of antitumor activity for animal models to successfully correlate the systemic exposure of inhibitors against ATM (KU-55933, KU-59403; KuDOS Pharma- agents tolerated in humans compared with mice. A greater under- ceuticals, AstraZeneca) and ATR (Schisandrin B, AZD6738) have standing of the development and progression of pediatric disease is advanced to early-phase trials in adult patients with cancer. crucial for optimizing study designs for control groups, stratifica- Simultaneous targeting of proteins that cross-talk (e.g., ATM/ATR tion, and biomarker assay development. The observation that and Wee1 at the G2–M checkpoint) could show results that are activity of an agent in pediatric leukemia may be limited to patients more robust that targeting a single checkpoint protein. In addi- with specific mutations (38), or Rb status in solid tumors (16), tion, combining aurora kinase or Wee1 kinase inhibition with would require recruitment of select patient populations. disruption of microtubule dynamics might achieve higher sensi- Finally, studies that are closed prematurely when adult trials do tivity of cancer cells. The design of these combination strategies not reach their primary endpoint can limit availability of new drugs will rely on the outcome of trials that are completed or underway. before pediatric studies are complete. This was seen when Combination clinical studies with immunotherapy are ongoing MLN8237 (alisertib) failed to reach its primary endpoint for adult in adults. Of note, the development of other novel agents that lymphomas and lung cancers during early-phase studies in pedi- indirectly affect cell division are ongoing. The anti-tropomyosin atric patients. Despite promising results for pediatric use, drug drugs represent a novel class of anti-actin agents that target development was halted. Table 2 lists the current regulatory tropomyosins that are important for tubulin stability in the status and indications of the agents discussed in this review. This kinetochore and are being studied in combination with antimi- highlights the vulnerability of pediatric drug development to totics (84). outcomes in adult trials. Current clinical studies to determine the

Table 2. Developmental status for selected cell-cycle inhibitors Inhibitor Developmental status Indication Date Palbociclib (IBRANCE) FDA approved HR-positive, HER2-negative advanced or metastatic March 2017 breast cancer Ribociclib (LEE011) FDA approved Same as Palbociclib March 2017 Abemaciclib Investigational drug Metastatic breast cancer July 2017 Flavopiridol (Alvocidib) Orphan designation B-cell chronic lymphocytic leukemia (B-CLL); April 2007 Orphan designation Prolymphocytic leukemia arising from CLL April 2007 Orphan designation Acute myeloid leukemia April 2014 AT9283 Investigational drug Acute myeloid leukemia April 2014 Investigational drug Solid tumors April 2006 Alisertib (MLN8237) Orphan designation Small cell lung cancer July 2013 Orphan designation/Withdrawn Peripheral T-cell lymphoma May 2012 AZD-1775 Investigational drug Hematological malignancies and solid tumors December 1992 Ispinesib Investigational drug Hematological malignancies and solid tumors June 1998 Paclitaxel (Taxol) FDA approved Ovarian cancer April 1998 FDA approved Breast cancer FDA approved Non–small cell lung cancer (NSCLC) June 1998 Nab-paclitaxel (Abraxane) FDA approved Metastatic breast cancer January 2005 FDA approved NSCLC October 2012 FDA approved Late-stage pancreatic cancer September 2013 Docetaxel (Taxotere) FDA approved Metastatic breast cancer June 1998 FDA approved NSCLC December 1999 FDA approved Metastatic prostate cancer May 2004 FDA approved Gastric adenocarcinoma March 2006 FDA approved Head and neck cancer October 2006 Vincristine sulfate liposome (Marqibo) FDA approved Adult Philadelphia chromosome-negative (Ph-) August 2012 acute lymphoblastic leukemia Brentuximab vedotin (Adcetris) FDA approved Hodgkin lymphoma August 2011 FDA approved Systemic anaplastic large cell lymphoma August 2011 Abbreviations: HR, hormone receptor; HER2, human epidermal growth factor receptor 2.

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most efﬁcacious and safe approaches are critical for the advance- Grant Support ment of drugs for pediatric use and provide valuable information This work is supported by the Clinical and Translational Research (CTR) and new hope to improve the quality of life to children with these Pilot Award to V.B. Sampson under grant number NIHU54-GM104941. rare diseases.

Disclosure of Potential Conﬂicts of Interest Received July 10, 2017; revised August 24, 2017; accepted September 19, No potential conﬂicts of interest were disclosed. 2017; published OnlineFirst November 2, 2017.

References 1. Malumbres M. Cyclin-dependent kinases. Genome Biol 2014;15:122. 20. Wood AC, Krytska K, Ryles HT, Infarinato NR, Sano R, Hansel TD, et al. 2. Bendris N, Lemmers B, Blanchard JM. Cell cycle, cytoskeleton dynamics Dual ALK and CDK4/6 inhibition demonstrates synergy against neuro- and beyond: the many functions of cyclins and CDK inhibitors. Cell Cycle blastoma. Clin Cancer Res 2017;23:2856–68. 2015;14:1786–98. 21. Geoerger B, Bourdeaut F, DuBois SG, Fischer M, Geller JI, Gottardo NG, 3. Kim SJ, Nakayama S, Miyoshi Y, Taguchi T, Tamaki Y, Matsushima T, et al. et al. A phase I study of the CDK4/6 inhibitor ribociclib (LEE011) in Determination of the specific activity of CDK1 and CDK2 as a novel pediatric patients with malignant rhabdoid tumors, neuroblastoma, and prognostic indicator for early breast cancer. Ann Oncol 2008;19:68–72. other solid tumors. Clin Cancer Res 2017;23:2433–41. 4. Zeestraten EC, Maak M, Shibayama M, Schuster T, Nitsche U, Matsushima 22. Gelbert LM, Cai S, Lin X, Sanchez-Martinez C, Del Prado M, Lallena MJ, T, et al. Specific activity of cyclin-dependent kinase I is a new potential et al. Preclinical characterization of the CDK4/6 inhibitor LY2835219: in- predictor of tumour recurrence in stage II colon cancer. Br J Cancer vivo cell cycle-dependent/independent anti-tumor activities alone/in com- 2012;106:133–40. bination with gemcitabine. Invest New Drugs 2014;32:825–37. 5. Zhao Y, Zhang Y, Yang Z, Li A, Dong J. Simultaneous knockdown of BRAF 23. Patnaik A, Rosen LS, Tolaney SM, Tolcher AW, Goldman JW, Gandhi L, and expression of INK4A in melanoma cells leads to potent growth et al. Efficacy and safety of abemaciclib, an inhibitor of CDK4 and CDK6, inhibition and apoptosis. Biochem Biophys Res Commun 2008;370: for patients with breast cancer, non-small cell lung cancer, and other solid 509–13. tumors. Cancer Discov 2016;6:740–53. 6. Placke T, Faber K, Nonami A, Putwain SL, Salih HR, Heidel FH, et al. 24. Senderowicz AM. Flavopiridol: the first cyclin-dependent kinase inhibitor Requirement for CDK6 in MLL-rearranged acute myeloid leukemia. Blood in human clinical trials. Invest New Drugs 1999;17:313–20. 2014;124:13–23. 25. Whitlock JA, Krailo M, Reid JM, Ruben SL, Ames MM, Owen W, et al. Phase I 7. Whittaker SR, Mallinger A, Workman P, Clarke PA. Inhibitors of cyclin- clinical and pharmacokinetic study of flavopiridol in children with refrac- dependent kinases as cancer therapeutics. Pharmacol Ther 2017;173: tory solid tumors: a Children's Oncology Group Study. J Clin Oncol 83–105. 2005;23:9179–86. 8. Bretones G, Delgado MD, Leon J. Myc and cell cycle control. Biochim 26. Gorlick R, Kolb EA, Houghton PJ, Morton CL, Neale G, Keir ST, et al. Initial Biophys Acta 2015;1849:506–16. testing (stage 1) of the cyclin dependent kinase inhibitor SCH 727965 9. Hutcheson J, Witkiewicz AK, Knudsen ES. The RB tumor suppressor at the (dinaciclib) by the pediatric preclinical testing program. Pediatr Blood intersection of proliferation and immunity: relevance to disease immune Cancer 2012;59:1266–74. evasion and immunotherapy. Cell Cycle 2015;14:3812–9. 27. Walsby E, Lazenby M, Pepper C, Burnett AK. The cyclin-dependent kinase 10. Kelleher FC, O'Sullivan H. FOXM1 in sarcoma: role in cell cycle, pluripo- inhibitor SNS-032 has single agent activity in AML cells and is highly tency genes and stem cell pathways. Oncotarget 2016;7:42792–804. synergistic with cytarabine. Leukemia 2011;25:411–9. 11. Ingham M, Schwartz GK. Cell-cycle therapeutics come of age. J Clin Oncol 28. Tong WG, Chen R, Plunkett W, Siegel D, Sinha R, Harvey RD, et al. Phase I 2017;35:2949–2959. and pharmacologic study of SNS-032, a potent and selective Cdk2, 7, and 9 12. Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade M, Trachet E, et al. Specific inhibitor, in patients with advanced chronic lymphocytic leukemia and inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated multiple myeloma. J Clin Oncol 2010;28:3015–22. antitumor activity in human tumor xenografts. Mol Cancer Ther 2004;3: 29. Keen N, Taylor S. Aurora-kinase inhibitors as anticancer agents. Nat Rev 1427–38. Cancer 2004;4:927–36. 13. Barton KL, Misuraca K, Cordero F, Dobrikova E, Min HD, Gromeier M, et al. 30. Glover DM, Leibowitz MH, McLean DA, Parry H. Mutations in aurora PD-0332991, a CDK4/6 inhibitor, significantly prolongs survival in a prevent centrosome separation leading to the formation of monopolar genetically engineered mouse model of brainstem glioma. PLoS ONE spindles. Cell 1995;81:95–105. 2013;8:e77639. 31. Macurek L, Lindqvist A, Lim D, Lampson MA, Klompmaker R, Freire R, et al. 14. Huillard E, Hashizume R, Phillips JJ, Griveau A, Ihrie RA, Aoki Y, et al. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Cooperative interactions of BRAFV600E kinase and CDKN2A locus defi- Nature 2008;455:119–23. ciency in pediatric malignant astrocytoma as a basis for rational therapy. 32. Krenn V, Musacchio A. The Aurora B kinase in chromosome Bi-orientation Proc Natl Acad Sci U S A 2012;109:8710–5. and spindle checkpoint signaling. Front Oncol 2015;5:225. 15. Flaherty KT, Lorusso PM, Demichele A, Abramson VG, Courtney R, 33. Quartuccio SM, Schindler K. Functions of Aurora kinase C in meiosis and Randolph SS, et al. Phase I, dose-escalation trial of the oral cyclin-depen- cancer. Front Cell Dev Biol 2015;3:50. dent kinase 4/6 inhibitor PD 0332991, administered using a 21-day 34. Khan J, Ezan F, Cremet JY, Fautrel A, Gilot D, Lambert M, et al. Over- schedule in patients with advanced cancer. Clin Cancer Res 2012;18: expression of active Aurora-C kinase results in cell transformation and 568–76. tumour formation. PLoS One 2011;6:e26512. 16. Rader J, Russell MR, Hart LS, Nakazawa MS, Belcastro LT, Martinez D, et al. 35. Howard S, Berdini V, Boulstridge JA, Carr MG, Cross DM, Curry J, et al. Dual CDK4/CDK6 inhibition induces cell-cycle arrest and senescence in Fragment-based discovery of the pyrazol-4-yl urea (AT9283), a multi- neuroblastoma. Clin Cancer Res 2013;19:6173–82. targeted kinase inhibitor with potent aurora kinase activity. J Med Chem 17. Tao YF, Wang NN, Xu LX, Li ZH, Li XL, Xu YY, et al. Molecular mechanism of 2009;52:379–88. G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 36. Petersen W, Liu J, Yuan L, Zhang H, Schneiderjan M, Cho YJ, et al. Dasatinib inhibitor, in leukemia cells. Cancer Cell Int 2017;17:35. suppression of medulloblastoma survival and migration is markedly 18. Pikman Y, Alexe G, Roti G, Conway AS, Furman A, Lee ES, et al. Synergistic enhanced by combining treatment with the aurora kinase inhibitor drug combinations with a CDK4/6 inhibitor in T-cell acute lymphoblastic AT9283. Cancer Lett 2014;354:68–76. leukemia. Clin Cancer Res 2017;23:1012–24. 37. Moreno L, Marshall LV, Pearson AD, Morland B, Elliott M, Campbell- 19. Hart LS, Rader J, Raman P, Batra V, Russell MR, Tsang M, et al. Preclinical Hewson Q, et al. A phase I trial of AT9283 (a selective inhibitor of aurora therapeutic synergy of MEK1/2 and CDK4/6 inhibition in neuroblastoma. kinases) in children and adolescents with solid tumors: a Cancer Research Clin Cancer Res 2017;23:1785–96. UK study. Clin Cancer Res 2015;21:267–73.

OF8 Cancer Res; 2017 Cancer Research

Targeting Cell-Cycle Regulation for Pediatric Cancer

38. Vormoor B, Veal GJ, GriffinMJ,BoddyAV,IrvingJ,MintoL,etal.A 58. Sawin KE, Mitchison TJ, Wordeman LG. Evidence for kinesin-related phase I/II trial of AT9283, a selective inhibitor of aurora kinase in proteins in the mitotic apparatus using peptide antibodies. J Cell Sci 1992; children with relapsed or refractory acute leukemia: challenges to run 101:303–13. early phase clinical trials for children with leukemia. Pediatr Blood 59. Weil D, Garcon L, Harper M, Dumenil D, Dautry F, Kress M. Targeting the Cancer 2017;64. kinesin Eg5 to monitor siRNA transfection in mammalian cells. Biotech- 39. Maris JM, Morton CL, Gorlick R, Kolb EA, Lock R, Carol H, et al. Initial niques 2002;33:1244–8. testing of the aurora kinase A inhibitor MLN8237 by the Pediatric Pre- 60. Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ. clinical Testing Program (PPTP). Pediatr Blood Cancer 2010;55:26–34. Small-molecule inhibitor of mitotic spindle bipolarity identified in a 40. Sells TB, Chau R, Ecsedy JA, Gershman RE, Hoar K, Huck J, et al. MLN8054 phenotype-based screen. Science 1999;286:971–4. and alisertib (MLN8237): discovery of selective oral aurora A inhibitors. 61. Myers SM, Collins I. Recent findings and future directions for interpolar ACS Med Chem Lett 2015;6:630–4. mitotic kinesin inhibitors in cancer therapy. Future Med Chem 2016;8: 41. Manfredi MG, Ecsedy JA, Chakravarty A, Silverman L, Zhang M, Hoar KM, 463–89. et al. Characterization of Alisertib (MLN8237), an investigational small- 62. Carol H, Lock R, Houghton PJ, Morton CL, Kolb EA, Gorlick R, et al. Initial molecule inhibitor of aurora A kinase using novel in vivo pharmacody- testing (stage 1) of the kinesin spindle protein inhibitor ispinesib by the namic assays. Clin Cancer Res 2011;17:7614–24. pediatric preclinical testing program. Pediatr Blood Cancer 2009;53: 42. Kelly KR, Nawrocki ST, Espitia CM, Zhang M, Yang JJ, Padmanabhan S, 1255–63. et al. Targeting Aurora A kinase activity with the investigational agent 63. Souid AK, Dubowy RL, Ingle AM, Conlan MG, Sun J, Blaney SM, et al. A alisertib increases the efficacy of cytarabine through a FOXO-dependent pediatric phase I trial and pharmacokinetic study of ispinesib: a Children's mechanism. Int J Cancer 2012;131:2693–703. Oncology Group phase I consortium study. Pediatr Blood Cancer 2010; 43. Muscal JA, Scorsone KA, Zhang L, Ecsedy JA, Berg SL. Additive effects of 55:1323–8. vorinostat and MLN8237 in pediatric leukemia, medulloblastoma, and 64. Blagden SP, Molife LR, Seebaran A, Payne M, Reid AH, Protheroe AS, neuroblastoma cell lines. Invest New Drugs 2013;31:39–45. et al. A phase I trial of ispinesib, a kinesin spindle protein inhibitor, 44. Mosse YP, Lipsitz E, Fox E, Teachey DT, Maris JM, Weigel B, et al. Pediatric with docetaxel in patients with advanced solid tumours. Br J Cancer phase I trial and pharmacokinetic study of MLN8237, an investigational 2008;98:894–9. oral selective small-molecule inhibitor of Aurora kinase A: a Children's 65. Calvo E C, Til E, et al. Phase I study of ispinesib in combination with Oncology Group Phase I Consortium study. Clin Cancer Res 2012; capecitabine in patients with advanced solid tumors. The 2005 Interna- 18:6058–64. tional Conference on Molecular Targets and Cancer Therapeutics; Novem- 45. Wetmore C, Boyett J, Li S, Lin T, Bendel A, Gajjar A, et al. Alisertib is active as ber 2005; Philadelphia, PA. [Accessed July 20, 2006]. single agent in recurrent atypical teratoid rhabdoid tumors in 4 children. 66. Chandrasekaran G, Tatrai P, Gergely F. Hitting the brakes: targeting Neuro Oncol 2015;17:882–8. microtubule motors in cancer. Br J Cancer 2015;113:693–8. 46. Do K, Doroshow JH, Kummar S. Wee1 kinase as a target for cancer therapy. 67. Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat Cell Cycle 2013;12:3159–64. Rev Cancer 2010;10:194–204. 47. Puigvert JC, Sanjiv K, Helleday T. Targeting DNA repair, DNA metabolism 68. Sampson VB, Vetter NS, Zhang W, Patil PU, Mason RW, George E, et al. and replication stress as anti-cancer strategies. FEBS J 2016;283:232–45. Integrating mechanisms of response and resistance against the tubulin 48. Krajewska M, Heijink AM, Bisselink YJ, Seinstra RI, Sillje HH, de Vries EG, binding agent Eribulin in preclinical models of osteosarcoma. Oncotarget et al. Forced activation of Cdk1 via wee1 inhibition impairs homologous 2016;7:86594–607. recombination. Oncogene 2013;32:3001–8. 69. Horton TM, Ames MM, Reid JM, Krailo MD, Pendergrass T, Mosher R, et al. 49. Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M, et al. A Phase 1 and pharmacokinetic clinical trial of paclitaxel for the treatment Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensi- of refractory leukemia in children: a Children's Oncology Group study. tizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther Pediatr Blood Cancer 2008;50:788–92. 2009;8:2992–3000. 70. Pappo AS, Lyden E, Breitfeld P, Donaldson SS, Wiener E, Parham D, 50. Kreahling JM, Foroutan P, Reed D, Martinez G, Razabdouski T, Bui MM, et al. Two consecutive phase II window trials of irinotecan alone or in et al. Wee1 inhibition by MK-1775 leads to tumor inhibition and combination with vincristine for the treatment of metastatic rhabdo- enhances efficacy of gemcitabine in human sarcomas. PLoS ONE 2013; myosarcoma: the Children's Oncology Group. J Clin Oncol 2007;25: 8:e57523. 362–9. 51. Muller K, Scheithauer H, Pietschmann S, Hoffmann M, Rossler J, Graf N, 71. Snyder JP, Nettles JH, Cornett B, Downing KH, Nogales E. The binding et al. Reirradiation as part of a salvage treatment approach for progressive conformation of Taxol in beta-tubulin: a model based on electron crys- non-pontine pediatric high-grade gliomas: preliminary experiences from tallographic density. Proc Natl Acad Sci U S A 2001;98:5312–6. the German HIT-HGG study group. Radiat Oncol 2014;9:177. 72. Flores JP, Saif MW. Novel oral taxane therapies: recent Phase I results. Clin 52. Caretti V, Hiddingh L, Lagerweij T, Schellen P, Koken PW, Hulleman E, et al. Investig 2013;3:333–41. WEE1 kinase inhibition enhances the radiation response of diffuse intrinsic 73. Hurwitz CA, Relling MV, Weitman SD, Ravindranath Y, Vietti TJ, Strother pontine gliomas. Mol Cancer Ther 2013;12:141–50. DR, et al. Phase I trial of paclitaxel in children with refractory solid tumors: a 53. Chaudhuri L, Vincelette ND, Koh BD, Naylor RM, Flatten KS, Peterson KL, Pediatric Oncology Group Study. J Clin Oncol 1993;11:2324–9. et al. CHK1 and WEE1 inhibition combine synergistically to enhance 74. Blaney SM, Seibel NL, O'Brien M, Reaman GH, Berg SL, Adamson PC, et al. therapeutic efficacy in acute myeloid leukemia ex vivo. Haematologica Phase I trial of docetaxel administered as a 1-hour infusion in children with 2014;99:688–96. refractory solid tumors: a collaborative pediatric branch, National Cancer 54. Qi W, Zhang W, Edwards H, Chu R, Madlambayan GJ, Taub JW, et al. Institute and Children's Cancer Group trial. J Clin Oncol 1997;15: Synergistic anti-leukemic interactions between panobinostat and MK- 1538–43. 1775 in acute myeloid leukemia ex vivo. Cancer Biol Ther 2015;16: 75. Song BS, Seo J, Kim DH, Lim JS, Yoo JY, Lee JA. Gemcitabine and docetaxel 1784–93. for the treatment of children and adolescents with recurrent or refractory 55. Tibes R, Bogenberger JM, Chaudhuri L, Hagelstrom RT, Chow D, Buechel osteosarcoma: Korea Cancer Center Hospital experience. Pediatr Blood ME, et al. RNAi screening of the kinome with cytarabine in leukemias. Cancer 2014;61:1376–81. Blood 2012;119:2863–72. 76. Rai SS, Wolff J. Localization of the vinblastine-binding site on beta-tubulin. 56. Ford JB, Baturin D, Burleson TM, Van Linden AA, Kim YM, Porter CC. J Biol Chem 1996;271:14707–11. AZD1775 sensitizes T cell acute lymphoblastic leukemia cells to cytar- 77. Dupuis LL, Lu X, Mitchell HR, Sung L, Devidas M, Mattano LA Jr, et al. abine by promoting apoptosis over DNA repair. Oncotarget 2015;6: Anxiety, pain, and nausea during the treatment of standard-risk childhood 28001–10. acute lymphoblastic leukemia: a prospective, longitudinal study from the 57. Do K, Wilsker D, Ji J, Zlott J, Freshwater T, Kinders RJ, et al. Phase I study of Children's Oncology Group. Cancer 2016;122:1116–25. single-agent AZD1775 (MK-1775), a Wee1 kinase inhibitor, in patients 78. Walterhouse D, Watson A. Optimal management strategies for rhabdo- with refractory solid tumors. J Clin Oncol 2015;33:3409–15. myosarcoma in children. Paediatr Drugs 2007;9:391–400.

www.aacrjournals.org Cancer Res; 2017 OF9

Mills et al.

79. Canellos GP, Rosenberg SA, Friedberg JW, Lister TA, Devita VT. Treatment 82. Flerlage JE, Metzger ML, Wu J, Panetta JC. Pharmacokinetics, immunoge- of Hodgkin lymphoma: a 50-year perspective. J Clin Oncol 2014;32:163–8. nicity, and safety of weekly dosing of brentuximab vedotin in pediatric 80. Yokoyama M, Kusano Y, Takahashi A, Inoue N, Ueda K, Nishimura N, et al. patients with Hodgkin lymphoma. Cancer Chemother Pharmacol 2016; Incidence and risk factors of febrile neutropenia in patients with non- 78:1217–23. Hodgkin B-cell lymphoma receiving R-CHOP in a single center in Japan. 83. Rugo HS, Rumble RB, Macrae E, Barton DL, Connolly HK, Dickler MN, Support Care Cancer. 2017 May 27. [Epub ahead of print]. et al. Endocrine therapy for hormone receptor-positive metastatic breast 81. Shah NN, Merchant MS, Cole DE, Jayaprakash N, Bernstein D, Delbrook C, cancer: American Society of Clinical Oncology Guideline. J Clin Oncol et al. Vincristine sulfate liposomes injection (VSLI, Marqibo(R)): results 2016;34:3069–103. from a phase I study in children, adolescents, and young adults with 84. Currier MA, Stehn JR, Swain A, Chen D, Hook J, Eiffe E, et al. Identiﬁcation refractory solid tumors or leukemias. Pediatr Blood Cancer 2016;63: of cancer-targeted tropomyosin inhibitors and their synergy with micro- 997–1005. tubule drugs. Mol Cancer Ther 2017;16:1555–65.

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Recent Advances of Cell-Cycle Inhibitor Therapies for Pediatric Cancer

Christopher C. Mills, EA. Kolb and Valerie B. Sampson

Cancer Res Published OnlineFirst November 2, 2017.

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