Received: 17 June 2021 Revised: 21 July 2021 Accepted: 29 July 2021 DOI: 10.1002/ctm2.533 LETTER TO EDITOR The oral KIF11 inhibitor 4SC-205 exhibits antitumor activity and potentiates standard and targeted therapies in primary and metastatic neuroblastoma models Dear Editor, According to functional genomics, neuroblastoma cells Neuroblastoma remains incurable for most patients seem to be one of the cell types that are more dependent with high-risk disease.1 Perturbation of transcription fac- on the expression of KIF11 for survival being particularly tors (MYCN and PHOX2B), kinases (ALK, MEK), and cell sensitive to its pharmacological inhibition.5 Concurring cycle regulators (CDK4/6, CHECK1), among other factors, with these observations, the silencing of KIF11 caused a make neuroblastoma cells highly proliferative, which is reduction in cell viability (Figure S3A-C) and a 3–4 fold associated with poor patient outcomes.2,3 To circumvent reduction in the growth of established neuroblastoma sub- the limitations of the classical microtubule poisons such as cutaneous xenografts (Figure 2A–C and S3D-R). KIF11 vinca alcaloyds used in the treatment of neuroblastoma,1 inhibitors have moved forward toward phase 1 and 2 clini- we sought to explore alternative mitotic regulators as new cal trials in adult tumors,6,7 with very limited development therapeutic targets for high-risk neuroblastoma patients. for childhood cancer. Herein, we provide a complete pre- One of these mitotic spindle-specific proteins is kinesin clinical characterization of the potent and highly selective family member 11 (KIF11), also known as kinesin spindle KIF11 inhibitor, 4SC-205 (Figure 2D), the first oral KIF11 protein, kinesin-5, or Eg5, which is essential for bipolar inhibitor that has been evaluated in phase I clinical trials spindle formation and mitotic progression in human cells.4 in adult patients (NCT01065025). Compared to other KIF11 Transcriptomic analyses showed that the expression of inhibitors, 4SC-205 can be administrated daily, thus being multiple kinesins, including KIF11 was higher in the high- able to hit the target in a more sustained manner. Neu- risk neuroblastoma compared with low- and intermediate- roblastoma cells treated with 4SC-205 (Figure 2E; Table risk groups (Figures 1A and S1; Table S1). Overall sur- S5) displayed all the expected phenotypic features resulting vival was significantly poorer in patients with high KIF11 from KIF11 inhibition such as the inability to form bipo- expression (Figure 1B–D; Table S2). KIF11 high expression lar spindles (Figures 2F and S4A), cell cycle arrest during was identified as an independent prognostic factor of sur- mitosis (Figure S4B-H), and induction of apoptosis (Fig- vival, together with risk assessment (HR = 3.051; Table S3) ure S5), thereby confirming the high KIF11 specificity of and found to be higher in patients with amplification of this compound. While similar effects were observed in 3D MYCN, 1p36 loss, or 17q23 gain (Figure 1E). At the protein spheroid cultures (Figure S6A-C), 4SC-205 did not affect level, KIF11 expression was detected in the cytoplasm of the viability of differentiated cells (Figure S6D-G). neuroblastic cells (Figure 1F) and showed higher expres- When used in vivo, 4SC-205 treated mice showed a sion compared to low- or intermediate-risk neuroblastoma remarkable shrinkage of the original SK-N-BE(2) subcu- samples (p < 0.05) and in tumors with segmental chro- taneous xenograft (Figures 2G and 2H) or tumor growth mosome alterations such as 1p36, 11q deletion, and gain delay in SK-N-AS xenografts (Figures 2J, 2K, and Figure of 17q23 (Table S4). Kaplan–Meier analysis confirmed that S7). Increased phosphorylation of histone H3 and apop- high KIF11 protein expression was associated with shorter totic hallmarks (i.e., processing of PARP) confirmed that event-free and overall survival (Figures 1G and H). While the antitumor effect of 4SC-205 was comparable to that of there is a positive correlation between KIF11 and MYCN genetic KIF11 silencing in vivo (Figures 2I,L). Transcrip- mRNA expression levels, MYCN is neither sufficient, nor tomic analysis of the 4SC-205-treated tumors confirmed necessary for KIF11 expression (Figure S2). the expected genetic changes of arrested cells in mitosis This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics Clin. Transl. Med. 2021;11:e533. wileyonlinelibrary.com/journal/ctm2 1 of 8 https://doi.org/10.1002/ctm2.533 2 of 8 LETTER TO EDITOR F I G U R E 1 KIF11 expression is an independent prognostic factor of survival in neuroblastoma. (A) KIF11 mRNA expression levels comparing low-/intermediate- with high-risk neuroblastoma tumors (GSE62564, n = 498). (B-D) Kaplan–Meier overall survival curve in a cohort of 498 patients based on KIF11 mRNA expression (B) or stratified in low- and intermediate-risk (C) or high-risk (D) neuroblastoma subcohorts. (E) KIF11 mRNA expression in neuroblastoma patients with different genomic alterations (GSE3960, n = 101). (F) Representative images of KIF11 immunohistochemistry in low-and high-risk neuroblastoma tissues. Scale bar indicates 50 μm. (G and H) Kaplan–Meier curves of event-free survival (G) and overall survival (H) based on KIF11 protein expression LETTER TO EDITOR 3 of 8 F I G U R E 2 Genetic and pharmacological inhibition of KIF11 reduces tumor growth in subcutaneous neuroblastoma xenografts. (A) Analysis of tumor volume of SK-N-BE(2) cells transduced with an inducible shKIF11 lentiviral construct comparing the effects of KIF11 silencing (+Dox) versus control (-Dox). (B) Waterfall plot comparing the change in tumor volume at day 28 post-injection versus day 18, when doxycycline was added into the drinking water. (C) Western blot analysis of excised tumors at termination of the experiment. Turbo-RFP (tRFP) reporter expression was used as a control for shRNA transgene induction. (D) 4SC-205 chemical structure. (E) Dose-response curves of neuroblastoma cell lines treated with increasing concentrations of 4SC-205 for 48 h. IC50 values are represented as the average of three independent experiments ± SEM. (F) Mitotic spindle immunofluorescence of SK-N-BE(2) cells transfected with siKIF11 or treated with 4SC-205 (25 nM) for 24 h. KIF11: red, α-TUBULIN: green, DAPI: blue. Scale bar, 5 μm. (G) Individual tumor growth of xenografts derived from SK-N-BE(2) comparing vehicle (n = 10) versus 40 mg/kg 4SC-205 (n = 10). (H) Waterfall plot comparing the change in tumor volume at day 16 post-treatment versus day 4. (I) Western blot analysis of cell-cycle and apoptosis-related proteins in SK-N-BE(2) resected tumors. (J) Tumor growth of subcutaneous xenograft derived from SK-N-AS treated with vehicle (n = 10) or 40 mg/kg 4SC-205 (n = 10). (K) Tumor volume fold change at day 7 post-treatment versus day 0. (L) Western blot analysis of resected tumors at the end of the experiment. (M) Gene set enrichment analysis of SK-N-BE(2) xenografts treated with vehicle or 4SC-205. Graph represents normalized enrichment score (NES) values of enriched sets with p < 0.05. (N and O) Heatmap representing top 20 differentially deregulated genes of G2/M checkpoint and E2F targets (N), and mTORC signaling-related genes (O) 4 of 8 LETTER TO EDITOR F I G U R E 3 4SC-205 impairs PDOX growth and prolongs the survival of mice bearing neuroblastoma liver metastasis. (A) Schematic representation of PDOX generation and characterization. (B) Immunohistochemistry of neuroblastoma markers in FFPE tumor sections from the original tumor (upper panels) and after implantation in mice (lower panels). H&E: Hematoxylin and eosin staining. Scale bar represents 110 μm. (C and D) Chromosomal copy number variations (CNV) of the original tumor and after implantation in mice. The most relevant molecular pathogenic alterations found in the original tumor and PDOX are highlighted. (E) Schematic illustration of the treatment schedule. Mice bearing PDOX were treated for 3 weeks with either vehicle (n = 7) or 4SC-205 (40 mg/kg, n = 11). (F) Tumor weights at the end of the experiment. (G) Rep- resentative picture of the dissected tumors (T: tumor; K: kidney). Scale bar: 1 cm. (H) Representative images of phosphorylated histone H3. Scale bar: 10 μm. (I). Quantification of phospho-histone H3 positive cells in histological sections from vehicle- and 4SC-205-treated tumors. Graph rep- resents the average percentage of positive cells ± SEM from vehicle-or 4SC-205 (10 representative fields/tumor) -treated tumors (n = 5/group). (J) Scheme of the experimental design. SK-N-BE(2) cells were injected into the tail vein, and 21 days later, mice were randomized into vehicle (n = 13) and 40 mg/kg 4SC-205 groups (n = 15). The mice received oral administration of 4SC-205 three times a week for five consecutive weeks. (K) Scatter dot plots representing the average quantification of tumor bioluminescence ± SEM at the indicated days post-treatment. **p < 0.01, two-tailed student’s t-test. (L) Representative images of luciferase activity in eight mice from the vehicle and 4SC-205 treatment groups at 28 days posttreatment. (M) Kaplan–Meier survival curve of mice with neuroblastoma liver metastases treated with either vehicle or 40 mg/kg 4SC-205 for 5 weeks. Statistical differences were calculated using the Gehan–Breslow–Wilcoxon test. *p < 0.05, ***p < 0.001, two-tailed Student’s t-test LETTER TO EDITOR 5 of 8 F I G U R E 4 4SC-205 potentiates the effect of chemotherapy and neuroblastoma-targeted therapies. (A) Scheme of the experimental design for the combination of 4SC-205 and standard chemotherapies. Images are representative of crystal violet staining of SK-N-BE(2) cells treated with CDDP (1000 nM), doxorubicin (15 nM), topotecan (10 nM), 4SC-205 (17.5 nM) and their corresponding combinations (Combo). (B) Heatmaps showing the percentage of cellular fraction affected by drug combination treatments. (C) Graphs represent the average effect on cell viability from three independent experiments ± SEM (n = 3/condition). (D) Combinatorial analysis performed using SynergyFinder 2.0 software. (E) Schematic representation of the experimental design combining 4SC-205 and neuroblastoma-targeted therapies. Representative 6 of 8 LETTER TO EDITOR and tumor cells with reduced proliferation or viability (Fig- inhibitors alone, with most of the combination doses show- ure 2M–O). We next tested 4SC-205 in a patient-derived ing additive effects (Figures 4E–J and S10D-G; Table S7). orthotopic xenografts (PDOX) derived from a very high- In summary, our study provides a rationale for the future risk neuroblastoma patient. VH-NB608 PDOX retained therapeutic integration in clinical trials of 4SC-205, an most of the histological and molecular features of the orig- structurally distinct oral KIF11 inhibitor that shows potent inal tumor (Figure 3A–D). 4SC-205-treated mice displayed antitumor activity in multiple preclinical neuroblastoma a 14.75-fold reduction in tumor weight compared to the models and sensitizes neuroblastoma cells to standard vehicle group (Figures 3E and 3F). Mice treated with 4SC- chemotherapy and specific neuroblastoma-targeted thera- 205 presented small tumors located in the adrenal gland, pies. whereas vehicle-treated mice had large tumors with the kidney completely surrounded by the tumor (Figure 3G). AC K N OW L E D G M E N T S Furthermore, 4SC-205 tumors had a larger fraction of cells We are very thankful to Prof. Thomas U. Mayer for pro- with phosphorylation of histone H3, thereby indicating a viding us with contact with the 4SC. We would like to specific targeting of KIF11 in these tumors, and suggest- acknowledge the members of the Laboratory Animal Ser- ing that tumors were still sensitive to the inhibitor after vice and High Technology Unit for technical support. We 3 weeks (Figure 3H,I). are grateful to the CNAG-CRG for technical and bioinfor- Half of neuroblastoma patients present metastases at matics assistance with transcriptome analyses. We would the time of diagnosis.8 Therefore, we proceeded to test the like to thank Editage (www.editage.com) for English lan- efficacy of 4SC-205 in a neuroblastoma liver metastasis guage editing. model. In response to treatment, a clear delay in metastatic outgrowth was observed in 4SC-205-treated mice (Fig- CONFLICT OF INTEREST ure 3J–L and S8). As a consequence, the median lifespan Dr. Moreno participates in data monitoring committees of the animals was significantly expanded by ∼27% (Fig- of clinical trials sponsored by Novartis, Actuate Thera- ure 3M; vehicle: 33 days vs. 4SC-205: 42 days). Noticeable, peutics, Shionogi, Incyte, the University of Southampton 4SC-205 administration minimally affected mice weight and the Royal Marsden NHS Foundation Trust; and had (<10%) during the course of the treatment (Figure S9). To a consulting role for Novartis and Shionogi. Dr. Lucas achieve a better therapeutic effect and provide a rationale Moreno is also a member of the Executive Committee of for further development of 4SC-205 in clinical trials, we the European neuroblastoma research cooperative group combined 4SC-205 with chemotherapies, such as platine (SIOPEN) which receives royalties for the sales of dinu- derivatives (cisplatin), doxorubicin, and topotecan, which tuximab beta. Rolf Krauss (RK) is an employee of 4SC. are currently used as standard treatment for patients with Alberto Villanueva (AV) is co-founder of Xenopat S.L. No high-risk neuroblastoma. In all cases, the combination of potential conflict of interest was disclosed by the rest of the 4SC-205 with the chemotherapies showed additive effects authors. (Figures 4A–D and S10A-C; Table S6). Pediatric precision medicine programs have discovered a small number of AU T H O R CO N T R I B U T I O N S recurrent alterations such as ALK activating mutations or Miguel F. Segura and Anna Santamaria conceived and hyperactivation of the ERK Pathway,9,10 which constitute designed the study. Marc Masanas, Nuria Masiá, Leti- the basis for the development of targeted therapies against cia Suárez-Cabrera, Mireia Olivan, Aroa Soriano, Blanca high-risk neuroblastoma tumors. Thus, we combined 4SC- Majem, Carlos Jimenez, Ariadna Boloix, Ignasi Toloedano, 205 with two ALK inhibitors (ceritinib and lorlatinib) or Gabriela Guillén, Alexandra Navarro, and Alberto Vil- with the MEK1/2 inhibitor selumetinib. The combination lanueva carried out the experiments. Marc Masanas, Laura of 4SC-205 with ALK or MEK inhibitors showed a ∼2– Devis-Jauregui, Rebeca Burgos-Panadero, Pau Rodriguez- 3-fold reduction in cell proliferation compared with the Sodupe, and Aroa soriano analyzed the data. David crystal violet staining images of the ALK-mutated SH-SY5Y cell line treated with the ALK inhibitors ceritinib (62.5 nM) or lorlatinib (50 nM) with or without 4SC-205 (15 nM). (F) Heatmaps show the percentage of the cellular fraction affected by the drug combination treatments at the indicated doses. The graph represents the average of three independent experiments (n = 3/condition) ± SEM at 62.5 nM and 50 nM of ceritinib or lorlatinib, respectively. (G) Combinatorial analysis of 4SC-205 and ceritinib/lorlatinib in SH-SY5Y and KELLY cells. (H) Crystal violet staining images of SK-N-BE(2) cells treated with selumetinib (250 nM) and 4SC-205 (20 nM) and their respective combinations. (I) Heatmap showing the fraction of cells affected after the combination of 4SC-205 and selumetinib at the indicated concentrations for 48 h. Graph represents the average percentage of cell proliferation (n = 3/condition) ± SEM at 250 nM of selumetinib. (J) Combinatorial analysis of 4SC-205 and selumetinib in SK-N-BE(2) and SK-N-AS. *p < 0.05, **p < 0.01, ***p < 0.001, two tailed Student’s t-test LETTER TO EDITOR 7 of 8 Llobet-Navas, Josep Sánchez de Toledo, Josep Roma, Rosa 4 Molecular Mechanisms and Experimental Therapy in Noguera, Lucas Moreno, and Soledad Gallego provided Oncology-Oncobell Program, Bellvitge Biomedical Research intellectual support for result interpretation and critical Institute (IDIBELL), L’Hospitalet de Llobregat, Spain revision. Rolf Krauss provided the compound used in this 5 Group of Translational Research in Pediatric Solid study. Marc Masanas, Miguel F. Segura, and Anna Santa- Tumors Department of Pathology, Medical School, maria wrote the initial manuscript. All the authors read University of Valencia-INCLIVA Biomedical Health and approved the final manuscript. Research Institute, Valencia, Spain 6 Low Prevalence Tumors. Centro de Investigación D A T A AVA I L A B I L I T Y S T A T E M E N T Biomédica en Red de Cáncer (CIBERONC), Instituto de The authors confirm that all data supporting the findings Salud Carlos III, Madrid, Spain of this study are available within the article and the corre- 7 Quantitative Genomic Medicine Laboratory, qGenomics, sponding web servers. Further information from WES data Barcelona, Spain analyses is available from the corresponding authors upon 8 Department of Surgery, Universitat Autònoma de reasonable request. Barcelona (UAB), Barcelona, Spain 9 Department of Pathology, Vall d’Hebron University Marc Masanas1 Hospital, Universitat Autònoma de Barcelona, Barcelona, Nuria Masiá2 Spain Leticia Suárez-Cabrera2 10 Group of Chemoresistance and Predictive Factors, Mireia Olivan2,3 Subprogram Against Cancer Therapeutic Resistance Aroa Soriano1 (ProCURE), ICO, Oncobell Program, IDIBELL, Blanca Majem2 L’Hospitalet del Llobregat, Barcelona, Spain Laura Devis-Jauregui4 11 Xenopat S.L., Business Bioincubator, Bellvitge Health Rebeca Burgos-Panadero5,6 Science Campus, Barcelona, Spain Carlos Jiménez1 12 Catalan Institute of Oncology (ICO), Barcelona, Spain Pau Rodriguez-Sodupe7 13 Pediatric Oncology and Hematology Department, Ariadna Boloix1 Hospital Universitari Vall d’Hebron - Universitat Ignasi Toledano2 Autònoma de Barcelona (UAB), Barcelona, Spain Gabriela Guillén1,8 14 4SC, Martinsried, Germany Alexandra Navarro9 David Llobet-Navas4,6 Correspondence Alberto Villanueva10,11 Miguel F. Segura, Group of Translational Research in Josep Sánchez de Toledo1,12 Child and Adolescent Cancer, Passeig Vall d’Hebron Josep Roma1 119–129, Collserola Building. Lab 207, 08035 Barcelona, Rosa Noguera5,6 Spain. Lucas Moreno1,13 Email: miguel.segura@vhir.org Rolf Krauss14 Anna Santamaria, Cell Cycle and Cancer Laboratory, Soledad Gallego1,13 Biomedical Research Group in Urology, Passeig Vall Anna Santamaria2 d’Hebron 119–129, Collserola Building. Lab 210, 08035 Miguel F. Segura1 Barcelona, Spain. Email: anna.santamaria@vhir.org 1 Group of Translational Research in Child and Adolescent Cancer, Vall d’Hebron Research Institute (VHIR) - Funding information Universitat Autònoma de Barcelona (UAB), Barcelona, The financial support for this research was provided by Spain Instituto de Salud Calos III (PI20/00530 to Miguel F. 2 Cell Cycle and Cancer Laboratory, Biomedical Research Segura; PI20/01107 to Rosa Noguera; PI17/02248 and Group in Urology, Vall d’Hebron Research Institute (VHIR) CPII18/00027 to Anna Santamaria; PI19/01320 to Alberto - Universitat Autònoma de Barcelona (UAB), Barcelona, Villanueva); Ministerio de Educación, Cultura y Deporte Spain (Grant no. FPU16/01099 to Marc Masanas). This work 3 Translational Oncology Laboratory, Anatomy Unit, was also supported by the Asociación NEN (Nico contra Department of Pathology and Experimental Therapy, el cancer infantil 2017–PVR00157). School of Medicine, Universitat de Barcelona (UB), L’Hospitalet de Llobregat, Spain 8 of 8 LETTER TO EDITOR ORCID amethasone in relapsed/refractory multiple myeloma. Cancer. Miguel F. Segura https://orcid.org/0000-0003-0916-3618 2017;123(23):4617–4630. 7. Garcia-Saez I, Skoufias DA. Eg5 targeting agents: from new anti- mitotic based inhibitor discovery to cancer therapy and resis- REFERENCES tance. Biochem Pharmacol. 2021;184:114364. 1. Matthay KK, Maris JM, Schleiermacher G, et al. Neuroblastoma. 8. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Nat Rev Dis Primers. 2016;2:16078. Lancet North Am Ed. 2007;369(9579):2106–2120. 2. Stafman LL, Beierle EA. Cell proliferation in neuroblastoma. 9. George RE, Sanda T, Hanna M, et al. Activating mutations in Cancers. 2016;8:13. ALK provide a therapeutic target in neuroblastoma. Nature. 3. London WB, Shimada H, d’Amore E, et al. Age, tumor grade, 2008;455(7215):975–978. and mitosis-karyorrhexis index (MKI) are independently pre- 10. 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