Preclinical spheroid models identify BMX as a therapeutic target for metastatic MYCN nonamplified neuroblastoma
Santhoshkumar Sundaramoorthy, Daniele Filippo Colombo, Rajendran Sanalkumar, Liliane Broye, Katia Balmas Bourloud, Gaylor Boulay, Luisa Cironi, Ivan Stamenkovic, Raffaele Renella, Fabien Kuttler, Gerardo Turcatti, Miguel N. Rivera, Annick Mühlethaler-Mottet, Anaïs Flore Bardet, Nicolò Riggi
Santhoshkumar Sundaramoorthy, Daniele Filippo Colombo, Rajendran Sanalkumar, Liliane Broye, Katia Balmas Bourloud, Gaylor Boulay, Luisa Cironi, Ivan Stamenkovic, Raffaele Renella, Fabien Kuttler, Gerardo Turcatti, Miguel N. Rivera, Annick Mühlethaler-Mottet, Anaïs Flore Bardet, Nicolò Riggi
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Research Article Therapeutics

Preclinical spheroid models identify BMX as a therapeutic target for metastatic MYCN nonamplified neuroblastoma

Abstract

The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest–derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.

Authors

Santhoshkumar Sundaramoorthy, Daniele Filippo Colombo, Rajendran Sanalkumar, Liliane Broye, Katia Balmas Bourloud, Gaylor Boulay, Luisa Cironi, Ivan Stamenkovic, Raffaele Renella, Fabien Kuttler, Gerardo Turcatti, Miguel N. Rivera, Annick Mühlethaler-Mottet, Anaïs Flore Bardet, Nicolò Riggi

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Figure 5

nMNA NB cell lines and primary spheroids are highly sensitive to BMX pharmacological inhibition.

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nMNA NB cell lines and primary spheroids are highly sensitive to BMX pha...
(A) Box plots depicting the sensitivity toward the BMX inhibitor QL-XII-47 for cancer cell lines across 31 different tumor types. Data were obtained from the Genomics of Drug Sensitivity in Cancer (GDSC) screening program and are presented as IC50 (μM). (B) Violin plots illustrating the differential sensitivity of NB cell lines toward the BMX inhibitor QL-XII-47 based on their MYCN amplification status. Data were obtained and presented as in A. (C) Box plots showing BMX mRNA expression levels for a panel of NB cell lines segregated based on their MYCN amplification status. The expression values of BMX were extracted from the Jagannathan data set available at R2 genomics platform. (D) Bar plots showing the viability of NB1 and NB4 spheroids treated with vehicle (DMSO) or BMX-IN-1 at 5 μM and 10 μM for 4 days. (E) Time-dependent analysis of spheroid viability as in D. (F) Representative images of NB1 (upper rows) and NB4 (lower rows) spheroids treated with DMSO as control, Gambogic acid (1 μM), and BMX-IN-1 (10 μM) for 7 days. Live cells were stained in green using Calcein AM, and dead cells in red using Ethidium homodimer-1. Objective, 4×/0.2. The entire well area was captured with 4 fields of view, and the representative images shown are cropped on the location of spheres in the wells. Scale bar: 1,000 μm. (G) Box plots showing the quantification of the assay shown in F, using normalized mean intensity of mean Ethidium homodimer-1 staining of spheroids treated with BMX-IN-1 (10 μM), DMSO, and Gambogic acid (1 μM). (H) Graph depicting the changes in sphere forming ability of NB1 and NB4 single cells sorted by FACS treated with 10 μM of BMX-IN-1 or vehicle (DMSO). (D–F, and H) Mean ± SEM values of 3 technical replicates are shown. Statistical analyses are performed using the unpaired t test (E, F, and H) and 1-way ANOVA followed by post hoc pairwise comparisons (D and G) to compute adjusted P values. **P < 0.001, ***P < 0.0001.