Additional file 1 of Metastatic clear-cell renal cell carcinoma: a frequent NOTCH1 mutation predictive of response to anti-NOTCH1 CB-103 treatment
Creators: name: Bui, Thi Oanh
name: Angeli, Eurydice
name: El Bouchtaoui, Morad
name: Gapihan, Guillaume
name: Dao, Van Tu
name: Paris, Justine
name: Leboeuf, Christophe
name: Soussan, Michael
name: Villarese, Patrick
name: Ziol, Marianne
name: Van Glabeke, Emmanuel
name: Le, Thi Huong
name: Feugeas, Jean-Paul
name: Janin, Anne
name: Bousquet, Guilhem
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Mathematical Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Virology

description: Additional file 1: Fig S1. Copy number gains and losses in 4 metastatic RCC samples using Oncoscan® (A) compared with 433 metastatic RCC samples from our meta-analysis on genomic data of clear-cell RCC (B). Some of the abnormalities were not previously described, including 9q11.2 and 15q11.1–11.2 amplifications (red arrows on panel A). Fig S2. NOTCH1 protein structure and signaling pathway. (A) NOTCH1 protein structure, the mature NOTCH1 receptor is a heterodimer composed of an extracellular subunit (NOTCH1-EC) and a transmembrane and intracellular subunit (NOTCH1-TMIC). NOTCH1-EC includes epidermal growth factor (EGF)-like repeats, involved in ligand binding, three LIN-12/NOTCH repeats (LNR), which prevent receptor activation in the absence of ligands, and the heterodimerization domain (HD) involved in non-covalent interactions between NOTCH1-EC and NOTCH1-TMIC. NOTCH1-TMIC comprises the transmembrane domain (TM) and the intracellular domain (ICD) (NOTCH1-ICD). NOTCH1-ICD comprises an RBPJ-associated molecule (RAM) domain, seven ankyrin (ANK) repeats, nuclear localization signals (NLS), a transactivation domain (TAD), and a PEST domain, in turn involved in proteasomal degradation of active NOTCH1-ICD. Most NOTCH1 mutations are located in the HD and PEST domains (red arrows), and the pL1575P_c4724T_C NOTCH1 mutation (highlighted in yellow) is located in the HDN domain. (B) Newly synthesized NOTCH1 precursor is cleaved by a furin-like convertase (Furin) in the Golgi apparatus to generate the mature receptor. NOTCH1 signaling occurs when a JAGGED or DELTA ligand expressed on a signal-sending cell interacts with NOTCH1 on a signalreceiving cell. This interaction triggers two sequential cleavages of NOTCH1: the first, by way of an a disintegrin and metalloproteinase (ADAM) metalloproteinase, generates the substrate for the second cleavage by γ-secretase, which releases the active NOTCH1-ICD. NOTCH1-ICD translocates to the nucleus where it forms a transcriptional activation complex by interacting with the transcription factor CSL/RBP-Jk, mastermind-like proteins, and other coactivators (CoA), leading to the expression of NOTCH1 target genes. In physiological conditions, NOTCH1 expression is controlled by ubiquitination and proteasomal degradation of NOTCH1-ICD. Fig S3. ddPCR allelic discrimination for the pL1575P_c4724T_C NOTCH1 mutation in 5 XRCC tumor xenografts at the first (in gray) and the fifth (in black) passages. Fig S4. Response to sunitinib treatment in first-line setting for two patients who provided metastatic samples for xenograft in murine models. (A) Patient corresponding to the XRCC4 model. (B) Patient corresponding to the XRCC5 model. Fig S5. In vivo anti-tumor effect of CB-103 in the XRCC5 xenograft model. (A) CB-103 monotherapy, sunitinib monotherapy, or the combination of CB-103 and sunitinib significantly inhibit tumor growth after 30 days of treatment (n = 6 per treatment group). This is associated with a significant gradual increase in the percentage of necrotic areas (B), and significant decrease in microvessel density (C). For cell proliferation, the decrease is only significant with the combination of CB-103 and sunitinib compared to untreated mice (C). After 30 days of treatment with CB-103 monotherapy, the pL1575P_c4724T_C NOTCH1 mutation (D) has virtually disappeared. (*P &lt; 0.05, ***P &lt; 0.001). Fig S6. Oncoscan® technology and the Molecular Inversion Probe: Target Generation and Hybridization Procedures. a) Annealing: Probe and gDNA hybridization; b) Gap filling with A/T or G/C nucleotides; c) Exonuclease selection for gap filled probes; d) Cleavage at site 1 for probe opening and inversion; e) Probe amplification and biotinylation; f) Cleavage at site 2 to release the tag sequence; f) Array hybridization followed by staining with phycoerythrin through the biotin-streptavidin interaction; g) Array scanning. Blue and gray colors indicate presence and absence of the phycoerythrin fluorescence signal respectively. Fig S7. Tumor growth inhibition coefficient in XRCC models. For a drug or a drug combination, the coefficient of inhibition is calculated as (a’-a)/a, a being the slope of the curve before the start of treatment (Day 0), and a’ the slope of the curve between Day 0 and Day 30 of treatment. If this growth inhibition coefficient is found to be less than 0, the tumor is considered sensitive to the drug administered; if it is above 0, the tumor is considered resistant to the drug. Table S1. Characteristics of the four patients included in the initial study using Oncoscan® technology. Table S2. List of genes located in locus 9p11.2, 15q11.1, and 15q11.2. Table S3. Point mutation identified on RCC metastases before treatment. Table S4. NOTCH1 targeted therapeutics. Table S5. Growth inhibition coefficient for drugs tested in XRCC model. Table S6. Characteristics of the five patient-derived xenograft models.