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Figure S7. Additional data on gefitinib and afatinib toxicity analysis in WT and Fanca-deficient mice.

Purpose:Fanconi anemia rare disease is characterized by bone marrow failure and a high predisposition to solid tumors, especially head and neck squamous cell carcinoma (HNSCC). Patients with Fanconi anemia with HNSCC are not eligible for conventional therapies due to high toxicity in healthy cells, predominantly hematotoxicity, and the only treatment currently available is surgical resection. In this work, we searched and validated two already approved drugs as new potential therapies for HNSCC in patients with Fanconi anemia.Experimental Design:We conducted a high-content screening of 3,802 drugs in a FANCA-deficient tumor cell line to identify nongenotoxic drugs with cytotoxic/cytostatic activity. The best candidates were further studied in vitro and in vivo for efficacy and safety.Results:Several FDA/European Medicines Agency (EMA)-approved anticancer drugs showed cancer-specific lethality or cell growth inhibition in Fanconi anemia HNSCC cell lines. The two best candidates, gefitinib and afatinib, EGFR inhibitors approved for non–small cell lung cancer (NSCLC), displayed nontumor/tumor IC50 ratios of approximately 400 and approximately 100 times, respectively. Neither gefitinib nor afatinib activated the Fanconi anemia signaling pathway or induced chromosomal fragility in Fanconi anemia cell lines. Importantly, both drugs inhibited tumor growth in xenograft experiments in immunodeficient mice using two Fanconi anemia patient–derived HNSCCs. Finally, in vivo toxicity studies in Fanca-deficient mice showed that administration of gefitinib or afatinib was well-tolerated, displayed manageable side effects, no toxicity to bone marrow progenitors, and did not alter any hematologic parameters.Conclusions:Our data present a complete preclinical analysis and promising therapeutic line of the first FDA/EMA-approved anticancer drugs exerting cancer-specific toxicity for HNSCC in patients with Fanconi anemia.

Figure S5. FACS gating strategy to analyze white blood cell population in WT and Fanca-deficient mice.

Figure S8. Additional data on genotoxicity in blood samples from WT or Fanca-deficient mice, by gefitinib and afatinib treatment.

Figure S2. Additional data on gefitinib and afatinib in vivo mouse xenograft experiments. Weight control.

Figure S3. Additional immunohistochemistry images from tumor xenografts.

Figure S6. Additional data on gefitinib and afatinib toxicity analysis in WT and Fanca-deficient mice.

Figure S1. Additional data from high content screening and validation

Supplementary Text

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10β. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases - trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10β protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10β is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10β undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10β, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.

Purpose: Fanconi anemia rare disease is characterized by bone marrow failure and a high predisposition to solid tumors, especially head and neck squamous cell carcinoma (HNSCC). Patients with Fanconi anemia with HNSCC are not eligible for conventional therapies due to high toxicity in healthy cells, predominantly hematotoxicity, and the only treatment currently available is surgical resection. In this work, we searched and validated two already approved drugs as new potential therapies for HNSCC in patients with Fanconi anemia. Experimental Design: We conducted a high-content screening of 3,802 drugs in a FANCA-deficient tumor cell line to identify nongenotoxic drugs with cytotoxic/cytostatic activity. The best candidates were further studied in vitro and in vivo for efficacy and safety. Results: Several FDA/European Medicines Agency (EMA)-approved anticancer drugs showed cancer-specific lethality or cell growth inhibition in Fanconi anemia HNSCC cell lines. The two best candidates, gefitinib and afatinib, EGFR inhibitors approved for non–small cell lung cancer (NSCLC), displayed nontumor/tumor IC50 ratios of approximately 400 and approximately 100 times, respectively. Neither gefitinib nor afatinib activated the Fanconi anemia signaling pathway or induced chromosomal fragility in Fanconi anemia cell lines. Importantly, both drugs inhibited tumor growth in xenograft experiments in immunodeficient mice using two Fanconi anemia patient–derived HNSCCs. Finally, in vivo toxicity studies in Fanca-deficient mice showed that administration of gefitinib or afatinib was well-tolerated, displayed manageable side effects, no toxicity to bone marrow progenitors, and did not alter any hematologic parameters. Conclusions: Our data present a complete preclinical analysis and promising therapeutic line of the first FDA/EMA-approved anticancer drugs exerting cancer-specific toxicity for HNSCC in patients with Fanconi anemia.

A 120 days feeding trial was conducted to evalúate diets in which fish meal (FM) was replaced with meat and bone meal (MBM) or tuna byproduct meal (TBM) on growth performance, apparent digestibility and hematological parameters of juvenile spotted rose snapper (SRS) L. guttatus. Three isonitrogenous compounds (47.6-49.0%) and isoenergetic (20.9-22.9 kJ g-¹) diets were formulated. A control diet contained FM as a main protein source (D-FM) and two diets with 35% of fish meal protein replaced by MBM or TBM protein (D-MBM, D-TBM). Each diet was fed to triplícate groups of 20 SrS juvenile (initial weight 8.2 ± 0.02 g) to apparent satiation three times a day. Growth performance, hematological parameters and apparent digestibility of SRS fed D-MBM or D-TBM diets were not significantly different from D-FM diet. However, the whole body crude protein was significantly higher in D-MBM group than D-TBM group, and the values were comparable to D-FM group. Based on these results, the meat and bone meal is an economical and viable option, as tuna byproduct meal in practical diets for juvenile spotted rose snapper.