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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

The use of elastomeric particles obtained from old tires to produce anti-vibration mats to reduce vibrations in railways is studied. In the first part of this work a numerical simulation for dimensioning the element has been performed. The behavior for a wavy surface was deducted obtaining good approximation to the empirical results. In the second part the methodology used to assess the Insertion Loss, IL, provided by the anti-vibration system is described. The analytical superstructure/ground model developed within the frame of the project is presented. The model is based on an elementary superstructure model with no elastomeric material and is conveniently modified to introduce an under ballast mat. Finally the study shows the validation of the mechanical behavior of prototype mats under dynamic efforts according to standards [1-6]. The results show that the behavior of crushed rubber is suitable to be used as anti-vibration systems

Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca(-/-) mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect of Fanca(-/-) progenitors, either when total BM or when purified Lin(-)Sca-1(+) cells were subjected to in vitro stimulation. Liquid cultures of Fanca(-/-) BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca(-/-) progenitors, purified Lin(-)Sca-1(+) cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCA genes. Lin(-)Sca-1(+) cells from Fanca(-/-) mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors of Fanca(-/-) mice.