Novel Somatic Genetic Variants as Predictors of Resistance to EGFR-Targeted Therapies in Metastatic Colorectal Cancer Patients

Background: About 40% of RAS/BRAF wild-type metastatic colorectal cancer (mCRC) patients undergoing anti-EGFR-based therapy have poor outcomes. Treatment failure is not only associated with poorer prognosis but higher healthcare costs. Our aim was to identify novel somatic genetic variants in the primary tumor and assess their effect on anti-EGFR response. Patients and Methods: Tumor (somatic) and blood (germline) DNA samples were obtained from two well-defined cohorts of mCRC patients, those sensitive and those resistant to EGFR blockade. Genetic variant screening of 43 EGFR-related genes was performed using targeted next-generation sequencing (NGS). Relevant clinical data were collected through chart review to assess genetic results. Results: Among 61 patients, 38 were sensitive and 23 were resistant to treatment. We identified eight somatic variants that predicted non-response. Three were located in insulin-related genes (I668N and E1218K in IGF1R, T1156M in IRS2) and three in genes belonging to the LRIG family (T152T in LRIG1, S697L in LRIG2 and V812M in LRIG3). The remaining two variants were found in NRAS (G115Efs*46) and PDGFRA (T301T). We did not identify any somatic variants related to good response. Conclusions: This study provides evidence that novel somatic genetic variants along the EGFR-triggered pathway could modulate the response to anti-EGFR drugs in mCRC patients. It also highlights the influence of insulin-related genes and LRIG genes on anti-EGFR efficacy. Our findings could help characterize patients who are resistant to anti-EGFR blockade despite harboring RAS/BRAF wild-type tumors

Discovery of First-In-Class inhibitors of a DNA damage repair pathway

[eng] Cancer is the second leading cause of death globally. The gold standard for cancer has historically been surgery, radiotherapy and chemotherapy. Although the efficacy of chemotherapy has been broadly confirmed, drug resistance is still one of the main causes of disease relapse and treatment failure. For this reason, new therapeutic strategies directed at targeting the key factors that transform normal cells and tissues into malignancies have been developed. Nevertheless, eventual resistance to targeted and immunological therapies has also emerged, leading to the necessity of additional targeted therapeutic approaches. Recent advances in genome sequencing have revealed vulnerabilities within cancer cells that may be exploited to selectively target the tumour with therapeutics. As one of the mechanisms that cancer cells use to develop chemo-resistance is through the acquisition of a better DNA damage capacity, several targeted disruptors of DNA damage response (DDR) pathways have been developed. This has been shown to re-sensitize cancer cells to chemotherapy, being PARP inhibitors the most well-known case. After detecting that the restoration of a specific DDR pathway is able to abolish sensitivity and lead to chemo- resistance, we decided to screen for inhibitors of this pathway by means of molecular docking and posterior cellular validation. Thanks to the availability of atomic-resolution structures of some of the proteins, we looked for compounds that disrupted a critical PPI within the pathway that resulted in a post-translational modification. The capacity of these compounds to inhibit the post-translational modification was validated in cell models and their ability to interact with one of the proteins intervening in the PPI was also tested by surface plasmon resonance. The best candidates (F27 and F2.14) were then virtually optimized in two successive rounds and also validated. F27 was able to chemo-sensitize cancer cells to cisplatin in clonogenic assays. F27 and F2.14 also showed greater cytotoxicity in cells mutated for a specific gene, what suggests that a future drug inhibitor of this pathway could be applied to specifically treat tumours with this mutation using the synthetic lethality concept. In parallel, we studied potential applications of our candidates. One of these applications was in terms of synthetic lethality. Many synthetic lethal interactions with our target DDR pathway have been reported. Initially, we validated several of them in our laboratory. Then, in order to identify new potential synthetic lethal genes, we analysed gene co-expression correlations included in cBioPortal between more than 22,000 genes and several upstream genes from this DDR pathway in different cancer types. We identified several genes that were commonly found co-expressed. Some of these synthetic lethal interactions had already been reported, and the rest represented potential new synthetic lethal genes. Furthermore, in order to estimate the potential market size of an inhibitor of this pathway we studied the annual cases of patients with solid tumours and haematologic malignancies with alterations on these synthetic lethal genes in Europe and North America. Another application that we tested was the potential synergy of our inhibitors and immunotherapy. We hypothesized that the dysregulation in several genes from this pathway that had been found altered in cancer may result in high expression of immune checkpoint ligands. In order to find correlations between inhibitory immune checkpoint ligands and these genes, we initially analysed their co-expression in different cancer types and we found several of them co-expressed with genes from this pathway. Afterwards, we validated these results using cell lines deficient for our target pathway, but we could only observe a small increase in the expression of two of these immune checkpoint ligands. In parallel, we found that F2.14 increased the expression of two immune checkpoint ligands in U2OS and HeLa cells.[cat] El càncer és la segona causa de mort a nivell mundial. El tractament clàssic pel càncer ha sigut la cirurgia, radioteràpia i quimioteràpia. Tot i que l’eficàcia de la quimioteràpia està àmpliament confirmada, l’aparició de resistència al tractament és una de les causes principals de recurrència i fallada terapèutica. Per aquest motiu, s’han desenvolupat noves estratègies de tractament dirigides a factors clau que transformen cèl·lules normals i teixits en tumorals. No obstant això, la resistència a aquest tipus de teràpies dirigides i a la immunoteràpia també ha aparegut, la qual cosa requereix el desenvolupament d’aproximacions terapèutiques addicionals. Avenços recents en seqüenciació genòmica han revelat certes vulnerabilitats en cèl·lules tumorals que podrien ser aprofitades per a tractar selectivament el tumor. Com que un dels mecanismes que utilitzen aquestes cèl·lules per desenvolupar quimioresistència és adquirint una millor capacitat de reparar el dany en el DNA, s’han desenvolupat diversos disruptors de les rutes de resposta al dany del DNA. Això ha permès induir de nou la sensibilitat de les cèl·lules tumorals a la quimioteràpia, essent els inhibidors de la PARP els més coneguts. Després de detectar que el restabliment d’una ruta específica de resposta al dany en el DNA permet abolir la sensibilitat i induir a quimioresistència, vam decidir realitzar un cribratge d’inhibidors d’aquesta ruta per mitjà del docking molecular i la seva validació posterior en cèl·lules. Gràcies a la disponibilitat de la resolució atòmica d’estructures de proteïnes, vam cercar compostos que impedissin una interacció proteïna-proteïna crítica per la ruta que resulta en una modificació post- traduccional. La capacitat d’aquests compostos per inhibir aquesta modificació post- traduccional es va validar en models cel·lulars i la seva capacitat per interaccionar amb una de les proteïnes que intervé en aquesta interacció també es va testar per ressonància de plasmó de superfície. Els millors candidats (F27 i F2.14) van ser llavors optimitzats virtualment en dues rondes successives i van ser també validats. L’F27 era capaç de quimiosensibilitzar cèl·lules tumorals al cisplatí en assajos clonogènics. Els compostos F27 i F2.14 també van mostrar major citotoxicitat en cèl·lules mutades per un gen específic, la qual cosa suggereix que un futur fàrmac inhibidor d’aquesta ruta es podria aplicar per tractar específicament tumors amb aquesta mutació utilitzant el concepte de letalitat sintètica. En paral·lel, vam estudiar aplicacions potencials dels nostres candidats. Una d’aquestes aplicacions era en termes de letalitat sintètica. Vàries interaccions letal sintètiques amb la nostra ruta diana han estat publicades. Inicialment, vam validar alguna d’elles en el nostre laboratori. Posteriorment, per tal d’identificar potencials nous gens letal sintètics, vam analitzar correlacions de gens co-expressats incloses en la base de dades del cBioPortal, entre més de 22.000 gens i varis gens upstream de la nostra ruta diana en diferents tipus tumorals. Vam identificar varis gens que es trobaven freqüentment co-expressats. Alguns d’aquests ja s’havien publicat, i d’altres, representaven potencials nou gens letal sintètics. Addicionalment, per tal d’estimar la mida potencial de mercat d’un inhibidor d’aquesta ruta, vam estudiar els casos anuals de pacients amb tumors sòlids i càncers hematològics amb alteracions en aquests gens de letalitat sintètica a Europa i Amèrica del Nord. Una altra aplicació que vam analitzar va ser la sinèrgia potencial dels nostres inhibidors i la immunoteràpia. Vam establir com a hipòtesi que la desregulació de certs gens d’aquesta ruta que s’han trobat alterats en càncer pot resultar en una elevada expressió de lligands inhibidors de punts de control immunitaris. Per tal de trobar correlacions entre aquests lligands i els gens de la ruta diana, vam analitzar primer la seva co-expressió en diferents tipus de càncer i en vam trobar varis de co-expressats. Tot seguit, vam validar aquests resultats emprant línies cel·lulars deficients per la nostra ruta diana, però només vam poder detectar un petit increment en l’expressió de dos d’aquests inhibidors de punts de control immunitaris. En paral·lel, vam observar que l’F2.14 incrementava l’expressió de dos inhibidors de punts de control immunitari en cèl·lules U2OS i HeLa

High content drug screening for Fanconi anemia therapeutics

Fanconi anemia is a rare disease clinically characterized by malformations, bone marrow failure and an increased risk of solid tumors and hematologic malignancies. The only therapies available are hematopoietic stem cell transplantation for bone marrow failure or leukemia, and surgical resection for solid tumors. Therefore, there is still an urgent need for new therapeutic options. With this aim, we developed a novel high-content cell-based screening assay to identify drugs with therapeutic potential in FA. A TALEN-mediated FANCA-deficient U2OS cell line was stably transfected with YFP-FANCD2 fusion protein. These cells were unable to form fluorescent foci or to monoubiquitinate endogenous or exogenous FANCD2 upon DNA damage and were more sensitive to mitomycin C when compared to the parental wild type counterpart. FANCA correction by retroviral infection restored the cell line's ability to form FANCD2 foci and ubiquitinate FANCD2. The feasibility of this cell-based system was interrogated in a high content screening of 3802 compounds, including a Prestwick library of 1200 FDA-approved drugs. The potential hits identified were then individually tested for their ability to rescue FANCD2 foci and monoubiquitination, and chromosomal stability in the absence of FANCA. While, unfortunately, none of the compounds tested were able to restore cellular FANCA-deficiency, our study shows the potential capacity to screen large compound libraries in the context of Fanconi anemia therapeutics in an optimized and cost-effective platform

CDK5RAP3, a New BRCA2 Partner That Regulates DNA Repair, Is Associated with Breast Cancer Survival

BRCA2 is essential for homologous recombination DNA repair. BRCA2 mutations lead to genome instability and increased risk of breast and ovarian cancer. Similarly, mutations in BRCA2-interacting proteins are also known to modulate sensitivity to DNA damage agents and are established cancer risk factors. Here we identify the tumor suppressor CDK5RAP3 as a novel BRCA2 helical domain-interacting protein. CDK5RAP3 depletion induced DNA damage resistance, homologous recombination and single-strand annealing upregulation, and reduced spontaneous and DNA damage-induced genomic instability, suggesting that CDK5RAP3 negatively regulates double-strand break repair in the S-phase. Consistent with this cellular phenotype, analysis of transcriptomic data revealed an association between low CDK5RAP3 tumor expression and poor survival of breast cancer patients. Finally, we identified common genetic variations in the CDK5RAP3 locus as potentially associated with breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. Our results uncover CDK5RAP3 as a critical player in DNA repair and breast cancer outcomes