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Structural and functional characterization of 4-1BBN/CEGFR after conjugation with p-SCN-Bn-deferoxamine (Df).

Oligonucleotides used in this study

Purpose:The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity.Experimental Design:Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo.Results:In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer.Conclusions:These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.

PDF file - 72K, RANK overexpression induces mammary stem markers

PDF file - 172K, RANK overexpression promotes growth, invasive changes and metastasis in cells with non-functional BRCA1

PDF file - 114K, FL-RANK cells grow in the absence of EGF and do not form tumors in vivo.

PDF file - 91K, RANK is expressed and active in MCF10A cells

PDF file - 89K, RANK/RANKL expression significantly associates with tumor subtype and lymph node metastasis

PDF file - 113K, RANK expression induces EMT in MCF10A and in HMECS immortalized with telomerase

PDF file - 167K

Background: Individuals with a phenotype of early-onset severe obesity associated with intellectual disability can have molecular diagnoses ranging from monogenic to complex genetic traits. Severe overweight is the major sign of a syndromic physical appearance and predicting the influence of a single gene and/or polygenic risk profile is extremely complicated among the majority of the cases. At present, considering rare monogenic bases as the principal etiology for the majority of obesity cases associated with intellectual disability is scientifically poor. The diversity of the molecular bases responsible for the two entities makes the appliance of the current routinely powerful genomics diagnostic tools essential. Objective: Clinical investigation of these difficult-to-diagnose patients requires pediatricians and neurologists to use optimized descriptions of signs and symptoms to improve genotype correlations. Methods: The use of modern integrated bioinformatics strategies which are conducted by experienced multidisciplinary clinical teams. Evaluation of the phenotype of the patient’s family is also of importance. Results: The next step involves discarding the monogenic canonical obesity syndromes and considering infrequent unique molecular cases, and/or then polygenic bases. Adequate management of the application of the new technique and its diagnostic phases is essential for achieving good cost/efficiency balances. Conclusion: With the current clinical management, it is necessary to consider the potential coincidence of risk mutations for obesity in patients with genetic alterations that induce intellectual disability. In this review, we describe an updated algorithm for the molecular characterization and diagnosis of patients with a syndromic obesity phenotype.

La localización extranodal gastrointestinal del linfoma de Hodgkin comprende el 5% de todos los linfomas. Dentro de este grupo, el linfoma anal primario representa menos del 0,05%, siendo por tanto una entidad extremadamente rara. Por otro lado, los tumores neuroendocrinos son un grupo heterogéneo de neoplasias relativamente poco frecuentes, pero de localización fundamentalmente digestiva. La asociación entre un linfoma de Hodgking de localización anal y un tumor neuroendocrino intestinal no ha sido descrita previamente en la literatura, pero no es en absoluto raro que los tumores neuroendocrinos puedan coexistir con otro tipo de neoplasias. Los autores presentan el caso infrecuente de presentación de linfoma Hodgkin de localización anal asociado a un tumor neuroendocrino intestinal en una paciente mujer de 74 años, describiéndose la clínica, resultados de pruebas complementarias y tratamiento recibido.

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. FGFR1 has been associated with tumorigenesis in a variety of tumor types, including lung cancer. As a therapeutic approach, their inhibition has been attempted and was initially focused on FGFR1-amplified tumors, though with limited success. Preliminary data of our group suggests that N-Cadherin play a key role for the oncogenicity of FGFR1 and predict FGFR-targeted therapy efficacy in Non-small cell lung cancer (NSCLC). However, it is possible that other biomarkers, together with N-Cadherin, can determine the response to anti-FGFR therapy. Therefore, it is essential the identification of new biomarkers that could help to predict more accurately those patients that could benefit from anti-FGFR therapy. Materials and Methods: We have treated 15 NSCLC PDX models with high FGFR1 expression levels and variable N-Cadherin expression levels, with the FGFR inhibitor (FGFRi) AZD4547. Based on their sensitivity to treatment, the PDX models were classified into two groups: Responders and Non-responders. All PDX models were characterized at the proteomic, genomic, and transcriptomic levels by western blot, whole exome sequencing, and RNA-Seq, respectively, and we used this information for defining the responder versus the non-responder group. Subsequently, the GDSC repository was used to validated our data in a cohort of lung adenocarcinoma (LUAD) cell lines. Results: Responder and non-responder groups revealed a distinct pattern of genomic alterations and gene expression profile. As expected, GSEA identified an enrichment in Responders group of pathways related to FGFR1 signaling and ECM proteins, among them N-Cadherin stands out. Moreover, we observed an enrichment of KRAS mutations and genes related to the KRAS signature and to the intrinsic resistance to FGFRi in the non-responder group. To validate our findings and using the GDSC repository, we observed an enriched resistance to inhibitors of FGFR1 signaling in a cohort of KRAS mutant LUAD cell lines. Furthermore, we confirm a tendency for increased sensitivity in high N-Cadherin expressing cell lines, but only in KRAS wildtype context. Conclusions: In conclusion, previous research had underlined N-Cadherin as a potential biomarker of response to FGFRi, while not successful in all contexts. Transcriptome study of PDXs classified as responder or non-responder to FGFRi revealed a differential gene expression pattern, with an upregulation of N-Cadherin pathway observed in the Responder group, while an enrichment of KRAS signaling and KRAS mutations in non-Responders. Furthermore, we validated our finding using a cell line repository that confirm that N-Cadherin could predict FGFR-targeted therapy efficacy but only in the KRAS wildtype context. Citation Format: Santiago G. Borrego, Cristina Cirauqui, David Gómez-Sánchez, Haiyun Wang, Alicia Luengo, Chiara Ambrogio, Luis Paz-Ares, Irene Ferrer. N-Cadherin acts as a predictive biomarker for anti-FGFR therapy in KRAS wild-type NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4489.

Drug-tolerant “dormant” cells (DTC) have emerged as one of the major non-genetic mechanisms driving resistance to targeted therapy in lung cancer, although the sequence of events leading to entry and exit from dormancy remain poorly described. Here, we performed real-time monitoring of the cell cycle dynamics during the adaptive response to Epidermal Growth Factor Receptor tyrosine kinase inhibitors (EGFR-TKi) in a panel of EGFR-mutated lung cancer cell lines. We identified a rare population of S/G2 cycling cells (referred to as early escapers) that emerged in the first hours of treatment amongst stably arrested and progressively dying G1 cells. We determined that early escapers evolved from a non-proliferative differentiated alveolar type 1 (AT1) phenotype which was invariably associated with cytoskeletal remodeling through Rho/ROCK pathway activation. Using a panel of Rho-pathway inhibitors, we found that the farnesyltransferase inhibitor tipifarnib induced a complete clearance of EGFR-TKi-induced DTC thus fully preventing relapse in vitro. Using a xenograft model and a PDX model of EGFRL858R/T790M lung cancer, co-treatment with tipifarnib prevented relapse to osimertinib for up to 6 months with no evidence of toxicity. Among the farnesylated proteins regulated during osimertinib treatment, concomitant inhibition of RHOE and LaminB1 was sufficient to recapitulated FTIs’ effect. Osimertinib and tipifarnib co-treatment completely suppressed the emergence of AT1 phenotype, prevented mitosis of S/G2-treated cells and increased the apoptotic response through activation of ATF4-CHOP-dependent Integrated Stress Response (ISR) pathway. Our data strongly support the use of tipifarnib in combination with osimertinib in patients to effectively and durably prevent relapse.

SCLC is an extremely aggressive subtype of lung cancer without approved targeted therapies. Here we identified YES1 as a novel targetable oncogene driving SCLC maintenance and metastasis.Association between YES1 levels and prognosis was evaluated in SCLC clinical samples. In vitro functional experiments for proliferation, apoptosis, cell cycle, and cytotoxicity were performed. Genetic and pharmacologic inhibition of YES1 was evaluated in vivo in cell- and patient-derived xenografts and metastasis. YES1 levels were evaluated in mouse and patient plasma-derived exosomes.Overexpression or gain/amplification of YES1 was identified in 31% and 26% of cases, respectively, across molecular subgroups, and was found as an independent predictor of poor prognosis. Genetic depletion of YES1 dramatically reduced cell proliferation, three-dimensional organoid formation, tumor growth, and distant metastasis, leading to extensive apoptosis and tumor regressions. Mechanistically, YES1-inhibited cells revealed alterations in the replisome and DNA repair processes, that conferred sensitivity to irradiation. Pharmacologic blockade with the novel YES1 inhibitor CH6953755 or dasatinib induced marked antitumor activity in organoid models and cell- and patient-derived xenografts. YES1 protein was detected in plasma exosomes from patients and mouse models, with levels matching those of tumors, suggesting that circulating YES1 could represent a biomarker for patient selection/monitoring.Our results provide evidence that YES1 is a new druggable oncogenic target and biomarker to advance the clinical management of a subpopulation of patients with SCLC.

Background Immunotherapy is a promising cancer treatment, but surrogate biomarkers of clinical efficacy have not been fully validated. The aim of this work was to evaluate several biomarkers as predictors of response to nivolumab monotherapy in patients with non–small-cell lung cancer. Patients and Methods Blood samples was collected at baseline, at 2 months after treatment start, and at disease progression. Lactate dehydrogenase level (LDH), neutrophils, and leukocyte values were obtained from medical record. Interleukin (IL)-8, IL-11, and kynurenine/tryptophan levels were determined by enzyme-linked immunosorbent assay. Total protein was extracted from circulating CD8+ T cells, and BCL-2 interacting mediator of cell death (BIM) protein expression tested by western blotting. Results Baseline LDH levels were significantly higher in non-responder patients than in those who responded (P = .045). The increase in indoleamine 2,3 dioxygenase activity was related to progression of disease, mainly in patients who did not respond to nivolumab treatment (P = .001). Increased levels of circulating IL-8 were observed in initially responding patients at time of progression, and it was related to lower overall survival (hazard ratio, 7.49; P = .025). A highest expression of BIM in circulating CD8+ T cells could be related to clinical benefit. The Student t test and Mann-Whitney U test were used to compare groups for continuous variables. Time to events was estimated using the Kaplan-Meier method, and compared by the log-rank test. Conclusions Changes in plasma LDH and IL-8, indoleamine 2,3 dioxygenase activity, and BIM expression in CD8+ T cells could be used to monitor and predict clinical benefit from nivolumab treatment in these patients.

Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent histology. While immunotherapy with checkpoint inhibitors has shown outstanding results in NSCLC, the precise identification of responders remains a major challenge. Most studies attempting to overcome this handicap have focused on adenocarcinomas or squamous cell carcinomas. Among NSCLC subtypes, the molecular and immune characteristics of lung large cell carcinoma (LCC), which represents 10% of NSCLC cases, are not well defined. We hypothesized that specific molecular aberrations may impact the immune microenvironment in LCC and, consequently, the response to immunotherapy. To that end, it is particularly relevant to thoroughly describe the molecular genotype–immunophenotype association in LCC–to identify robust predictive biomarkers and improve potential benefits from immunotherapy. We established a cohort of 18 early-stage, clinically annotated, LCC cases. Their molecular and immune features were comprehensively characterized by genomic and immune-targeted sequencing panels along with immunohistochemistry of immune cell populations. Unbiased clustering defined two novel subgroups of LCC. Pro-immunogenic tumors accumulated certain molecular alterations, showed higher immune infiltration and upregulated genes involved in potentiating immune responses when compared to pro-tumorigenic samples, which favored tumoral progression. This classification identified a set of biomarkers that could potentially predict response to immunotherapy. These results could improve patient selection and expand potential benefits from immunotherapy.

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.

El linfoma MALT es una forma poco frecuente de linfoma no Hodking de células B de bajo grado, que se desarrolla a expensas del tejido linfoide de las membranas mucosas. La localización más frecuente a nivel gastrointestinal es el estómago relacionando con la infección por Helicobacter pylori. El linfoma MALT colónico es una entidad extremadamente rara y cuya incidencia no supera el 5% de los casos en países asiáticos, con series porcentualmente menores al 2,5%, siendo la ubicación de este tipo de linfoma la más inusual de todo el tracto digestivo en comparación con otras neoplasias malignas que afectan al colon. Los autores exponen el caso infrecuente de presentación de linfoma tipo MALT de localización colónica en un paciente varón de 51 años, describiendo la clínica, los resultados de pruebas complementarias y el tratamiento recibido por el paciente.

Lung cancer is the leading cause of cancer-related deaths worldwide. KRAS is the most frequent mutated driver gene in NSCLC but today has no effective treatments. Therefore, there is an urgent unmet need to find a tailored treatment alternative in the clinic. We need experimental models which really predict the efficacy of these alternative treatments. PDX and organoids are models that recapitulate more accurately the complexity of human cancer, therefore they are the best models we can use to address such clinically relevant questions. We aim to generate and characterize both NSCLC PDX, and PDX-derived organoids (PDXDOs) collections to perform preclinical assays, address to find alternative therapies for KRAS-mutant lung cancer. Among these therapeutic strategies we evaluated the efficacy of CDK4 inhibitors (CDK4i) in combination with ERK1/2 inhibitors (ERK1/2i). We studied the mechanism of action of the drugs in combination and identified possible predictive response biomarkers by RNAseq. We have established and characterized a collection of 45 NSCLC PDX models derived from early-stage patients, eight of them with KRAS mutations. All retained the principal histologic and molecular characteristics of their donors during the passages. We have also generated a collection of 15 PDXDOs, which maintained the same characteristics of their PDX donors. We evaluate the efficacy of CDK4i in monotherapy and in combination with ERK1/2i in KRAS-mutated models. In vitro, using PDXDOs, the therapeutic combination tested reduced viability and downregulated signaling pathways involved in cell progression, this effect was observed in all PDXDO models tested, including those that were primary resistant to CDK4i in monotherapy. Similarly, in vivo, using the PDX models, the therapeutic combination tested produced a significant reduction in tumor growth compared to the drugs in monotherapy. The transcriptomic analysis showed CDK4i inhibited cell-cycle processes, whereas the combination therapy resulted in an increased inhibition and also reversed the CDK4i-mediated activation of PI3K or WNT pathways, which would explain the synergistic effect of the combination. In conclusion, we have established a collection of 45 NSCLC PDX models and 15 PDXDOs, which allow to evaluate (in vitro and in vivo) the therapeutic efficacy of different drug combinations targeting different driver genes mutated such KRAS, which today has no effective treatments. These preclinical models will be really useful to integrate drug screening with biomarker discovery. Using our collections, we have demonstrated the combination of CDK4i with ERK1/2i was more effective than the drugs in monotherapy, even for those cases which primary showed CDK4i-resistance. Therefore, we propose this therapeutic combination as a good therapeutic strategy to test in the clinic. Citation Format: Patricia Yagüe, David Gomez, Álvaro C Ucero, Laura García, Patricia Plaza, Nuria Carrizo, José Luis Solorzano, Luis Paz-Ares, Irene Ferrer. Evaluation of novel therapeutic strategies for KRAS mutated NSCLC patients using our own collections of PDX and PDX-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3101.

Lung cancer is the leading cause of cancer-related deaths worldwide. Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) activating mutations have significantly improved patient´s treatment with these alterations. However, 15-20% of EGFR-mutated patients do not respond to the therapy, even harboring the same mutations than responders. Little is known about the mechanisms involved in this primary resistance. This work aims to characterize molecular aberrations in clones with intrinsic resistance to first and third generation EGFR-TKIs generated from non-small cell lung cancer (NSCLC) cell lines. Three Erlotinib-sensitive (HCC827, PC9 and H4006) and one Osimertinib-sensitive (H1975) NSCLC cell lines were used to generate primary resistance models through a pulsatile method in which cells were treated with a high concentration of the TKI during a short period of time. Survivor colonies were individually expanded in the presence of the inhibitor until resistant cell lines were established. Exome, transcriptome and methylome were then analyzed in selected clones. Erlotinib- and Osimertinib-resistant clones exhibited cross-resistance to other EGFR-targeted therapies, such as Afatinib and Cetuximab, but not to the chemotherapeutics Carboplatin or Cisplatin. Among derived clones, we found several genetic alterations in genes associated with TKI acquired resistance, including NTRK1, ARID2, ERBB3, RET, and other genes. MET amplification was exclusively associated with resistant HCC827-derived clones. We also identified non-genetic alterations that include increased expression and over-activation of well-known TKI resistance-associated proteins, such as AXL, FGFR1, or AKT, as well as induction of epithelial-to-mesenchymal transition (EMT) and stemness-related genes. Transcriptome and methylome analyses revealed EMT plasticity among resistant clones that could reflect different EMT states conferring resistance in pre-treated tumors. Molecular inhibition of MET, a recognized resistance mechanism, sensitizes MET-amplified resistant clones to Erlotinib. Combination treatments or genetic inhibitions will be further performed to validate our findings. Taken together, our results suggest that multiple genetic and non-genetic alterations already present before TKI treatment could account for primary resistance. We will employ integrative analysis of our multi-omic data to identify novel targets that allow to overcome EGFR-TKI resistance. Further clinical validation will help to prospectively select the best treatment option for EGFR-mutated patients for the sake of precision medicine. Citation Format: Juan Manuel Coya, Eva Álvarez, David Gómez-Sánchez, Aranzazu Rosado, Irene Ferrer, Luis Paz-Ares. Comprehensive molecular characterization of mechanisms involved in primary resistance to EGFR tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 414.

32 p.-4 fig., Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity., Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo., Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer., Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions., This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).

Although two growth factor receptors, EGFR and HER2, are amongst the best targets for cancer treatment, no agents targeting HER3, their kinase-defective family member, have so far been approved. Because emergence of resistance of lung tumors to EGFR kinase inhibitors (EGFRi) associates with compensatory up-regulation of HER3 and several secreted forms, we anticipated that blocking HER3 would prevent resistance. As demonstrated herein, a neutralizing anti-HER3 antibody we generated can clear HER3 from the cell surface, as well as reduce HER3 cleavage by ADAM10, a surface metalloproteinase. When combined with a kinase inhibitor and an anti-EGFR antibody, the antibody completely blocked patient-derived xenograft models that acquired resistance to EGFRi. We found that the underlying mechanism involves posttranslational downregulation of HER3, suppression of MET and AXL upregulation, as well as concomitant inhibition of AKT signaling and upregulation of BIM, which mediates apoptosis. Thus, although HER3 is nearly devoid of kinase activity, it can still serve as an effective drug target in the context of acquired resistance. Because this study simulated in animals the situation of patients who develop resistance to EGFRi and remain with no obvious treatment options, the observations presented herein may warrant clinical testing. Funding: D.R. and I.M. thank the Lombroso Foundation for fellowships. This work was performed in the Marvin Tanner Laboratory for Research on Cancer. Y.Y. is the incumbent of the Harold and Zelda Goldenberg Professorial Chair in Molecular Cell Biology. Our studies are supported by the European Research Council (Oncombine; Grant #740469) and the Dr. Miriam and Sheldon Adelson Medical Research Foundation (Grant #15). Sí

[Background] Fibroblast growth factor receptor (FGFR)1 and FGFR4 have been associated with tumorigenesis in a variety of tumour types. As a therapeutic approach, their inhibition has been attempted in different types of malignancies, including lung cancer, and was initially focused on FGFR1-amplified tumours, though with limited success., [Methods] In vitro and in vivo functional assessments of the oncogenic potential of downregulated/overexpressed genes in isogenic cell lines were performed, as well as inhibitor efficacy tests in vitro and in vivo in patient-derived xenografts (PDXs). mRNA was extracted from FFPE non-small cell lung cancer samples to determine the prognostic potential of the genes under study., [Findings] We provide in vitro and in vivo evidence showing that expression of the adhesion molecule N-cadherin is key for the oncogenic role of FGFR1/4 in non-small cell lung cancer. According to this, assessment of the expression of genes in different lung cancer patient cohorts showed that FGFR1 or FGFR4 expression alone showed no prognostic potential, and that only co-expression of FGFR1 and/or FGFR4 with N-cadherin inferred a poorer outcome. Treatment of high-FGFR1 and/or FGFR4-expressing lung cancer cell lines and patient-derived xenografts with selective FGFR inhibitors showed high efficacy, but only in models with high FGFR1/4 and N-cadherin expression., [Interpretation] Our data show that the determination of the expression of FGFR1 or FGFR4 alone is not sufficient to predict anti-FGFR therapy efficacy; complementary determination of N-cadherin expression may further optimise patient selection for this therapeutic strategy., This work was funded by the Community of Madrid, the ISCIII co-funded by FEDER from Regional Development European Funds (European Union), the Spanish Ministry of Economy and Competitiveness, the Mutua Madrileña Foundation, the Ministry of Health and Social Welfare of Junta de Andalucía, the AECC scientific foundation and the Spanish Ministry of Education, Culture and Sport.

In patients with non-small cell lung cancer (NSCLC), the most frequent oncogene driver mutation in Western countries is Kirsten rat sarcoma viral oncogene homolog (KRAS), and KRAS-mutant NSCLC is associated with smoking. There are various sources of biological heterogeneity of KRAS-mutant NSCLC, including different genotypes that may be associated with specific clinical outcomes, the presence of other co-mutations that exhibit different biological features and drug sensitivity patterns, and mutant allelic content. The efficacy of chemotherapy in patients with KRAS-mutant NSCLC is generally poor and numerous novel therapeutic strategies have been developed. These approaches include targeting KRAS membrane associations, targeting downstream signalling pathways, the use of KRAS synthetic lethality, direct targeting of KRAS, and immunotherapy. Of these, immunotherapy may be one of the most promising treatment approaches for patients with KRAS-mutant NSCLC. Recent data also suggest the potential for distinct efficacy of immunotherapy according to the presence of other co-mutations. In view of the biological heterogeneity of KRAS-mutant NSCLC, treatment will likely need to be individualised and, in future, may require the use of rational combinations of treatment, many of which are currently under investigation.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFR(T790M) and EGFR(C797S). It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFR(T790M) and EGFR(C797S) tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3–dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor–resistant tumors, with EGFR(T790M) and EGFR(C797S) mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

[Objectives] Lung adenocarcinoma accounts for approximately half of lung cancer cases. Twenty to 50% of tumors of this type harbor mutations affecting epidermal growth factor receptor (EGFR) expression or activity, which can be therapeutically targeted. EGFR inhibitors in this context exhibit high efficacy and are currently used in the clinical setting. However, not all adenocarcinomas harboring EGFR mutations respond to therapy, so predictive biomarkers of therapeutic outcomes, as well as novel therapies sensitizing these tumors to EGFR inhibition, are needed., [Materials and methods] We performed in vitro gene overexpression/silencing and tumorigenic surrogate assays, as well as in vitro and in vivo combination treatments with Fibroblast Growth Factor Receptor (FGFR)/EGFR inhibitors. At the clinical level, we determined FGFR4 expression levels in tumors from patients treated with EGFR inhibitors and correlated these with treatment response., [Results] We describe a cooperative interaction between EGFR and FGFR4, which results in their reciprocal activation with pro-oncogenic consequences in vitro and in vivo. This cooperation is independent of EGFR activating mutations and increases resistance to different EGFR inhibitors. At the therapeutic level, we provide evidence of the synergistic effects of the combination of EGFR and FGFR inhibitors in high FGFR4-expressing, EGFR-activated tumors in vitro and in vivo. Correlated with these results, we found that patients treated with EGFR inhibitors relapse earlier when their tumors exhibit high FGFR4 expression., [Conclusions] We propose a novel predictive biomarker for EGFR-targeted therapy, and a highly efficacious combinatory therapeutic strategy to treat EGFR-dependent; this may may extend the use of appropriate inhibitors beyond EGFR-mutated adenocarcinoma patients., L.P.A. was funded by the Comunidad de Madrid, CAM, (B2017/BMD3884), ISCIII (PI14/01964, PIE15/00076, PI17/00778 and DTS17/00089) and CIBERONC (CD16/12/00442), and co-funded by FEDER from Regional Development European Funds (European Union). I.F. is funded by the AECC (AIO2015) and Consejería de Igualdad, Salud y Políticas Sociales de la Junta de Andalucía (PI-0029-2013) and ISCIII (PI16/01311), and co-funded by FEDER from Regional Development European Funds (European Union). AC was supported by grants from the Spanish Ministry of Economy and Competitiveness Plan Estatal de I + D+I 2013–2016, ISCIII (PI15/00045) and CIBERONC (CD16/12/00275), and co-funded by FEDER from Regional Development European Funds (European Union). S.M.P. is funded by the Consejería de Salud y Bienestar Social (PI-0046-2012), the Fundación Mutua Madrileña (2014) and ISCIII (PI17/00033). A.Q is funded by the ISCIII (FI12/00429). L.O. is funded by the Ministerio de Educación, Cultura y Deporte (FPU13/02595).

[Rationale]: The characterization of new genetic alterations is essential to assign effective personalized therapies in non–small cell lung cancer (NSCLC). Furthermore, finding stratification biomarkers is essential for successful personalized therapies. Molecular alterations of YES1, a member of the SRC (proto-oncogene tyrosine-protein kinase Src) family kinases (SFKs), can be found in a significant subset of patients with lung cancer., [Objectives]: To evaluate YES1 (v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1) genetic alteration as a therapeutic target and predictive biomarker of response to dasatinib in NSCLC., [Methods]: Functional significance was evaluated by in vivo models of NSCLC and metastasis and patient-derived xenografts. The efficacy of pharmacological and genetic (CRISPR [clustered regularly interspaced short palindromic repeats]/Cas9 [CRISPR-associated protein 9]) YES1 abrogation was also evaluated. In vitro functional assays for signaling, survival, and invasion were also performed. The association between YES1 alterations and prognosis was evaluated in clinical samples., [Measurements and Main Results]: We demonstrated that YES1 is essential for NSCLC carcinogenesis. Furthermore, YES1 overexpression induced metastatic spread in preclinical in vivo models. YES1 genetic depletion by CRISPR/Cas9 technology significantly reduced tumor growth and metastasis. YES1 effects were mainly driven by mTOR (mammalian target of rapamycin) signaling. Interestingly, cell lines and patient-derived xenograft models with YES1 gene amplifications presented a high sensitivity to dasatinib, an SFK inhibitor, pointing out YES1 status as a stratification biomarker for dasatinib response. Moreover, high YES1 protein expression was an independent predictor for poor prognosis in patients with lung cancer., [Conclusions]: YES1 is a promising therapeutic target in lung cancer. Our results provide support for the clinical evaluation of dasatinib treatment in a selected subset of patients using YES1 status as predictive biomarker for therapy., Supported by Fundación para la Investigación Médica Aplicada (FIMA), Spanish Ministry of Economy and Innovation and Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional grants 12/02040, PI13/00806, PI16/01821, PI16/00280, RTICC RD12/0036/0040, and SAF2015-6455-R; Centro de Investigación Biomédica en Red de Cáncer grants CB16/12/00390, CB16/12/00390, CB16/12/00442, and CB16/12/00443; Asociación Española Contra el Cáncer (AECC) Scientific Foundation grant GCB14-2170; Bristol-Myers Squibb (preclinical research agreement); AECC and a fellowship from the Basque Government (I.G.); the Castilla-León Government grant CSI049 U16 , the Ministry of Economy and Competitiveness grant SAF2015-64556-R , the Fundación Ramón Areces, Worldwide Cancer Research grant 14-1248 , and AECC Scientific Foundation grant GC16173472GARC (X.R.B.); and the Spanish Ministry of Science, Innovation and Universities (MCIU, RTI 2018-094507-B-100), La Caixa Foundation and Caja Navarra Foundation (F.L.).