Search

11 p.-6 fig., Identification of genomic alterations that influence the immune response within the tumor microenvironment is mandatory in order to identify druggable vulnerabilities. In this article, by interrogating public genomic datasets we describe copy number variations (CNV) present in breast cancer (BC) tumors and corresponding subtypes, associated with different immune populations. We identified regulatory T-cells associated with the Basal-like subtype, and type 2 T-helper cells with HER2 positive and the luminal subtype. Using gene set enrichment analysis (GSEA) for the Type 2 T-helper cells, the most relevant processes included the ERBB2 signaling pathway and the Fibroblast Growth Factor Receptor (FGFR) signaling pathway, and for CD8+ T-cells, cellular response to growth hormone stimulus or the JAK-STAT signaling pathway. Amplification of ERBB2, GRB2, GRB7, and FGF receptor genes strongly correlated with the presence of type 2 T helper cells. Finally, only 8 genes were highly upregulated and present in the cellular membrane: MILR1, ACE, DCSTAMP, SLAMF8, CD160, IL2RA, ICAM2, and SLAMF6. In summary, we described immune populations associated with genomic alterations with different BC subtypes. We observed a clear presence of inhibitory cells, like Tregs or Th2 when specific chromosomic regions were amplified in basal-like or HER2 and luminal groups. Our data support further evaluation of specific therapeutic strategies in specific BC subtypes, like those targeting Tregs in the basal-like subtype., AO’s lab is supported by the Instituto de Salud Carlos III(ISCIII, PI19/00808). ACEPAIN. CRIS Cancer Foundation and Diputación de Albacete. This research is also supported by PI18/01020from the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (FEDER) “A way to achieve Europe” (ERDF). EM is supported by a “Juan de la Cierva Incorporación” contract of the Spanish Ministry of Science and Innovation with Ref. IJC2019-041728-I.

2021 The Authors., Melanoma is the deadliest skin cancer with a very poor prognosis in advanced stages. Although targeted and immune therapies have improved survival, not all patients benefit from these treatments. The mitogen-activated protein kinase ERK5 supports the growth of melanoma cells in vitro and in vivo. However, ERK5 inhibition results in cell-cycle arrest rather than appreciable apoptosis. To clarify the role of ERK5 in melanoma growth, we performed transcriptomic analyses following ERK5 knockdown in melanoma cells expressing BRAFV600E and found that cellular senescence was among the most affected processes. In melanoma cells expressing either wild-type or mutant (V600E) BRAF, both genetic and pharmacologic inhibition of ERK5 elicited cellular senescence, as observed by a marked increase in senescence-associated β-galactosidase activity and p21 expression. In addition, depletion of ERK5 from melanoma cells resulted in increased levels of CXCL1, CXCL8, and CCL20, proteins typically involved in the senescence-associated secretory phenotype. Knockdown of p21 suppressed the induction of cellular senescence by ERK5 blockade, pointing to p21 as a key mediator of this process. In vivo, ERK5 knockdown or inhibition with XMD8–92 in melanoma xenografts promoted cellular senescence. Based on these results, small-molecule compounds targeting ERK5 constitute a rational series of prosenescence drugs that may be exploited for melanoma treatment., The work in E. Rovida’s lab was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, IG-15282 and IG-21349), by Ente Fondazione Cassa di Risparmio di Firenze (ECRF), and Universita degli Studi di Firenze (Fondo di Ateneo ex-60%). A. Tubita was supported by a “Carlo Zanotti” Fondazione Italiana per la Ricerca sul Cancro (FIRC)-AIRC fellowship (ID-23847).

Antibody–drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as [vic-]trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression., This work was supported by Breast Cancer Research Foundation (BCRF-20-008), Instituto de Salud Carlos III (project reference numbers AC15/00062, CB16/12/00449 and PI19/01181), the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Fundacion Mutua Madrilena and Asociación ~ Española Contra el Cáncer. S. Duro-Sanchez is supported by the Spanish Ministerio de Universidades by the grant Formacion de Profesorado Universitario (FPU20/05388). A. Esteve-Codina is funded by ISCIII /MINECO (PT17/0009/0019) and co-funded by FEDER.

© The Author(s) 2021., Despite advances in its treatment, lung cancer still represents the most common and lethal tumor. Because of that, efforts to decipher the pathophysiological actors that may promote lung tumor generation/progression are being made, with the final aim of establishing new therapeutic options. Using a transgenic mouse model, we formerly demonstrated that the sole activation of the MEK5/ERK5 MAPK route had a pathophysiological role in the onset of lung adenocarcinomas. Given the prevalence of that disease and its frequent dismal prognosis, our findings opened the possibility of targeting the MEK5/ERK5 route with therapeutic purposes. Here we have explored such possibility. We found that increased levels of MEK5/ERK5 correlated with poor patient prognosis in lung cancer. Moreover, using genetic as well as pharmacological tools, we show that targeting the MEK5/ERK5 route is therapeutically effective in lung cancer. Not only genetic disruption of ERK5 by CRISPR/Cas9 caused a relevant inhibition of tumor growth in vitro and in vivo; such ERK5 deficit augmented the antitumoral effect of agents normally used in the lung cancer clinic. The clinical correlation studies together with the pharmacological and genetic results establish the basis for considering the targeting of the MEK5/ERK5 route in the therapy for lung cancer., This work was supported by grants from the Instituto de Salud Carlos III (PI15/01180 and PI19/00840, co-funded by European Union ERDF, “A way to make Europe”) to A. Esparís-Ogando. A. Sánchez-Fdez was supported by the Cancer Center Network Program from the ISCIII (RD12/0036/0003) and by a predoctoral fellowship from the Scientific Foundation of the Spanish Association Against Cancer (AECC). Our laboratory is supported by grants from the Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R to A. Pandiella), the AECC and the CRIS Cancer Foundation. Our Cancer Research Institute, and the work carried out at our laboratory receive support from the European Community through the regional development funding program (FEDER). We are grateful to Nathanael S. Gray (Dana-Farber Cancer Institute, Boston, Massachusetts) for initially providing us with the compound JWG-071, and Dr. J. Lizcano (UAB, Barcelona, Spain) for advice on its in vivo use.

© The Author(s)., [Background]: Treatment of renal cancer has significantly improved with the arrival to the clinic of kinase inhibitors and immunotherapies. However, the disease is still incurable in advanced stages. The fact that several approved inhibitors for kidney cancer target receptor tyrosine kinases (RTKs) suggests that these proteins play a critical role in the pathophysiology of the disease. Based on these precedents, we decided to explore whether RTKs other than those targeted by approved drugs, contribute to the development of kidney cancer., [Methods]: The activation status of 49 RTKs in 44 paired samples of normal and tumor kidney tissue was explored using antibody arrays, with validation by western blotting. Genetic and pharmacologic approaches were followed to study the biological implications of targeting the epidermal growth factor receptor (EGFR) and its ligand Transforming Growth Factor-α (TGFα)., [Results]: Activation of the EGFR was found in a substantial number of tumors. Moreover, kidney tumors expressed elevated levels of TGFα. Down-regulation of EGFR or TGFα using RNAi or their pharmacological targeting with blocking antibodies resulted in inhibition of the proliferation of in vitro cellular models of renal cancer. Importantly, differences in the molecular forms of TGFα expressed by tumors and normal tissues were found. In fact, tumor TGFα was membrane anchored, while that expressed by normal kidney tissue was proteolytically processed., [Conclusions]: The EGFR-TGFα axis plays a relevant role in the pathophysiology of kidney cancer. This study unveils a distinctive feature in renal cell carcinomas, which is the presence of membrane-anchored TGFα. That characteristic could be exploited therapeutically to act on tumors expressing transmembrane TGFα, for example, with antibody drug conjugates that could recognize the extracellular region of that protein., AP: Ministry of Economy and Competitiveness of Spain (BFU2015–71371-R), the Instituto de Salud Carlos III through CIBERONC, Junta de Castilla y León (CSI146P20), the Scientific Foundation of the Spanish Association Against Cancer (AECC), ALMOM, ACMUMA and the CRIS Cancer Foundation. JCM is funded by the Instituto de Salud Carlos III through a Miguel Servet program (CP12/03073 and CPII17/00015) and receives research support from the same institution (PI18/00796). LGS is recipient of a predoctoral contract (BES-2016-077748). IRP is recipient of a predoctoral contract (CSI030–18). SGA is recipient of a predoctoral contract from the MINECO (BES-2013-065223). Work carried out in our laboratory receives support from the European Community through the Regional Development Funding Program (FEDER).

BRCA1/2 protein-deficient or mutated cancers comprise a group of aggressive malignancies. Although PARPis have shown considerably efficacy in their treatment, the widespread use of these agents in clinical practice is restricted by various factors, including the development of acquired resistance due to the presence of compensatory pathways. BETis can completely disrupt the HR pathway by repressing the expression of BRCA1 and could be aimed at generation combination regimes to overcome PARPi resistance and enhance PARPi efficacy. Due to the poor pharmacokinetic profile and short half-life, the first-in-class BETi JQ1 was loaded into newly developed nanocarrier formulations to improve the effectivity of olaparib for the treatment of BRCAness cancers. First, polylactide polymeric nanoparticles were generated by double emulsion. Moreover, liposomes were prepared by ethanol injection and evaporation solvent method. JQ1-loaded drug delivery systems display optimal hydrodynamic radii between 60 and 120 nm, with a very low polydispersity index (PdI), and encapsulation efficiencies of 92 and 16% for lipid- and polymeric-based formulations, respectively. Formulations show high stability and sustained release. We confirmed that all assayed JQ1 formulations improved antiproliferative activity compared to the free JQ1 in models of ovarian and breast cancers. In addition, synergistic interaction between JQ1 and JQ1-loaded nanocarriers and olaparib evidenced the ability of encapsulated JQ1 to enhance antitumoral activity of PARPis, We gratefully acknowledge the financial support from the Ministerio de Ciencia e Innovación y Agencia Estatal de la Investigación, Spain (Grant Nos. PID2020-117788RB-I00, r MCIN/AEI/10.13039/501100011033 and RED2018-102387-T Programa Redes Consolider) and Instituto de Salud Carlos III (grant number PI16/01121). Alberto Ocaña’s lab is supported by the Instituto de Salud Carlos III (ISCIII, PI19/00808), the CRIS Cancer Foundation and ACEPAIN.

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar y los autores pertenecientes a la UAM, Sarcomas are a heterogeneous group of tumors in which the role of ERK5 is poorly studied. To clarify the role of this MAPK in sarcomatous pathology, we used a murine 3-methyl-cholanthrene (3MC)-induced sarcoma model. Our data show that 3MC induces pleomorphic sarcomas with muscle differentiation, showing an increased expression of ERK5. Indeed, this upregulation was also observed in human sarcomas of muscular origin, such as leiomyosarcoma or rhabdomyosarcoma. Moreover, in cell lines derived from these 3MC-induced tumors, abrogation of Mapk7 expression by using specific shRNAs decreased in vitro growth and colony-forming capacity and led to a marked loss of tumor growth in vivo. In fact, transcriptomic profiling in ERK5 abrogated cell lines by RNAseq showed a deregulated gene expression pattern for key biological processes such as angiogenesis, migration, motility, etc., correlating with a better prognostic in human pathology. Finally, among the various differentially expressed genes, Klf2 is a key mediator of the biological effects of ERK5 as indicated by its specific interference, demonstrating that the ERK5–KLF2 axis is an important determinant of sarcoma biology that should be further studied in human pathology, This work has been supported with Grant RTI2018-094093-B-I00 funded by MCIN/AEI/10.13039/501100011033, “ERDF A way of making Europe” to RSP. Also supported with funds from Fundación Leticia Castillejo Castillo, Roche España and ACEPAIN to RSP and MJRH. RSP and MJRH’s Research Institute and the work carried out in their laboratory, received partial support from the European Community through the FEDER. JJ and EAL hold a predoctoral research contract cofounded by the European Social Fund and UCLM. OR holds a contract for accessing the Spanish System of Science, Technology, and Innovation (SECTI) funded by the University of Castilla-La Mancha (UCLM) and received partial support from the European Social Fund (FSE) through its Operative Program for Castilla-La Mancha (2007–2013)

Introduction: A number of different drugs have been developed to target the PI3 kinase/AKT/mTOR pathway. Most randomized trials in breast cancer have been performed in patients unselected for pathway activation although aberrations of components of this pathway are common. Here we explore the predictive value of PIK3CA and AKT mutations in randomized trials of drugs targeting this signaling route. Methods: We searched PubMed for randomized trials of drugs targeting the PI3 kinase/AKT/mTOR pathway in metastatic breast cancer. The search was supplemented by a manual search of recent ASCO and ESMO meetings. Eligible studies needed to report efficacy data based on presence or absence of PIK3CA or AKT status measured either in tumor specimens or in circulating DNA. Hazard ratios (HR) for progression-free survival (PFS) in PIK3CA or AKT mutant and wild-type groups were extracted and pooled in a meta-analysis using generic inverse variance and random effects modeling. Sensitivity analyses were performed based on the mechanism of action of the experimental drug. Results: Of the 15 studies identified, 10 studies comprising of 3615 patients were eligible for analysis. Studies included pan-PI3kinase inhibitors (n=5), alpha-specific PI3kinase inhibitors (n=1), AKT inhibitors (n=2) and mTOR inhibitors (n=2). Approximately 38% of patients had either PIK3CA or AKT mutations. The addition of drugs targeting the PI3 kinase/AKT/mTOR pathway was associated with a significantly longer improvement in PFS in patients with PIK3CA or AKT mutations than in those with wild-type status (HR 0.61, 95% CI 0.50-0.75 versus HR 0.83, 95% CI 0.67-1.02, p for difference = 0.04). Similar results were observed in sensitivity analyses (see Table). Conclusion: Benefit from drugs targeting the PI3 kinase/AKT/mTOR pathway in breast cancer appears to occur exclusively in those with PIK3CA or AKT mutations. Future trials of these drugs should be designed only in biomarker selected patients. TableGroupPIK3CA or AKT mutationPIK3CA and AKT wildtypep for differenceHR95% CIHR95% CIExcluding AKT inhibitors0.640.52-0.790.810.63-1.030.16Excluding pan-PI3 kinase inhibitors0.580.47-0.710.710.51-1.150.24Excluding AKT and alpha-specific PI3 kinase inhibitors0.690.48-1.000.980.83-1.150.09Excluding mTOR inhibitors0.620.49-0.800.920.80-1.060.008 Citation Format: Fahad A Almugbel, Ramy Saleh, Alberto Ocana, Atanasio Pandiella, Eitan Amir. PIK3CA status as a predictor of benefit from drugs targeting the PI3K/AKT/mTOR pathway: A meta-analysis of randomized trials [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-11.

Small molecule inhibitors (TKIs) of HER2 have demonstrated clinical benefit in HER2-positive breast tumors. One of them, lapatinib, is used once advanced tumors become refractory to the HER2 antibody trastuzumab. Another one, neratinib, has shown benefit in high-risk early-stage breast cancer after trastuzumab-based therapies. A common characteristic is that patients are formerly treated with trastuzumab. We have explored whether trastuzumab previous therapy affects its antitumoral action. Long time exposure of the HER2+ cell line BT474 to trastuzumab resulted in trastuzumab-insensitive cells (BTRH cells). While treatment of wild type BT474 cells with lapatinib or neratinib resulted in decreased viability, BTRH cells were resistant to the action of these TKIs. Analogous results were obtained using trastuzumab-resistant cells derived from a PDX. Functional transcriptomic analyses and biochemical studies demonstrated that the TKIs caused DNA damage and apoptosis in wild type cells, but not in BTRH. Moreover, previous treatment with trastuzumab impairs response to small TKIs, by eliminating their proapoptotic action. Moreover, actioning on the apoptotic machinery using a chemical library of proapoptotic compounds led to the identification of clinical-stage drugs that may be used to fight trastuzumab-TKI resistance., Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the Scientific Foundation of the Spanish Association Against Cancer (AECC), the CRIS Cancer Foundation, ALMOM, ACMUMA and UCCTA. The Cancer Research Institute and the work carried out in our laboratory receive support from the European Community through the Regional Development Funding Program (FEDER). LDG was supported by a predoctoral contract of the Consejería de Educación of the Castilla and León Autonomous Government. LGS was supported by a MINECO predoctoral fellowship (BES-2016-077748).

The HER3 protein, that belongs to the ErbB/HER receptor tyrosine kinase (RTK) family, is expressed in several types of tumors. That fact, together with the role of HER3 in promoting cell proliferation, implicate that targeting HER3 may have therapeutic relevance. Furthermore, expression and activation of HER3 has been linked to resistance to drugs that target other HER receptors such as agents that act on EGFR or HER2. In addition, HER3 has been associated to resistance to some chemotherapeutic drugs. Because of those circumstances, efforts to develop and test agents targeting HER3 have been carried out. Two types of agents targeting HER3 have been developed. The most abundant are antibodies or engineered antibody derivatives that specifically recognize the extracellular region of HER3. In addition, the use of aptamers specifically interacting with HER3, vaccines or HER3-targeting siRNAs have also been developed. Here we discuss the state of the art of the preclinical and clinical development of drugs aimed at targeting HER3 with therapeutic purposes., This work was supported by the Ministry of Economy and Competitiveness of Spain (PID2020-115605RB-I00), the Instituto de Salud Carlos III through CIBERONC, Junta de Castilla y León (CSI146P20), ALMOM, ACMUMA, UCCTA, the CRIS Cancer Foundation and the Regional Development Funding Program (FEDER) “A way to make Europe”. LGS was recipient of a predoctoral contract (BES-2016–077748); and is at present contracted by the Cancer Research Foundation of the Salamanca University (FICUS).

During recent years, a number of new compounds against HER2 have reached clinics, improving the prognosis and quality of life of HER2-positive breast cancer patients. Nonetheless, resistance to standard-of-care drugs has motivated the development of novel agents, such as new antibody-drug conjugates (ADCs). The latter are a group of drugs that benefit from the potency of cytotoxic agents whose action is specifically guided to the tumor by the target-specific antibody. Two anti-HER2 ADCs have reached the clinic: trastuzumab-emtansine and, more recently, trastuzumab-deruxtecan. In addition, several other HER2-targeted ADCs are in preclinical or clinical development, some of them with promising signs of activity. In the present review, the structure, mechanism of action, and potential resistance to all these ADCs will be described. Specific attention will be given to discussing novel strategies to circumvent resistance to ADCs., This work was supported by grants from the Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R and PID2020-115605RB-I00), the Instituto de Salud Carlos III through CIBERONC, Junta de Castilla y León (CSI146P20), ALMOM, ACMUMA, UCCTA and the CRIS Cancer Foundation (A.P.), and by the Instituto de Salud Carlos III (PI19/00808), ACEPAIN, and CRIS Cancer Foundation (A.O.). Work carried out in A.P.’s and A.O.’s laboratories received support from the European Community through the Regional Development Funding Program (FEDER). L.G.-S. was the recipient of a predoctoral contract (BES-2016-077748) and is presently contracted with the Cancer Research Foundation of Salamanca University (FICUS).

In patients with trastuzumab-resistant HER2-positive breast cancer, the combination of everolimus (mTORC1 inhibitor) with trastuzumab failed to show a clinically significant benefit. However, the combination of mTOR inhibition and the antibody-drug conjugate (ADC) trastuzumab-emtansine (T-DM1) remains unexplored. We tested T-DM1 plus everolimus in a broad panel of HER2-positive breast cancer cell lines. The combination was superior to T-DM1 alone in four cell lines (HCC1954, SKBR3, EFM192A, and MDA-MB-36) and in two cultures from primary tumor cells derived from HER2-positive patient-derived xenografts (PDX), but not in BT474 cells. In the trastuzumab-resistant HCC1954 cell line, we characterized the effects of the combination using TAK-228 (mTORC1 and -2 inhibitor) and knockdown of the different mTOR complex components. T-DM1 did not affect mTOR downstream signaling nor induct autophagy. Importantly, mTOR inhibition increased intracellular T-DM1levels, leading to increased lysosomal accumulation of the compound. The increased efficacy of mTOR inhibition plus T-DM1 was abrogated by lysosome inhibitors (chloroquine and bafilomycin A1). Our experiments suggest that BT474 are less sensitive to T-DM1 due to lack of optimal lysosomal processing and intrinsic resistance to the DM1 moiety. Finally, we performed several in vivo experiments that corroborated the superior activity of T-DM1 and everolimus in HCC1954 and PDXderived mouse models. In summary, everolimus in combination with T-DM1 showed strong antitumor effects in HER2-positive breast cancer, both in vitro and in vivo. This effect might be related, at least partially, to mTOR-dependent lysosomal processing of T-DM1, a finding that might apply to other ADCs that require lysosomal processing., This work was supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219), Generalitat de Catalunya (2017 SGR 507). MINECO through gBFU2015-71371-R grant and the CRIS Cancer Foundation supported work in AP lab. D. Casadevall was supported by ISCIII (Rio Hortega Research Contract CM16/00023 and Juan Rodés Research Contract JR18/00003). F.J. Sánchez-Martín and S. Menéndez were supported by Department de Salut Generalitat de Catalunya (PERIS SLT002/16/00008 and PERIS SLT006/17/00040). M. Qin received financial support from the China Scholarship Council (CSC) for her doctoral fellowship. Work carried out in our laboratories receives support from the European Community through the Regional Development Funding Program (FEDER).

Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) that was approved in 2013 to treat HER2+ breast cancer. Despite its efficacy in the clinic, some patients exhibit intrinsic or acquired resistance to such ADC. To characterize mechanisms of resistance to T-DM1, we isolated several HER2+ resistant clones derived from the HCC1954 HER2+ cell line. The isolated clones were different as per their transcriptomic profiles. However, all the T-DM1-resistant clones showed decreased HER2 levels. Yet, the clones were still oncogenically dependent on HER2, as indicated by knock down experiments. The decrease in HER2 expression caused acquired resistance to T-DM1 and to other anti-HER2 therapies. Antibody array analyses showed that the epidermal growth factor receptor (EGFR) was expressed in these T-DM1-resistant HCC1954 clones. Indeed, therapies targeting EGFR, particularly cetuximab-DM1, demonstrated a strong anti-proliferative action on cells with acquired resistance to T-DM1 and HER2 loss. The expression of EGFR in cells resistant to T-DM1 offers the possibility of using therapies directed to this receptor to combat resistance to anti-HER2 drugs and loss of HER2 overexpression.

[Background]: Despite the incorporation of novel therapeutics, advanced triple negative breast cancer (TNBC) still represents a relevant clinical problem. Considering this, as well as the clinical efficacy of antibody-drug conjugates (ADCs), we aimed at identifying novel ADC targets that could be used to treat TNBC. [Methods]: Transcriptomic analyses were performed on TNBC and normal samples from three different studies. Plasma membrane proteins of three cell lines representative of the TNBC subtype were identified by cell surface biotinylation or plasma membrane isolation, followed by analyses of cell surface proteins using the Surfaceome online tool. Immunofluorescence and western studies were used to characterize the action of a CD98hc-directed ADC, which was prepared by in house coupling of emtansine to an antibody that recognized the ectodomain of CD98hc. Xenografted TNBC cells were used to analyze the antitumoral properties of the anti-CD98hc ADC. [Results]: Comparative genomic studies between normal breast and TNBC tissues, together with proteomic and bioinformatic analyses resulted in the elaboration of a catalog of potential ADC targets. One of them, the CD98hc transmembrane protein, was validated as an ADC target. An antibody recognizing the ectodomain of CD98hc efficiently internalized and reached the lysosomal compartment. An emtansine-based ADC derived from such antibody was prepared and showed antitumoral properties in TNBC in vitro and in vivo models. Mechanistically, the anti-CD98hc ADC blocked cell cycle progression, that was followed by cell death caused by mitotic catastrophe. [Conclusions]: This work describes a list of potential ADC targets in TNBC and validates one of them, the transmembrane protein CD98hc. The studies presented here also demonstrate the robustness of the multiomic approach herewith described to identify novel potential ADC targets., JCM is funded by the Instituto de Salud Carlos III through a Miguel Servet program (CP12/03073 and CPII17/00015) and receives research support from the same institution (PI15/00684 and PI18/00796) (Co-funded by European Regional Development Fund/European Social Fund "A way to make Europe"/"Investing in your future"). AP: Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R); Instituto de Salud Carlos III through CIBERONC; Junta de Castilla y León (CSI146P20); CRIS Cancer Foundation, ACMUMA, UCCTA, and ALMOM. Work carried out in our laboratories receives support from the European Community through the Regional Development Funding Program (FEDER).

© Pandiella et al., Ocoxin Oral Solution (OOS) and Viusid (VS) are nutritional supplements that include several natural products which affect different cellular functions, such as proliferation or the redox status. In addition, some of their constituent components have been described to exert an antiviral effect. Considering this, it was hypothesized that treatment with OOS and VS could protect from viral infections. In order to evaluate the impact of OOS and VS on viral infection, lentivirus and retrovirus whose genomes coded for green fluorescent protein were used. In addition, and as a second approach to measure viral infection, a hemagglutinin‑tagged form of the mitogen‑activated protein kinase ERK5 was also inserted in the retroviral vector. Viral particles produced in 293T cells were used to infect HeLa cells in the presence or absence of OOS or VS. It was observed that VS had a minimal effect on the capacity of either lentivirus or retrovirus to infect HeLa cells. However, OOS significantly reduced the infection of HeLa cells with both of these viruses. The effect was dose‑dependent, reaching a maximum at a 1:100 dilution of OOS. These results suggested that, in addition to its well‑known antitumoral properties, OOS may also inhibit infection with viruses. This effect is relevant since patients receiving oncological therapies are more susceptible to viral infections, and nutritional supplements such as OOS may help in reducing the severity of these potential pathogenic infections., AP is a staff employee of the Spanish Research Council (CSIC) and EDR is supported by a postdoctoral contract from the same institution. ES is an employee of Catalysis S.L. The work on ocoxin oral solution in preclinical models carried out by AP and EDR has received partial support from Catalysis S.L

© 2021 by the authors., In the last 20 years, antibody-drug conjugates (ADCs) have been incorporated into the oncology clinic as treatments for several types of cancer. So far, the Food and Drug Administration (FDA) has approved 11 ADCs and other ADCs are in the late stages of clinical development. Despite the efficacy of this type of drug, the tumors of some patients may result in resistance to ADCs. Due to this, it is essential not only to comprehend resistance mechanisms but also to develop strategies to overcome resistance to ADCs. To reach these goals, the generation and use of preclinical models to study those mechanisms of resistance are critical. Some cells or patient tumors may result in primary resistance to the action of an ADC, even if they express the antigen against which the ADC is directed. Isolated primary tumoral cells, cell lines, or patient explants (patient-derived xenografts) with these characteristics can be used to study primary resistance. The most common method to generate models of secondary resistance is to treat cancer cell lines or tumors with an ADC. Two strategies, either continuous treatment with the ADC or intermittent treatment, have successfully been used to develop those resistance models., AP: Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through CIBERONC, Junta de Castilla y León (CSI146P20), ALMOM, ACMUMA, UCCTA and the CRIS Cancer Foundation. AO: Instituto de Salud Carlos III (PI19/00808), ACEPAIN, and CRIS Cancer Foundation. Work carried out in AP and AO laboratories receives support from the European Community through the Regional Development Funding Program (FEDER). LGS was recipient of a predoctoral contract (BES-2016-077748); and is at present contracted by the Cancer Research Foundation of the Salamanca University (FICUS).

© 2021 by the authors., Alternative splicing is an essential biological process, which increases the diversity and complexity of the human transcriptome. In our study, 304 splicing pathway-related genes were evaluated in tumors from breast cancer patients (TCGA dataset). A high number of alterations were detected, including mutations and copy number alterations (CNAs), although mutations were less frequently present compared with CNAs. In the four molecular subtypes, 14 common splice genes showed high level amplification in >5% of patients. Certain genes were only amplified in specific breast cancer subtypes. Most altered genes in each molecular subtype clustered to a few chromosomal regions. In the Luminal subtype, amplifications of LSM1, CLNS1A, and ILF2 showed a strong significant association with prognosis. An even more robust association with OS and RFS was observed when expression of these three genes was combined. Inhibition of LSM1, CLNS1A, and ILF2, using siRNA in MCF7 and T47D cells, showed a decrease in cell proliferation. The mRNA expression of these genes was reduced by treatment with BET inhibitors, a family of epigenetic modulators. We map the presence of splicing-related genes in breast cancer, describing three novel genes, LSM1, CLNS1A, and ILF2, that have an oncogenic role and can be modulated with BET inhibitors., A.O.’s lab is supported by the Instituto de Salud Carlos III (ISCIII, PI19/00808); ACEPAIN; CRIS Cancer Foundation and Diputación de Albacete. This research is also supported by PI18/01020 from the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (FEDER) “A way to achieve Europe” (ERDF); N.L. MDM was supported by the Spanish Ministry of Education (FPU grant; Ref.: FPU18/01319). B.G. was financed by the 2018-2.1.17-TETKR-00001, 2020-1.1.6-JÖVO-2021-00013, and 2018-1.3.1-VKE-2018-00032 grants and by the Higher ˝ Education Institutional Excellence Programme (2020-4.1.1.-TKP2020) of the Ministry for Innovation and Technology in Hungary.

© The Author(s)., [Background]: Trastuzumab-based therapies are the therapeutic option for HER2 positive (HER2+) breast cancer. HER2 amplification is the only biomarker validated for trastuzumab-based therapies. However, a proportion of tumors become refractory during treatment course. For this reason, the finding of new biomarkers beyond HER2 overexpression to identify patients who would benefit most from trastuzumab regimens is of outstanding importance., [Methods]: Models of trastuzumab-resistant or hypersensitive cells were generated by exposure to trastuzumab. Cell surface, total HER2, and analyses of proteins involved in cell cycle or apoptosis were analyzed by western blotting. Cell proliferation was analyzed by cell counting, cell cycle and apoptosis was evaluated by FACS. Transcriptomic characterization of the cellular models was performed using bioinformatic online tools, and clinico-genomic analyses were performed using the PAMELA clinical trial data., [Results]: Besides differing in sensitivities to trastuzumab, the different cellular models also showed distinct response to other anti-HER2 drugs (lapatinib, neratinib, pertuzumab and T-DM1) used in the clinic. That differential effect was not due to changes in cell surface, total or activated HER2. Trastuzumab caused important induction of cell death in hypersensitive cells but not in parental or resistant cells. Transcriptomic analyses of these cellular models together with querying of online databases allowed the identification of individual genes and gene signatures that predicted prognosis and trastuzumab response in HER2+ breast cancer patients., [Conclusion]: The identification of trastuzumab response biomarkers may be used to select patients particularly sensitive to facilitate the use of trastuzumab-based therapies and refine follow-up guidelines in patients with HER2+ tumors., A Pandiella: Ministry of Economy and Competitiveness of Spain (BFU2015–71371-R), the Junta de Castilla y León (CSI146P20), the Instituto de Salud Carlos III through CIBERONC, the Scientific Foundation of the Spanish Association Against Cancer (AECC), ALMOM, ACMUMA and the CRIS Cancer Foundation. LDG is recipient of a predoctoral contract of the Consejería de Educación of the Castilla y León Autonomous Government. Work carried out in our laboratory receives support from the European Community through the Regional Development Funding Program (FEDER). FBM: Fundación Científica Asociación Española Contra el Cáncer AECC_Postdoctoral17–1062

© 2021 by the authors., The dysregulation of post-translational modifications (PTM) transversally impacts cancer hallmarks and constitutes an appealing vulnerability for drug development. In breast cancer there is growing preclinical evidence of the role of ubiquitin and ubiquitin-like SUMO and Nedd8 peptide conjugation to the proteome in tumorigenesis and drug resistance, particularly through their interplay with estrogen receptor signaling and DNA repair. Herein we explored genomic alterations in these processes using RNA-seq and mutation data from TCGA and METABRIC datasets, and analyzed them using a bioinformatic pipeline in search of those with prognostic and predictive capability which could qualify as subjects of drug research. Amplification of UBE2T, UBE2C, and BIRC5 conferred a worse prognosis in luminal A/B and basal-like tumors, luminal A/B tumors, and luminal A tumors, respectively. Higher UBE2T expression levels were predictive of a lower rate of pathological complete response in triple negative breast cancer patients following neoadjuvant chemotherapy, whereas UBE2C and BIRC5 expression was higher in luminal A patients with tumor relapse within 5 years of endocrine therapy or chemotherapy. The transcriptomic signatures of USP9X and USP7 gene mutations also conferred worse prognosis in luminal A, HER2-enriched, and basal-like tumors, and in luminal A tumors, respectively. In conclusion, we identified and characterized the clinical value of a group of genomic alterations in ubiquitination, SUMOylation, and neddylation enzymes, with potential for drug development in breast cancer., Work in Alberto Ocaña’s lab is supported by the Instituto de Salud Carlos III (ISCIII, PI19/00808); ACEPAIN; Diputación de Albacete; and the CRIS Cancer Foundation. Work in Atanasio Pandiella’s lab is supported by the Ministry of Economy and Competitiveness of Spain (BFU2015- 71371-R, the Junta de Castilla y León (CSI146P20), and the CRIS Foundation. Balázs Györffy is financed by the 2018-2.1.17-TET-KR-00001 grant and by the Higher Education Institutional Excellence Programme of the Ministry for Innovation and Technology (MIT) in Hungary, within the framework of the Bionic thematic programme of the Semmelweis University.

[Purpose]: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab., [Experimental Design]: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2–based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial., [Results]: NRG1 was expressed in HER2-positive breast cancer–derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group., [Conclusions]: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab., This work is supported by ISCIII (CIBERONC CB16/12/00481, CB16/12/00241, PI18/00382, PI18/00006, PI18/01219 and by Generalitat de Catalunya (2017 SGR 507). S. Menendez is supported by Department de Salut, Generalitat de Catalunya (PERIS SLT006/17/00040). MARBiobanc is supported by ISCiii/FEDER (PT17/0015/0011) and by “Xarxa de Bancs de tumors” sponsored by Pla Director d’ Oncologia de Catalunya (XBTC) and Fundacion Jimenez Díaz Biobanks Platform by PT13/0010/0012 grant. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R) and the CRIS Cancer Foundation provides support to A. Pandiella. Work carried out in our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER). J.C. Montero is funded by the ISCIII through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI18/00796). J. Albanell is supported by Breast Cancer Research Foundation (BCRF20-08), Instituto de Salud Carlos III Project Reference number AC15/00062 and the EC under the framework of the ERA-NET TRANSCAN-2 initiative co-financed by FEDER, Instituto de Salud Carlos III (CB16/12/00449 and PI19/01181), and Asociacion Espanola Contra el Cáncer (AECC).

© 2021 The Author(s)., Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy., This research has been funded by Spanish Ministry of Economy and Competitiveness - MINECO - (SAF 2015–65113-C2–1-R and RTI2018–094356-B-C21 to AEB, SAF2015–65113-C2–2 to LFP, SAF2017–88026-R to JL) with the co-funding of European Regional Development Fund (EU-ERDF), Canary Islands Government (CEI2018–23/ACIISI to BG, CEI2019–08/ACIISI to BG and LFP, ProID2021010037 to AEB, LFP and BG) and "Juan de la Cierva Incorporacion" Grant Program from the Ministry of Science, Innovation and Universities (IJC2018-035193-I to CR). This project has been also supported by Alfredo Martin-Reyes Foundation (Arehucas)-Canary Islands Foundation for Cancer Research (FICIC). HAT is recipient of a predoctoral program grant from ULPGC (2016). JCM was funded by the Instituto de Salud Carlos III through a Miguel Servet program (CPII17/00015).

© The Author(s)., [Background]: Although the anti-HER2 antibody trastuzumab augments patient survival in HER2+ breast cancer, a relevant number of patients progress to this treatment. In this context, novel drug combinations are needed to increase its antitumor activity. In this work, we have evaluated the efficacy of proteolysis targeting chimera (PROTAC) compounds based on BET inhibitors (BETi) to augment the activity of trastuzumab in HER2+ breast cancer models., [Methods]: BT474 and SKBR3 HER2+ breast cancer cell lines were used. The effects of trastuzumab and the BET-PROTAC MZ1 either alone or in combination, were evaluated using MTT proliferation assays, three-dimensional invasion and adhesion cultures, flow cytometry, qPCR and Western blot. In vivo studies were carried out in a xenografted model in mice. Finally, a Clariom_S_Human transcriptomic array was applied to identify deregulated genes after treatments., [Results]: MZ1 induced a higher antiproliferative effect compared to the BETi JQ1. The combination of MZ1 and -trastuzumab significantly decreased cell proliferation, the formation of three-dimensional structures and cellular invasion compared to either of the drugs alone. Evaluation of apoptosis resulted in an increase of cell death following treatment with the combination, and biochemical studies displayed modifications of apoptosis and DNA damage components. In vivo administration of agents alone or combined, to tumors orthotopically xenografted in mice, resulted in a decrease of the tumor volume only after MZ1-Trastuzumab combination treatment. Results from a transcriptomic array indicated a series of newly described transcription factors including HOXB7, MEIS2, TCERG1, and DNAJC2, that were associated to poor outcome in HER2+ breast cancer subtype and downregulated by the MZ1-trastuzumab combination., [Conclusions]: We describe an active novel combination that includes the BET-PROTAC MZ1 and trastuzumab, in HER2+ tumors. Further studies should be performed to confirm these findings and pave the way for their future clinical development., This work has been supported by Instituto de Salud Carlos III (PI19/00808), ACEPAIN, Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A. Ocaña), Ministry of Economy and Competitiveness of Spain (BFU2015–71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A. Pandiella). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER). MdM. Noblejas-López was supported by Spanish Ministry of Education (FPU grant; Ref.: FPU18/01319). E.M. Galan-Moya is funded by the implementation research program of the UCLM (UCLM resolution date: 31/07/2014), with a contract for accessing the Spanish System of Science, Technology and Innovation-SECTI (co-funded by the European Commission/FSE funds). This work has been supported by Junta de Comunidades de Castilla-La Mancha (SBPLY/19/180501/000173) (to E.M. Galan-Moya and A.Ocana). D. Tébar-García is funded by a contract from Junta de Comunidades de Castilla-La Mancha (SBPLY/19/180501/000173). J.C. Montero is funded by the Instituto de Salud Carlos III through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI15/00684 and PI18/00796 M). M. Burgos is funded by Castilla-La Mancha support grant for Biomedicine and Health Science Research ref.: II-2018_11.

© 2021 by the authors., Among the described druggable vulnerabilities, acting on the DNA repair mechanism has gained momentum, with the approval of PARP inhibitors in several indications, including breast cancer. However, beyond the mere presence of BRCA1/BRCA2 mutations, the identification of additional biomarkers that would help to select tumors with an extreme dependence on DNA repair machinery would help to stratify therapeutic decisions. Gene set enrichment analyses (GSEA) using public datasets evaluating expression values between normal breast tissue and breast cancer identified a set of upregulated genes. Genes included in different pathways, such as ATM/ATR, BARD1, and Fanconi Anemia, which are involved in the DNA damage response, were selected and confirmed using molecular alterations data contained at cBioportal. Nineteen genes from these gene sets were identified to be amplified and upregulated in breast cancer but only five of them NBN, PRKDC, RFWD2, UBE2T, and YWHAZ meet criteria in all breast cancer molecular subtypes. Correlation of the selected genes with prognosis (relapse free survival, RFS, and overall survival, OS) was performed using the KM Plotter Online Tool. In last place, we selected the best signature of genes within this process whose upregulation can be indicative of a more aggressive phenotype and linked with worse outcome. In summary, we identify genomic correlates within DNA damage pathway associated with prognosis in breast cancer., We would like to thank our funding institutions, Instituto de Salud Carlos III, Diputación de Albacete, AECC Foundation, Ministry of Economy and Competitiveness of Spain, the Spanish Cancer Centers Network Program, and the European Commission as the FEDER funding program responsible institution. We would like to especially thank our supporting foundations, such as the “Asociación Costuras en la Piel en Apoyo a la investigación de cáncer en Albacete” (ACEPAIN) and the Cris Cancer Foundation for their continuous efforts to support our work.

© 2021 by the authors., Targeting the innate immune system has attracted attention with the development of anti- CD47 antibodies. Anti-CD47 antibodies block the inhibition of the phagocytic activity of macrophages caused by the up-regulation of CD47 on tumor cells. In this study, public genomic data was used to identify genes highly expressed in breast tumors with elevated CD47 expression and analyzed the association between the presence of tumor immune infiltrates and the expression of the selected genes. We found that 142 genes positively correlated with CD47, of which 83 predicted favorable and 32 detrimental relapse-free survival (RFS). From those associated with favorable RFS, we selected the genes with immunologic biological functions and defined a CD47-immune signature composed of PTPRC, HLA-E, TGFBR2, PTGER4, ETS1, and OPTN. In the basal-like and HER2+ breast cancer subtypes, the expression of the CD47-immune signature predicted favorable outcome, correlated with the presence of tumor immune infiltrates, and with gene expression signatures of T cell activation. Moreover, CD47 up-regulated genes associated with favorable survival correlated with pro-tumoral macrophages. In summary, we described a CD47-immune gene signature composed of 6 genes associated with favorable prognosis, T cell activation, and pro-tumoral macrophages in breast cancer tumors expressing high levels of CD47., This work was supported by Instituto de Salud Carlos III (PI19/00808), ACEPAIN, Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A.O.). Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A.P.). M.d.M.N.-L. was supported by the Spanish Ministry of Education (FPU grant; Ref.: FPU18/01319). The work carried out in our laboratories received support from the European Community through the regional development funding program (FEDER). B.G. was supported by the grants NVKP_16-1-2016-0037, 2018-1.3.1-VKE-2018-00032, and KH-129581.

© 2020 by the authors., Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin., This research was funded by Instituto de Salud Carlos III (PI16/01121 and PI19/00808), Diputación de Albacete and CIBERONC (to A. Ocana); CRIS Cancer Foundation (to A. Ocana and A. Pandiella); and Junta de Comunidades de Castilla-La Mancha (SBPLY/19/180501/000173) (to E.M. Galan-Moya and A. Ocana). We would also like to thank the Cancer Association, ACEPAIN for supporting part of this work. E.M. Galan-Moya is funded by the Implementation Research Program of the UCLM (UCLM resolution date: 31/07/2014), with a contract accessing the Spanish System of Science, Technology and Innovation-Secti (co-funded by the European Commission/FSE funds). A. Alcaraz and M. Nuncia-Cantarero are funded by grants from the Junta de Comunidades de Castilla-La Mancha. S. Martinez-Canales is funded by a fellowship from the Regional Centre for Biomedical Research of the UCLM (CRIB-UCLM). M. Burgos is funded by a regional fellowship from Biomedicine and Health Science Research (II-2018_11).

© 2020 Fuentes-Antrás, Guevara-Hoyer, Baliu-Piqué, García-Sáenz, Pérez-Segura, Pandiella and Ocaña., Immunotherapy has become a cornerstone in the treatment of cancer and changed the way clinicians and researchers approach tumor vulnerabilities. Durable responses are commonly observed with immune checkpoint inhibitors in highly immunogenic tumors, while the infusion of T cells genetically engineered to express chimeric antigen receptors (CARs) has shown impressive efficacy in certain types of blood cancer. Nevertheless, harnessing our own immunity has not proved successful for most breast cancer patients. In the era of genomic medicine, cellular immunotherapies may provide a more personalized and dynamic tool against tumors displaying heterogeneous mutational landscapes and antigenic pools. This approach encompasses multiple strategies including the adoptive transfer of tumor-infiltrating lymphocytes, dendritic cells, natural killer cells, and engineered immune components such as CAR constructs and engineered T cell receptors. Although far from permeating the clinical setting, technical advances have been overwhelming in recent years, with continuous improvement in traditional challenges such as toxicity, adoptive cell persistence, and intratumoral trafficking. Also, there is an avid search for neoantigens that can be targeted by these strategies, either alone or in combination. In this work, we aim to provide a clinically-oriented overview of preclinical and clinical data regarding the use of cellular immunotherapies in breast cancer.

Treatment with anti-PD-L1 antibodies has shown efficacy in basal-like breast cancer. In this context, identification of pre-activated immune tumors is a main goal. Here we explore mutations in PD1 and PD-L1 high-expressing tumors to identify genomic correlates associated with outcome. To do so, RNA-seq and mutation data from 971 breast cancer patients from the TCGA dataset were used to identify most prevalent mutations in patients with high levels of PD1 and PD-L1. Transcriptomic signatures associated with the selected mutations were identified and analyzed in terms of outcome and immune cell infiltration. We identified co-occurrent mutations in RYR2 and AHNAK in 8% and 5% of basal-like tumors respectively, which conferred good prognosis in patients with high expression of PD1 and PD-L1 genes. The transcriptomic signature associated with these mutations, composed of CXCL9, GBP5, C1QA, IL2RG, CSF2RB, IDO1 and LAG3 genes, also conferred good prognosis and correlated with immune infiltrations within the tumors. The joint signature classified patients with favorable relapse-free survival (HR: 0.28, CI: 0.2&ndash, 0.38, p = 1.7 &times, 10&minus, 16) and overall survival (HR: 0.18, CI: 0.09&ndash, 0.34, p = 6.8 &times, 9), showing a stronger prediction capacity than previous reported signatures. In conclusion, we describe two novel mutations and their transcriptomic signature, both associated with a favorable outcome and immune infiltrates in PD1 and PD-L1 high-expressing basal-like tumors.

[Purpose]: Integrins, transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions, have been linked to several cancer-associated features. A less explored function of integrins in cancer is their role in leukocyte homing and activation. Understanding their relationship with immune cell infiltrates and immune checkpoints is an area of interest in cancer research. [Methods]: The expression of 33 different integrins was evaluated in relation with breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to assess the association of the identified integrin genes with immune cell infiltration, and the TCGA and METABRIC studies to assess correlations between integrin gene expression and genomic signatures of immune activation. [Results]: We identified 7 genes coding for integrin α and β subunits, i.e., ITGA4, ITGB2, ITGAX, ITGB7, ITGAM, ITGAL and ITGA8, which predict a favorable prognosis in Basal-like and HER2+ breast cancers. Their expression positively correlated with the presence of immune cell infiltrates within the tumor (dendritic cells, CD4+ T-cells, neutrophils, CD8+ T-cells and B-cells), with markers of T-cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast, we found that genes coding for integrins that predicted a detrimental outcome (IBSP, ITGB3BP, ITGB6, ITGB1 and ITGAV) were not associated with any of these parameters. [Conclusions]: We identified an integrin signature composed of 7 genes with potential to recognize immune infiltrated and activated Basal-like and HER2+ breast cancers with a favorable prognosis., This work was supported by the Instituto de Salud Carlos III (PI19/00808), ACEPAIN, Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A. Ocaña) and the Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A. Pandiella). Our laboratories receive support from the European Community through the regional development funding program (FEDER). BG was supported by a 2018 − 2.1.17-TET-KR-00001 grant and by the Higher Education Institutional Excellence Programme (2020 − 4.1.1.-TKP2020) of the Ministry for Innovation and Technology of Hungary, within the framework of the Bionic thematic programme of the Semmelweis University.

© 2020 by the authors., Targeted cancer therapies against oncogenic drivers are actively being developed and tested in clinical trials. Targeting an oncogenic driver may only prove effective if the mutation is present in most tumoral cells. Therefore, highly heterogeneous tumors may be refractory to these therapies. This makes tumor heterogeneity a major challenge in cancer therapy. Although heterogeneity has traditionally been attributed to genetic diversity within cancer cell populations, it is now widely recognized that human cancers are heterogeneous in almost all distinguishable phenotypic characteristics. Understanding the genetic variability and also the non-genetic influences of tumor heterogeneity will provide novel insights into how to reverse therapeutic resistance and improve cancer therapy., This work has been supported by CRIS Cancer Foundation, Instituto de Salud Carlos III (PI19/00808), ACEPAIN, ALMOM, Diputación de Albacete and CIBERONC.

© The Author(s)., Exploitation of the protein degradation machinery as a therapeutic strategy to degrade oncogenic proteins is experiencing revolutionary advances with the development of proteolysis targeting chimeras (PROTACs). PROTACs are heterobifunctional structures consisting of a ligand that binds a protein to be degraded and a ligand for an E3 ubiquitin ligase. The bridging between the protein of interest and the E3 ligase mediated by the PROTAC facilitates ubiquitination of the protein and its proteasomal degradation. In this review we discuss the molecular medicine behind PROTAC mechanism of action, with special emphasis on recent developments and their potential translation to the clinical setting., This work has been supported by Instituto de Salud Carlos III (PI19/00808), ACEPAIN; Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A. Ocaña). Ministry of Economy and Competitiveness of Spain (BFU2015–71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A. Pandiella). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER).

The inhibition of bromo- and extraterminal domains (BET) has shown an anti-proliferative effect in triple negative breast cancer (TNBC). In this article we explore mechanisms of resistance to BET inhibitors (BETi) in TNBC, with the aim of identifying novel ways to overcome such resistance. Two cellular models of acquired resistance to the BET inhibitor JQ1 were generated using a pulsed treatment strategy. MTT, colony formation, and cytometry assays revealed that BETi-resistant cells were particularly sensitive to PLK1 inhibition. Targeting of the latter reduced cell proliferation, especially in resistant cultures. Quantitative PCR analysis of a panel of mitotic kinases uncovered an increased expression of AURKA, TTK, and PLK1, confirmed by Western blot. Only pharmacological inhibition of PLK1 showed anti-proliferative activity on resistant cells, provoking G2/M arrest, increasing expression levels of cyclin B, pH3 and phosphorylation of Bcl-2 proteins, changes that were accompanied by induction of caspase-dependent apoptosis. JQ1-resistant cells orthotopically xenografted into the mammary fat pad of mice led to tumours that retained JQ1-resistance. Administration of the PLK1 inhibitor volasertib resulted in tumour regression. These findings open avenues to explore the future use of PLK1 inhibitors in the clinical setting of BETi-resistant patients., This work has been supported by Instituto de Salud Carlos III (PI16/01121 and PI19/00808), ACEPAIN; Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A. Ocaña). Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A. Pandiella). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER). J.C. Montero is a recipient of a Miguel Servet fellowship program (CP12/03073 and CPII17/00015) and receives research support from the ISCIII (grants PI15/00684 and PI18/00796). E.M. Galan-Moya is funded by the implementation research program of the UCLM (UCLM resolution date: July 31, 2014), with a contract for accessing the Spanish System of Science, Technology and Innovation-SECTI (co-funded by the European Commission/FSE funds) and Council of Communities of Castilla-La Mancha (JCCM) (SBPLY/19/180501/000173). M. Burgos is funded by the Spanish Ministry for Science and Innovation, ref: BFU2015-69874-R and Castilla-La Mancha support grant for Biomedicine and Health Science Research ref: II-2018_11.

There is an unmet need for new therapies in metastatic ovarian cancer and basal-like breast cancer since no curative therapies are currently available. Immunotherapy has shown to be active in several solid tumors, but particularly more in those where a pre-activated immune state does exist. In this work, we aim to identify biomarkers that could distinguish immune-activated tumors and predict response to therapies in ovarian and basal-like breast cancer, as well as their association with the level of tumor immune infiltration. We found that the combined expression of IFNG, CD30, CXCL13, and PRF1 correlated with better overall survival (OS) in advanced stage ovarian cancer. This was confirmed using an independent dataset from TCGA. Interestingly, we observed that this gene combination also predicted for better prognosis in ovarian tumors with low mutational load, which typically respond less to immunotherapy. Expression of IFNG, CD30, CXCL13, and PRF1 was associated with increased level of immune infiltrates (CD8+ T cells, dendritic cells, and neutrophils) within the tumor. Moreover, we found that these gene signature also correlated with an increased OS and with a higher level of tumor immune infiltrates (B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells) in basal-like breast cancer. In conclusion, our analysis identifies genes signatures with potential to recognize immune activated ovarian and basal-like breast cancers with favorable prognosis and with a remarkable predictive capacity in tumors with low mutational burden. The presented results led to a hypothesis being formulated, but prospective clinical studies are needed to support a potential clinical application., This work has been supported by Instituto de Salud Carlos III (PI16/01121 and PI19/00808), ACEPAIN; Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to AO). Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to AP). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER).

Antibody–drug conjugates (ADC) are multicomponent molecules constituted by an antibody covalently linked to a potent cytotoxic agent. ADCs combine high target specificity provided by the antibody together with strong antitumoral properties provided by the attached cytotoxic agent. At present, four ADCs have been approved and over 60 are being explored in clinical trials. Despite their effectiveness, resistance to these drugs unfortunately occurs. Efforts to understand the bases underlying such resistance are being carried out with the final purpose of counteracting them. In this review, we report described mechanisms of resistance to ADCs used in the clinic along with other potential ones that may contribute to resistance acquisition. We also discuss strategies to overcome resistance to ADCs., This work was supported by the Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R to A. Pandiella and BES-2013-065223 to S. GarcíaAlonso); the Junta de Castilla y Leon (CSI002U16); the Scienti fic Foundation of the AECC and the CRIS Foundation (to A. Pandiella). Work in A. Ocana~ laboratory is supported by the Instituto de Salud Carlos III (PI16/01121); ACEPAIN; Diputacion de Albacete; and the CRIS Cancer Foundation. The work carried out in our laboratories receives additional support from the European Community through the regional development funding program (FEDER).

In breast cancer, it is unclear the functional modifications at a transcriptomic level that are associated with the evolution from epithelial cells and ductal carcinoma in situ (DCIS) to basal-like tumors. By applying weighted gene co-expression network analysis (WGCNA), we identified 17 gene co-expression modules in normal, DCIS and basal-like tumor samples. We then correlated the expression pattern of these gene modules with disease progression from normal to basal-like tumours and found eight modules exhibiting a high and statistically significant correlation. M4 included genes mainly related to cell cycle/division and DNA replication like CCNA2 or CDK1. The M7 module included genes linked with the immune response showing top hub genes such as CD86 or PTPRC. M10 was found specifically correlated to DCIS, but not to basal-like tumor samples, and showed enrichment in ubiquitination or ubiquitin-like processes. We observed that genes in some of these modules were associated with clinical outcome and/or represented druggable opportunities, including AURKA, AURKB, PLK1, MCM2, CDK1, YWHAE, HSP90AB1, LCK, or those targeting ubiquitination. In conclusion, we describe relevant gene modules related to biological functions that can influence survival and be targeted pharmacologically., Instituto de Salud Carlos III (PI16/01121), CIBERONC, ACEPAIN; Diputación de Albacete and CRIS Cancer Foundation (to AO). Sociedad Española de Oncología Médica (SEOM), beca estancias en extranjero to AO. BAE (Beca Ampliación de Estudios) to AO. The work carried out in the EU laboratories receive support from the European Community through the regional development funding program (FEDER). CIBERONC to AO. BG was supported by the NVKP_16-1-2016-0037 grant of the National Research, Development and Innovation Office, Hungary. We thank Nenad Sestan for supporting us with grant MH106934.

Protein ubiquitination and degradation represent druggable vulnerabilities of cancer cells. We used gene expression and functional annotation analyses to identify genes in the ubiquitin pathway which are differentially expressed between normal breast and basal-like tumors. With this approach we identified 16 ubiquitin related genes overexpressed in basal-like breast cancers compared with normal breast. We then explored the association between these genes and outcomes using the KMPlotter online tool. Two genes, the ubiquitin-conjugating enzyme E2T (UBE2T) and the denticleless protein homolog (DTL) were overexpressed and linked with detrimental outcome in basal-like and luminal breast cancer patients. Furthermore, we found that UBE2T and DTL were amplified in around 12% of breast tumors based on data contained at cBioportal. In non-small cell lung adenocarcinomas, UBE2T and DTL were also amplified in around 7% of cases and linked with disease recurrence after surgical resection. No significant molecular alterations or a clear trend for clinical outcome was observed for these genes in ovarian serous cystadenocarcinoma, esophagus-stomach cancer or non-small squamous cell carcinoma. Our data suggest that UBE2T and DTL may have a role in the pathophysiology of breast and lung tumors, opening avenues for future clinical evaluation of agents targeting those proteins or their pathways.

Metastatic dissemination of tumor cells is responsible for the fatal outcome of breast cancer. Therefore, understanding the mechanisms involved in dissemination is essential for the development of new therapeutic strategies to prevent metastasis. One mechanism involved in metastatic dissemination of breast cancer cells is dependent on control of the production of matrix metalloproteinases by the neuregulins (NRGs). The NRGs are polypeptide factors that act by binding to the ErbB/HER subfamily of receptor tyrosine kinases. NRG-mediated activation of HER receptors causes an increase in the production of metalloprotease 13 (MMP13, also termed collagenase-3), which facilitates metastatic dissemination of breast tumors. In this context, we aimed to explore whether the clinically approved tyrosine kinase inhibitor dasatinib was able to neutralize this mechanism of metastatic dissemination. Here, we show that dasatinib restricted NRG-induced MMP13 upregulation, both in vitro and in vivo, and in vivo metastatic dissemination of breast cancer cells. Chemical proteomics studies showed that the main cellular targets of dasatinib were SRC family kinases (SFKs). Moreover, genetic studies showed that knockdown of SRC or YES strongly inhibited NRG-induced MMP13 upregulation in vitro. Mechanistically, dasatinib treatment or knockdown of SRC also inhibited ERK1/2 kinases in vitro, which were required for NRG-induced MMP13 upregulation. These results open the possibility of clinically exploring the antitumoral action of dasatinib in those tumors in which the NRG–MMP13 signaling axis may play a relevant role in the control of tumor cell dissemination., AOR is the recipient of a predoctoral contract from the University of Salamanca. JCM is funded by the Instituto de Salud Carlos III through a Miguel Servet program (CP12/03073) and receives research support from the same institution (PI15/00684). The authors’ research in AP laboratory is supported by grants from the Ministry of Economy and Competitiveness of Spain (BFU2012-39151 and BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003), the Scientific Foundation of the Spanish Association Against Cancer (AECC), and the CRIS Cancer Foundation. AO laboratory is funded by the Instituto de Salud Carlos III (PI16/01121), Diputacion de Albacete, and ACEPAIN. The IBMCC-CIC and the work carried out at our laboratories receive support from the European Community through the Regional Development Funding Program (FEDER).

Tumoral cells not only depend on oncogenic abnormalities to maintain its malignant phenotype but on non-oncogenic vulnerabilities. Targeting epigenomics can modify specific cellular functions required for malignant transformation. The Bromodomain (BRD) family mediates their effect by recruiting proteins of the transcription machinery, recognizing acetylated-lysine residues in nucleosomal histones. Bromodomain and extra-terminal (BET) inhibitors have shown to produce growth inhibition in several tumors through the inhibition of the expression of several transcription factors. In this review we will discuss the current knowledge regarding BET inhibitors in breast cancer. Recent data demonstrates their antiproliferative effect in several cancer subtypes, including the triple negative subtype, or when combined with cell signaling inhibitors. We will also describe options for therapeutic combinations or potential mechanisms of resistance, with special emphasis on their future clinical development., Instituto de Salud Carlos III (PI13/01444), ACEPAIN; Diputación de Albacete and CRIS Cancer Foundation (to AO). BAE (Beca Ampliación de Estudios) to AO for his stay at Yale University, CT, USA. Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to AP). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER).

Background: A proportion of ER-positive, HER2-negative patients treated with both adjuvant endocrine and chemotherapy continue to remain high risk for recurrence. These individuals are the ideal patient population for future adjuvant trials. Methods: We used gene expression data from ER+/HER2- patients who received adjuvant endocrine and chemotherapy. We utilized a training cohort of multiple gene expression datasets (N=131; GSE16391, GSE21653, GSE17907, GSE19615, GSE16716, GSE45255) and a validation cohort (N=277; GSE25066) from similarly treated ER+ patients. All data sets were generated with Affymetrix HGU133 arrays. Cox proportional hazards regression was performed across all genes using all possible cutoff values between the lower and upper quartile. Genes were ranked according to the estimated hazard rates for relapse. The top three genes were combined into a final signature that used the mean expression of these genes as the predictive score. Statistical analyses were performed in the R environment. Results: The final signature capable to predict survival consisted of three genes, MX1, MBD4, and ZWINT. Each of these genes achieved high significance in a univariate analysis: MX1 HR=5.7 (p=1.8E-04) and HR=4.7 (p=1.3E-03), MBD4 HR=3.4 (p=2.8E-04) and HR=4.3 (p=4.4E-04), and ZWINT HR=4.4 (p=1E-03) and HR=3.7 (p=8.5E-03) in the training and validation cohorts, respectively. Using the mean expression of the three genes, the estimated hazard rates for survival were 20.5 (p=2.5E-05) and 17.5 (p=1E-04) in the two cohorts, respectively. Only one patient relapsed in each cohort. In a multivariate analysis including grade and lymph node status as covariates (size was not available), the three-gene signature retained significance (HR=13.2, p=0.011; and HR=13.9, p=0.01 in the two cohorts, respectively), while only lymph node status (HR=3.6, p=0.007) was significant in the validation cohort and no clinical variable was significant in the training cohort. Conclusions: High expression of a 3-gene signature can identify ER+ patients who remain at very high risk for recurrence despite current adjuvant endocrine and chemotherapy. Citation Format: Györffy B, Ocana A, Herman P, Hatzis C, Pandiella A, Pusztai L. Three-gene signature predictive of high residual risk of recurrence after adjuvant chemotherapy in ER-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-49.

[Introduction]: Therapeutic targeting of inhibitors of the immune response has reached the clinical setting. Inhibitors of the novel receptor LAG3, which negatively regulates T-cell activation, are under investigation. Here we explore the presence and prognostic role of LAG3 in cancer., [Methods]: A systematic search of electronic databases identified publications exploring the effect of LAG3 on overall survival (OS) and (for early-stage cancers) disease-free survival (DFS). Hazard ratios (HR) were pooled in a meta-analysis using generic inverse-variance and random effect modeling. Subgroup analyses were conducted based on disease site and tumor type., [Results]: Fifteen studies met the inclusion criteria. LAG3 was associated with better overall survival [HR 0.81, 95% confidence interval (CI) 0.66–0.99; P = 0.04], with subgroup analysis showing no significant differences between disease-site subgroups. The beneficial effect of LAG3 on OS was of greater magnitude in early-stage malignancies (HR 0.73, 95% CI 0.60–0.88) than in the metastatic setting (HR 1.20, 95% CI 0.70–2.05), but this difference was not statistically significant (subgroup difference p = 0.18). LAG3 did not have a significant association with DFS [HR 1.02, 95% confidence interval (CI) 0.77–1.37; P = 0.87], with subgroup analysis showing worse DFS in patients with lymphoma and improved DFS in those with breast cancer., [Conclusions]: High expression of LAG3 is associated with favorable overall survival in several solid tumors. A trend toward an association in early-stage disease suggests the importance of immune surveillance in this setting.

Antigen recognition by MHC class I molecules is a key step for the initiation of the immune response. We hypothesized that expression of these molecules could be a marker of immune-activated breast cancers. Data from KM Plotter were extracted to develop an exploratory cohort. Information from Cancer Genome Atlas (TCGA) and METABRIC was used to create two validation cohorts. Raw data were re-processed and analyzed using plyr R and Bioconductor. We predicted epitope-HLA binding to MHC I molecules by using NetMHC 4.0. Cox proportional hazards regression was computed to correlate gene expression and survival outcome. There was a weak but positive correlation between mutational burden and the expression of most MHC class I molecules. In the exploratory cohort, expression of HLA-A and HLA-B was associated with favorable relapse-free survival (RFS) and overall survival (OS) in the basal-like subgroup. This was confirmed in the METABRIC and TCGA dataset. Expression of HLA-A and HLA-B was associated with biomarkers of T cell activation (GZMA, GZMB, and PRF1) and improved the predictive capacity of known immunologic signatures. Several neopeptides expressed in breast cancer were also identified including FUK, SNAPC3, GC, ANO8, DOT1L, HIST1H3F, MYBPH, STX2, FRMD6, CPSF1, or SMTN, among others. Expression of HLA A and B is associated with T cell activation and identifies immune activated, basal-like breast cancers with favorable prognosis. Antigen recognition markers should be incorporated into the assessment of the tumor immune state of basal-like breast patients.

[Background] Triple negative breast cancer (TNBC) is an incurable disease where novel therapeutic strategies are needed. Proteolysis targeting chimeric (PROTAC) are novel compounds that promote protein degradation by binding to an ubiquitin ligase. In this work, we explored the antitumoral activity of two novel BET-PROTACs, MZ1 and ARV-825, in TNBC, ovarian cancer and in a BET inhibitor resistant model., [Methods] OVCAR3, SKOV3, BT549, MDA-MB-231 cell lines and the JQ1 resistant cell line MDA-MB-231R were evaluated. MTTs, colony-forming assay, three-dimensional cultures in matrigel, flow cytometry, and western blots were performed to explore the anti-proliferative effect and biochemical mechanism of action of MZ1 and ARV-825. In vivo studies included BALB/c nu/nu mice engrafted with MDA-MB-231R cells., [Results] The BET-PROTACs MZ1 and ARV-825 efficiently downregulated the protein expression levels of the BET protein BRD4, in MDA-MB-231 and MDA-MB-231R. MZ1 and ARV-825 also showed an antiproliferative effect on sensitive and resistant cells. This effect was corroborated in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR3) cell lines. MZ1 provoked G2/M arrest in MDA-MB-231. In addition, a profound effect on caspase-dependent apoptosis was observed in both sensitive and resistant cells. No synergistic activity was observed when it was combined with docetaxel, cisplatin or olaparib. Finally, in vivo administration of MZ1 rescued tumor growth in a JQ1-resistant xenograft model, reducing the expression levels of BRD4., [Conclusions] Using both in vitro and in vivo approaches, we describe the profound activity of BET-PROTACs in parental and BETi-resistant TNBC models. This data provides options for further clinical development of these agents in TNBC., This work has been supported by Instituto de Salud Carlos III (PI16/01121), ACEPAIN; Diputación de Albacete, CIBERONC and CRIS Cancer Foundation (to A. Ocaña). Ministry of Economy and Competitiveness of Spain (BFU2015–71371-R and BFU2015–69874-r), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to A. Pandiella). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER). J.C. Montero is funded by the Instituto de Salud Carlos III through a Miguel Servet program (CPII17/00015) and receives research support from the same institution (PI15/00684 and PI18/00796). E.M. Galan-Moya is funded by the implementation research program of the UCLM (UCLM resolution date: 31/07/2014), with a contract for accessing the Spanish System of Science, Technology and Innovation-SECTI (co-funded by the European Commission/FSE funds). M. Burgos is funded by the Spanish Ministry for Science and Innovation, ref.: BFU2015–69874-R and Castilla-La Mancha support grant for Biomedicine and Health Science Research ref.: II-2018_11.

© 2019 by the authors., Paclitaxel is one of the most widely used chemotherapeutic agents thanks to its effectiveness and broad spectrum of antitumor activity. However, it has a very poor aqueous solubility and a limited specificity. To solve these handicaps, a novel paclitaxel-trastuzumab targeted transport nanosystem has been developed and characterized in this work to specifically treat cancer cells that overexpress the human epidermal growth factor receptor-2 (HER2). Methods: Alginate and piperazine nanoparticles were synthetized and conjugated with paclitaxel:β-cyclodextrins complexes and trastuzumab. Conjugated nanoparticles (300 nm) were characterized and their internalization in HER2-overexpressing tumor cells was analyzed by immunofluorescence. Its specific antitumor activity was studied in vitro using human cell lines with different levels of HER2-expression. Results: In comparison with free paclitaxel:β-cyclodextrins complexes, the developed conjugated nanovehicle presented specificity for the treatment of HER2-overpressing cells, in which it was internalized by endocytosis. Conclusions: It seems that potentially avoiding the conventional adverse effects of paclitaxel treatment could be possible with the use of the proposed mixed nanovehicle, which improves its bioavailability and targets HER2-positive cancer cells., This work was supported by a UE ERC Starting Grant (MYCAP. Development of a technology to produce microcapsules, based on the formation of drops from viscous non-Newtonians liquids sprayed through fan-jet nozzles, to use in cancer therapy), by the Spanish Ministry of Economy and Competitiveness (BFU2012-39151, BFU2015-71371R, CTQ2016-78988-R, PI15/00684, and FEDER), the CRIS Cancer Foundation and the Scientific Foundation of the Spanish Association Against Cancer (AECC). CN is recipient of a predoctoral contract from the Junta de Castilla y León, co-funded by the European Social Foundation (EDU/602/2016) and AC was supported by a doctoral fellowship from Coordination for the Improvement of Higher Education Personnel (CAPES) from the Ministry of Education of Brazil.

© 2019 Morcillo-Garcia et al., [Purpose]: Epigenetic regulating proteins like histone methyltransferases produce variations in several functions, some of them associated with the generation of oncogenic processes. Mutations of genes involved in these functions have been recently associated with cancer, and strategies to modulate their activity are currently in clinical development., [Methods]: By using data extracted from the METABRIC study, we searched for mutated genes linked with detrimental outcome in invasive breast carcinoma (n = 772). Then, we used downstream signatures for each mutated gene to associate that signature with clinical prognosis using the online tool “Genotype-2-Outcome” (http://www.g-2-o.com). Next, we performed functional annotation analyses to classify genes by functions, and focused on those associated with the epigenetic machinery., [Results]: We identified KMT2D, SETD1A and SETD2, included in the lysine methyltransferase activity function, as linked with poor prognosis in invasive breast cancer. KMT2D which codes for a histone methyltransferase that acts as a transcriptional regulator was mutated in 6% of triple negative breast tumors and found to be linked to poor survival. Genes regulated by KMT2D included RAC3, KRT23, or KRT14, among others, which are involved in cell communication and signal transduction. Finally, low expression of KMT2D at the transcriptomic level, which mirror what happens when KMT2D is mutated and functionally inactive, confirmed its prognostic value., [Conclusion]: In the present work, we describe epigenetic modulating genes which are found to be mutated in breast cancer. We identify the histone methyltransferase KMT2D, which is mutated in 6% of triple negative tumors and linked with poor survival., Instituto de Salud Carlos III (PI16/01121), ACEPAIN; Diputación de Albacete and CRIS Cancer Foundation (to AO). Ministry of Economy and Competitiveness of Spain (BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and CIBERONC, the scientific foundation of the AECC and the CRIS Foundation (to AP). The work carried out in our laboratories receive support from the European Community through the regional development funding program (FEDER). E.M. Galan-Moya is funded by the implementation research program of the UCLM (UCLM resolution date: 31/07/2014), with a contract for accessing the Spanish System of Science, Technology and Innovation-Secti (co-funded by the European Commission/FSE funds).

The appearance of resistance to the anti-HER2 targeted drug trastuzumab constitutes, nowadays, an important challenge in the oncology clinic. To fight such resistance, we searched for potential vulnerabilities in cells resistant to that drug. To that end, we used cell lines primary resistant to trastuzumab, as well as cells made secondarily resistant to the drug upon continuous exposure. Using genomic and proteomic approaches, a deregulation in cell death pathways was identified in trastuzumab-resistant cells. More precisely, an increased response to the death factor TRAIL, caused by an increase in the cellular receptors for this factor, was observed. In parallel, a decrease in inhibitory components of the pathway was detected. This combination produces a more efficient assembly of the functional complex in the trastuzumab-resistant cells that translates in the observed increased response to TRAIL. Analysis of HER2 positive patient samples confirmed deregulation of this pathway in trastuzumab-resistant patients. Taken together our data identify a vulnerability of trastuzumab-resistant cells that could be used to design new targeted therapies in that context., This work was supported by the Ministry of Economy and Competitiveness of Spain (BFU2012-39151 and BFU2015-71371-R), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003), the Scientific Foundation of the Spanish Association Against Cancer (AECC), the Memoria de D. Samuel Solorzano Barruso Foundation and the CRIS Cancer Foundation. The Cancer Research Institute and the work carried out at our laboratory receive support from the European Community through the Regional Development Funding Program (FEDER).

Bryonia dioica Jacq. is a climbing perennial herb with tuberous roots which is widely used in traditional medicine in Algeria for the treatment of cancer. The present study aimed to evaluate the apoptogenic activity and phytochemical composition of the aqueous extract of B. dioica roots growing in Algeria. The cytotoxic effect of B. dioica aqueous decoction against breast cancer MDA‑MB‑231 cells was evaluated by an MTT assay. Apoptosis induction was assessed by an Annexin V‑fluorescien iosthiocyanate assay. Propidium iodide staining of cell DNA was used to assess the effects on the cell cycle. In addition to UV‑Visible (UV‑vis) analysis, the major compounds of the extracts were determined using liquid chromatography‑mass spectrometric analyses. Our results showed that the B. dioica aqueous extract induced cell death in a time‑dependent manner. The highest inhibitory effect was produced at concentrations of 50 µg/ml or higher after 72 h of treatment (91.15±0.71%). Furthermore, the extract induced apoptosis of MDA‑MB‑231 cells. At 250 µg/ml, 64.61% of the treated MDA‑MB‑231 cells were apoptotic. This was accompanied by cell cycle arrest at G2/M phase. The percentage of cells in G2/M increased from 15.7% (untreated cells) to 59.13% (50 µg/ml) and 58.51% (250 µg/ml). The UV‑vis absorption spectra of B. dioica aqueous extract showed two absorption bands characteristic of the flavonol skeleton; 350‑385 nm (Band I) and 250‑280 nm (Band II). Myricetin (2,5,7,3,4,5‑pentahydroxylflavonol) was found to be the major compound in the B. dioica aqueous extract. These findings suggest that B. dioica could be considered a promising source for developing novel therapeutics against breast cancer.

© 2020 by the authors., Nanoparticles (NPs) are promising drug delivery systems (DDS) for identifying and treating cancer. Active targeting NPs can be generated by conjugation with ligands that bind overexpressed or mutant cell surface receptors on target cells that are poorly or not even expressed on normal cells. Receptor-mediated endocytosis of the NPs occurs and the drug is released inside the cell or in the surrounding tissue due to the bystander effect. Antibodies are the most frequently used ligands to actively target tumor cells. In this context, antibody-based therapies have been extensively used in HER2+ breast cancer. However, some patients inherently display resistance and in advanced stages, almost all eventually progress. Functionalized NPs through conjugation with antibodies appear to be a promising strategy to optimize targeted therapies due to properties related to biocompatibility, suitable delivery control and efficiency of functionalization. This review is focused on the different strategies to conjugate antibodies into polymeric NPs. Recent antibody conjugation approaches applied to the improvement of breast cancer therapy are highlighted in this review., We gratefully acknowledge financial support from Ministerio de Ciencia e Innovación, Spain (Grant Nos. CTQ2017-84131-R and CTQ2016-81797-REDC Programa Redes Consolider) (to C. Alonso-Moreno), and Instituto de Salud Carlos III (PI19/00808), CIBERONC and CRIS Cancer Foundation (to A. Ocaña)

1027 Background: Basal-like breast cancer is a specific subtype of breast tumors with limited therapeutic options. Although treatment with the anti-PD-L1 antibody atezolizumab has recently shown clinical activity in this setting, not all patients do respond even expressing high levels of PD-L1. In the present article we explored the presence of mutations in breast cancer tumors with high expression of PD1 and PD-L1 with the aim to identify molecular correlates associated with outcome. Methods: We used RNA-seq and mutational data from 971 breast cancer patients using the TCGA dataset, to identify mutations in patients with high levels of PD1 and PD-L1. Data analysis was performed using DESeq R and MAFTools Bioconductor packages. Transcriptomic signatures from the identified mutations were associated with outcome using the Kapplan-Meyer Plotter tool. We correlated the identified transcripts with immune populations using TIMER online tool and correlation between genes with Cancertool online platform. Results: We identified co-occurrent mutations in RYR2 and AHNAK in 8% and 5% of basal like tumors, respectively, in patients with high levels of PD1 and PD-1. The transcriptomic signature of these mutations conferred good prognosis for relapse free survival (RFS) and overall survival (OS). CXCL9 for RYR2 and GBP5, C1QA, IL2RG, CSF2RB and IDO1 for AHNAK were the most relevant genes identified in these signatures. Expression of CXCL9, GBP5, IL2RG and IDO1 correlated with the presence of immune cell populations mainly dendritic cells. This signature, including CXCL9, GBP5, C1QA, IL2RG, CSF2RB and IDO1 classified patients with favorable RFS (HR 0.27 CI 0.2-0.30; p = 1.1e-16) and OS (HR 0.18 CI 0.09-0.34; p = 6.8e-9). This signature showed a stronger prediction capacity compared with already described immunologic signatures. Finally we identify that LAG3 was the only gene commonly present in both signatures and correlated positively with the expression of PD1 and PD-L1. Conclusions: We describe two novel mutations which transcriptomic signatures associated with favorable outcome in basal-like tumors that express elevated levels of PD1 and PD-L1. Future studies should be performed to confirm the role of these mutations and signatures in relation with clinical activity of PD1/PD-L1 inhibitors.

[Background]: An important proportion of HER2-positive metastatic breast cancer patients do not respond to trastuzumab. The combination of dasatinib and trastuzumab has shown to be synergistic in preclinical models., [Methods]: We conducted a phase II trial combining dasatinib 100 mg once daily with trastuzumab 2 mg/kg and paclitaxel 80 mg/m2 weekly. Primary objective was objective response rate (ORR) and secondary included safety, other efficacy parameters and pharmacodynamics in tumour tissue, blood samples and skin biopsies., [Results]: From June 2013 to December 2015, 29 patients were included. Median number of cycles was 12 (1–49). Only 6 patients discontinued due to adverse events. ORR was 79.3% (95% CI 60.3–92), clinical benefit rate 82.8% (95% CI 64.2–94.2). Median time to progression 23.9 months (95% CI 14.9–not reached [NR]), median progression-free survival 23.9 months (95% CI 10.3–NR). No grade 4 toxicity was seen. Grade 3 toxicities included: ejection fraction decrease, neutropenia, hyponatremia, fatigue and sensory neuropathy and one left ventricular systolic dysfunction. Phosphorylated (p)-SRC was reduced in peripheral blood mononuclear cells. Phosphorylated SRC, ERK and AKT were also reduced in epidermal keratinocytes., [Conclusions]: Dasatinib can be safely combined with trastuzumab and paclitaxel. The combination is active with an ORR of almost 80%. Trial registration: NCT01306942, EudraCT 2010-023304-27., The study was financially supported by Bristol-Myers Squibb which also supplied the dasatinib.