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© 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).

[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.

[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.

[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.

Identification of druggable vulnerabilities is a main objective in triple‐negative breast cancer (TNBC), where no curative therapies exist. Gene set enrichment analyses (GSEA) and a pharmacological evaluation using a library of compounds were used to select potential druggable combinations. MTT and studies with semi‐solid media were performed to explore the activity of the combinations. TNBC cell lines (MDAMB‐231, BT549, HS‐578T and HCC3153) and an additional panel of 16 cell lines were used to assess the activity of the two compounds. Flow cytometry experiments and biochemical studies were also performed to explore the mechanism of action. GSEA were performed using several data sets (GSE21422, GSE26910, GSE3744, GSE65194 and GSE42568), and more than 35 compounds against the identified functions were evaluated to discover druggable opportunities. Analyses done with the Chou and Talalay algorithm confirmed the synergy of dasatinib and olaparib. The combination of both agents significantly induced apoptosis in a caspase‐dependent manner and revealed a pleotropic effect on cell cycle: Dasatinib arrested cells in G0/G1 and olaparib in G2/M. Dasatinib inhibited pChk1 and induced DNA damage measured by pH2AX, and olaparib increased pH3. Finally, the effect of the combination was also evaluated in a panel of 18 cell lines representative of the most frequent solid tumours, observing a particularly synergism in ovarian cancer. Breast cancer, triple negative, dasatinib, olaparib, screening., 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). M. Noblejas‐López was supported by Spanish Ministry of Education (FPU grant; Ref: FPU18/01319). JC Montero is a recipient of a Miguel Servet fellowship program (CP12/03073) and receives research support from the ISCIII (grants PI15/00684). M. Burgos is funded by Castilla‐La Mancha support grant for Biomedicine and Health Science Research ref.: II‐2018_11. EM 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).

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a time-dependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl-Stat5 related malignancies.

Human Chronic Myelogenous Leukemia (CML) is a hematological stem cell disorder which is associated with activation of Bcr-Abl-Stat5 oncogenic pathway. Direct Bcr-Abl inhibitors are initially successful for the treatment of CML but over time many patients develop drug resistance. In the present study, the effects of CM363, a novel naphthoquinone (NPQ) derivative, were evaluated on human CML-derived K562 cells. CM363 revealed an effective cell growth inhibition (IC50 = 0.7 ± 0.5 μM) by inducing cancer cells to undergo cell cycle arrest and apoptosis. CM363 caused a dose- and time-dependent reduction of cells in G0/G1 and G2/M phases. This cell cycle arrest was associated with increased levels of cyclin E, pChk1 and pChk2 whereas CM363 downregulated cyclin B, cyclin D3, p27, pRB, Wee1, and BUBR1. CM363 increased the double-strand DNA break marker γH2AX. CM363 caused a timedependent increase of annexin V-positive cells, DNA fragmentation and increased number of apoptotic nuclei. CM363 triggered the mitochondrial apoptotic pathway as reflected by a release of cytochrome C from mitochondria and induction of the cleavage of caspase-3 and -9, and PARP. CM363 showed multikinase modulatory effects through an early increased JNK phosphorylation followed by inhibition of pY-Bcrl-Abl and pY-Stat5. CM363 worked synergistically with imatinib to inhibit cell viability and maintained its activity in imatinib-resistant cells. Finally, CM363 (10 mg/Kg) suppressed the growth of K562 xenograft tumors in athymic mice. In summary, CM363 is a novel multikinase modulator that offers advantages to circumvent imanitib resistance and might be therapeutically effective in Bcrl-Abl- Stat5 related malignancies., This research has been supported by the Spanish Ministry of Science and Innovation (SAF2009-13296) and MINECO (SAF2012-37344, SAF2014-53526R, and SAF2015-65113-C2-2-R) with the co-funding of European Regional Development Fund (ERDF). This Project has been also supported by Centro Atlántico del Medicamento S.A. (CEAMED; www.ceamedsa.com) and Alfredo Martín-Reyes Foundation (Arehucas)-Canary Islands Foundation for Cancer Research (FICIC).

Background: Attention Deficit and Hyperactivity Disorder (ADHD) is one of the most common behavioral disorders of childhood, its prevalence in Spain is estimated at 6.8%. Studies have shown an increase in the use of ADHA medications. The aim of this study was to learn the pattern and the evolution of ADHD medication consumption, and estimate the prevalence in Castile-La Mancha (Spain). Methods: The prevalence of ADHD is estimated by calculating the Defined Daily Dose per 1000 inhabitants and day (DHD) of the total medication for ADHD (Therapeutic group N06BA) during the years 1992 to 2015. The trend its shift points and the Annual Percentages of Change (APC) using regression join point. The prevalence estimated is calculated with a model ARIMA. Results: We estimated a prevalence of ADHD 13.22 cases per 1000 inhabitants per day for Castile-La Mancha in the population aged 5 to 19 years in 2015, the consumption total has increased a 98.93%. The consumption has increased by 10.3% APC from 1992 to 2015 with several years or join points (2000, 2009 and 2012). The prevalence estimated in 2020 is 14.11 cases per 1000 inhabitants per day.

Background Metastatic ovarian and basal-like breast cancers are incurable diseases with limited therapeutic options. In our study, we explored synergistic interactions of Chk1 inhibitors (rabusertib and SAR020106) with approved therapies in these indications. In addition, we evaluated the role of these agents to overcome platinum resistance. Methods We evaluate the effect of rabusertib and chemotherapic agents in ovarian (OVCAR3, OVCAR8, IGROV1, SKOV3) and breast (MDA-MB-231, HS578T, BT549, HCC3153) cancer cell lines. We also use the commercially available cell line A2780Rcis and a breast cancer model created in our laboratory, MDA-MB231Rcis. Results The combination of platinum agents, cisplatin and carboplatin was synergistic with Chk1 inhibitors rabusertib and SAR020106 in most of basal-like and ovarian cancer cell lines. The combination of rabusertib with gemcitabine was very synergistic in basal-like cell lines but not in ovarian cancer. Doxorubicin, another DNA damaging agent, was also synergistic in ovarian cell lines, but showed less activity in the breast models. Combinations with topotecan were slightly synergistic only in basal-like tumors. The combination of rabusertib with agents that target mitosis were not synergistic. The combination of Chk1 inhibitor with platinum compounds or gemcitabine affects colony formation and tumor invasiveness. Combination of platinum agents and gemcitabine with rabusertib induced cell death that was mainly mediated by caspases. The effect of rabusertib on platinum resistance showed addition of Chk1 inhibitor to cisplatin induces cell death in cisplatin resistant cells. Conclusions Inhibition of Chk1 has a strong synergistic interaction with DNA damaging agents, including platinum compounds, gemcitabine and olaparib, on both tumor types. The effect of rabusertib with platinum agents and gemcitabine on colony growth and long-term effect confirms the effect on proliferation, invasion and long-term survival in basal-like and ovarian cancer cell lines. Addition of rabusertib to cisplatin induces cell death in all the cell lines including the resistant cells, demonstrate that resistance to cisplatin can be overcome by inhibition of Chk1 kinase. Legal entity responsible for the study Alberto Ocana. Funding Instituto de Salud Carlos III (PI16/01121) Diputacion de Albacete and CIBERONC CRIS Cancer Foundation cancer association ACEPAIN research program of the UCLM regional fellowship for Biomedicine and Health science research (II-2018_11). Disclosure All authors have declared no conflicts of interest.

Background Triple-negative breast cancer (TNBC) is the most aggressive among breast cancer subtypes, as these tumors frequently develop resistance to the treatment used. External signals provided by the surrounding tumor microenvironment (TM), which in mammary tumors is mainly constituted by adipose tissue (AT), control this resistance. Therefore, therapies that targets not only the cancer bulk but also its surrounding TM may be more effective. Preliminary in vitro and in vivo studies using mesenchymal stem cells from TNBC patient’s AT (MSCTNBC) showed how a conditioned medium (CM) prepared from MSCTNBC (MSCTNBC-CM) promoted tumorigenicity, invasion, and chemoresistance. In the present work, molecular mechanism will be investigated to identify novel druggable targets in TNBC. Methods TNBC cells (MDA-MB-231, BT549, and HS578T) were exposed to MSCTNBC-CM. The activation profile of tyrosine kinase receptors (RTKs) was evaluated using a commercial array. The effect of the inhibitors in the absence and presence of MSCTNBC-CM on TNBC cells recurrence potential, invasion, and cell death were evaluated in vitro through clonogenic, matrix invasion, and flow cytometry assays. Impact on tumour growth was evaluated in a MSCTNBC-TNBC preclinical model (BALB-nu mice). Results RTKs activation profile in response to MSCTNBC-CM revealed that TM secreted factors activates Src protein family (SFK) in TNBC. The use of the SFK inhibitor Dasatinib, both in vitro and in vivo, showed a marked reduction of invasion and recurrence potential, an induction of cell death, and a lower of tumour growth. Conclusions In this study, we describe SFK as mediators in the communication within the tumour adipose niche and provides fundamental information to understand TNBC progression, as well as its behavior in response to chemotherapy. The specific blockade of the SFK signaling pathway with Dasatinib can interrupt this communication and revert TM protective effect, resulting in death and smaller tumour size. Our results open the gate to the development of new strategies targeting TM to treat TNBC patients. Legal entity responsible for the study The authors. Funding Diputacion de Albacete. Disclosure All authors have declared no conflicts of interest.

Background Triple negative breast cancer (TNBC) is an incurable disease, therefore novel therapies are needed. Bromo and extraterminal domain inhibitors (BETi) are currently in clinical development with promising activity in TNBC. However, resistance to these agents will limit their efficacy. Proteolysis targeting chimeric (PROTAC) are novel compounds that by binding to a ubiquitin ligase promotes protein degradation. Methods In our study we explore the antitumoral activity of two BET-PROTACs, MZ1 and ARV-825 in TNBC and ovarian cancer. We used MDAMB-231, BT549 and MDAMB-231 JQ1 resistant TNBC cells lines; and OVCAR3 and SKOV3 ovarian cancer cells lines. We evaluated the antiproliferation effect using MTT assay, colony-forming assay and three-dimensional cultures in Matrigel. To explore the molecular mechanism of drugs we used flow cytometry (cell cycle and cell death). The analysis of protein expression was determined by western blot. In vivo studies included BALB/c nu/nu mice that were injected with MDAMB-231 JQ1 resistant cells and later treated with BETi JQ1 and BET-PROTAC MZ1. Results Both PROTACs efficiently and maintained deleted the levels of BRD4 in MDA-MB-231 and in the resistant JQ1 cell line MDA-MB-231R. MZ1 and ARV-825 showed a profound antiproliferative effect in MDA-MD-231 sensitive and resistant cells using 2D and 3D cultures, and had a similar activity in other triple negative (BT549) and ovarian cancer (SKOV3, OVCAR) cell lines. MZ1 slightly arrested cells at G2/M in sensitive and resistant JQ1 MDA-MB-231 cells. In addition, a profound effect on apoptosis was observed, that was more evident in the sensitive cell line, effect that was caspase dependent. Given the potent antiproliferative activity of MZ1 no synergistic activity was observed when combined with docetaxel, cisplation or olaparib. Finally, administration of MZ1 was able to recue tumor growth in nude mice of MDA-MB-231 JQ1 resistant cells, by reducing the expression of BRD4. Conclusions We describe the profound activity of BET-PROTACs in TNBC cell lines and in an in vivo BETi resistant model. This data provides options for further clinical development of these agents in TNBC. Legal entity responsible for the study The authors. Funding Instituto de Salud Carlos III (PI16/01121), ACEPAIN; Diputacion de Albacete, CIBERONC, CRIS Cancer Foundation, implementation research program of the UCLM System of Science, Technology and Innovation-Secti (co-funded by the European Commission/FSE funds) and scientific foundation of the AECC. Disclosure All authors have declared no conflicts of interest.

Basal-like breast cancer and ovarian cancer are considered as unmet entities as patient outcome in advanced stages is short, even receiving the most appropriate treatment. These tumors are characterized by a high grade of genetic instability and a notable impairment of DNA repair processes. Thus, in response to DNA damage, they depend on checkpoint arrest proteins, such as Chek1, to stop division. Inactivation of Chk1 permits cell cycle progression without a proper DNA repair, therefore increasing genetic instability and aneuploidy and eventually leading to cell death. Chk1 inhibitors are currently in clinical development and, therefore, the identification of chemotherapies that could be used as clinical partners to potentiate their effect is a main goal. OVCAR3 and IGROV ovarian cancer cells as well as the basal-like MDA-MB-231 and HS578T cell lines were used to screen for agents that may increase the antiproliferative properties of Chk1 inhibitors. Combination of DNA-damaging agents, like platinum compounds or gemcitabine, with the Chk1 inhibitors LY2603618 and SAR020106 had a synergistic effect, not observed when combined with agents targeting mitosis, like taxotere, eribulin or vinorelbine. A similar antiproliferative effect was observed for PARP inhibitors such as olaparib, reinforcing the concept that inhibition of Chk1 allows cells to enter apoptosis when a relevant genetic instability is present. Clonogenic assays and 3D colony formation studies confirmed the effect observed on proliferation also on invasion and long-term survival. Analyses of cell cycle, cell death and intracellular mediators were performed by flow cytometry and Western blot. The effect of the association of LY2603618 with cisplatin and gemcitabine did not change the cell cycle. However, combination of both agents produced a marked induction of apoptosis. Experiments evaluating caspase activity demonstrated that the principal mechanism associated with the induction of apoptosis was caspase dependent. We also explored combination of agents that have a clear translation to the clinics including double combinations with chemotherapies like cisplatin and gemcitabine. We observed that these triple combinations were more effective than double or single agents. Finally, we wanted to evaluate the antiproliferative effect when adding LY2603618 to olaparib in addition to a DNA-damaging agent. Of note, we observed a higher effect than single-agent olaparib or the chemotherapy. Interestingly, inhibition of Chk1 also reverted the effect of cisplatin in platinum-resistant cells. Our findings support the clinical development of Chk1 inhibitors in combination with DNA-damaging agents in basal-like and ovarian cancer. Citation Format: Eva M. Galan-Moya, Ana Alcaraz-Sanabria, Cristina Nieto-Jimenez, Veronica Corrales-Sanchez, Miriam Nuncia-Cantarero, Juan C. Montero, Atanasio Pandiella, Alberto Ocaña. Pharmacologic screening identifies synthetic lethality interactions with Chk1 inhibitors in basal-like breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1608.

Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of monoclonal plasma cells in the bone marrow (BM). Despite recent advances in the treatment, MM represents an incurable disease for which development of new therapies is required. We report the antimyeloma effect of NVP-AEW541, a small molecule that belongs to the pyrrolo[2,3-d]pyrimidine class, identified as a selective inhibitor of the insulin-like growth factor-I receptor (IGF-IR) in vitro kinase activity. NVP-AEW541 had a potent cytotoxic effect on fresh cells and in a murine MM model. NVP-AEW541 partially abrogated the proliferative advantage conferred by the coculture with BM stromal cells and the presence of growth factors produced by the BM microenvironment. In addition, NVP-AEW541 potentiated the action of drugs, such as bortezomib, lenalidomide, dexamethasone or melphalan. Moreover the triple combination of NVP-AEW541, dexamethasone and bortezomib resulted in a significant increase in growth inhibition. Mechanistic studies indicated that NVP-AEW541 provoked a marked cell cycle blockade accompanied by pRb downregulation. Interestingly, NVP-AEW541 increased the levels of p27 associated with a reduction in the CDK2 activity. Finally, NVP-AEW541 induced cell death through caspase-dependent and -independent mechanisms. All these data, suggest the potential effect of IGF-IR kinase inhibitors as therapeutic agents for MM patients. © 2008 The Authors., This work was supported by a grant from the Ministry of Education and Science of Spain (BFU2006-01813/BMC), and by an Institutional Cancer Center Network program from the ISCIII. Our Cancer Research Institute receives support from the European Community through the regional development funding program (FEDER). PM was supported by the FISFEDER through projects to JSM, and a Spanish Myeloma Network Program (G03/136). EMO was supported by the ‘Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I)’ and by ‘Instituto de Salud Carlos III-Fondo de Investigación Sanitaria’ with expedient no. 400001. MG was supported by the ‘Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I)’ and by ‘Instituto de Salud Carlos III-Fondo de Investigación Sanitaria’ with expedient no. 05/0279. FH, CGE, JZ are employed by Novartis Pharma AG, Basel, Switzerland, the manufacturer of NVP-AEW541.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Protein kinases play a central role in the oncogenesis of colorectal tumors and are attractive druggable targets. Detection of activated kinases within a tumor could open avenues for drug selection and optimization of new kinase inhibitors. By using a phosphokinase arrays with human colorectal tumors we identified activated kinases, including the Epidermal Growth Factor Receptor (EGFR), components of the PI3K/mTOR pathway (AKT and S6), and STAT, among others. A pharmacological screening with kinase inhibitors against these proteins helped us to identify a new kinase inhibitor, termed EC-70124 that showed the highest anti-proliferative activity in cell lines. EC-70124 also inhibited cell migration and biochemical experiments demonstrated its effect targeting the PI3K/mTOR pathway. This drug also arrested cells at G2/M and induced apoptosis. Experiments in combination with standard chemotherapy used in the clinical setting indicated a synergistic effect. EC-70124 also reduced tumor growth in vivo and inhibited pS6 in the implanted tumors. In conclusion, by studying the kinase profile of colorectal tumors, we identified relevant activated pathways, and a new multi-kinase compound with significant antitumor properties., Ministry of Economy and Competitiveness of Spain (BFU2012-39151 and RD12/0036/0003 to AP), and the AECC (to AP). ISCIII, Fondo de Investigación Sanitaria (PI13/01444), CRIS Cancer Foundation, Diputación Albacete and ACEPAIN (to AO). JCM is a recipient of a Miguel Servet fellowship program. Our translational research laboratory was initially supported by AECC.

Advances in the treatment of breast cancer have resulted in increased survival. However, in the metastatic setting, the disease remains incurable. Therefore, understanding of the mechanisms that promote dissemination of breast cancer cells may favor the development of novel therapeutic strategies to fight those tumors. Here, we show that the ErbB ligands, Neuregulins (NRGs), promote metastatic dissemination of breast cancer cells by switching on a kinase-metalloproteinase network. Clinicopathological analyses demonstrated that NRG expression in breast tumors associated to lymph node invasion and poor patient outcome. Preclinical in vivo analyses showed that NRG expression favored in situ tumor growth, local spreading and metastatic dissemination. Genomic, biochemical and functional studies identified matrix metalloproteinases, particularly stromelysin 2 and collagenase 3, as key mediators of the NRG-induced dissemination properties of breast cancer cells. Mechanistic analyses demonstrated that NRG augmented metalloproteinase expression through a route controlled by ERK1/2 kinases. ERK1/2 increased collagenase 3 expression by controlling the activity of an SBF1-related transcription factor. In conclusion, we describe a pathway linked to breast cancer dissemination. The clinical availability of agents that target some of the components of this signalling pathway suggests that patients with tumors fed by NRGs or other factors able to activate the ERK-Collagenase 3 route may benefit from agents that act on that signalling axis., Ministry of Economy and Competitiveness of Spain (BFU2009-07728 and BFU2012- 39151), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD06/0020/0041 and RD12/0036/0003) and the Scientific Foundation of the Spanish Association Against Cancer (AECC). SSR was supported by a Juan de la Cierva contract. JCM is a recipient of a Miguel Servet fellowship program.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Disseminated triple negative breast cancer (TNBC) is an incurable disease with limited therapeutic options beyond chemotherapy. Therefore, identification of druggable vulnerabilities is an important aim. Protein kinases play a central role in cancer and particularly in TNBC. They are involved in many oncogenic functions including migration, proliferation, genetic stability or maintenance of stem-cell like properties. In this article we describe a novel multi-kinase inhibitor with antitumor activity in this cancer subtype. EC-70124 is a hybrid indolocarbazole analog obtained by combinatorial biosynthesis of Rebeccamycin and Staurosporine genes that showed antiproliferative effect and in vivo antitumoral activity. Biochemical experiments demonstrated the inhibition of the PI3K/mTOR and JAK/STAT pathways. EC-70124 mediated DNA damage leading to cell cycle arrest at the G2/M phase. Pathway analyses identified several deregulated functions including cell proliferation, migration, DNA damage, regulation of stem cell differentiation and reversion of the epithelial-mesenchymal transition (EMT) phenotype, among others. Combination studies showed a synergistic interaction of EC-70124 with docetaxel, and an enhanced activity in vivo. Furthermore, EC-70124 had a good pharmacokinetic profile. In conclusion these experiments demonstrate the antitumor activity of EC-70124 in TNBC paving the way for the future clinical development of this drug alone or in combination with chemotherapy., Ministry of Economy and Competitiveness of Spain (BFU2012-39151 and RD12/0036/0003 to AP), and the AECC (to AP). Instituto de Salud Carlos III (PI13/01444) ACEPAIN and CRIS Cancer Foundation (to AO). JCM is a recipient of a Miguel Servet fellowship program (CP12/03073).

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Triple negative breast cancer (TNBC) is an aggressive form of breast cancer. Despite response to chemotherapy, relapses are frequent and resistance to available treatments is often observed in the metastatic setting. Therefore, identification of new therapeutic strategies is required. Here we have investigated the effect of the mithramycin analog EC-8042 (demycarosil-3D-β-D-digitoxosyl mithramycin SK) on TNBC. The drug caused a dose-dependent inhibition of proliferation of a set of TNBC cell lines in vitro, and decreased tumor growth in mice xenografted with TNBC cells. Mechanistically, EC-8042 caused an arrest in the G2 phase of the cell cycle, coincident with an increase in pCDK1 and Wee1 levels in cells treated with the drug. In addition, prolonged treatment with the drug also causes apoptosis, mainly through caspaseindependent routes. Importantly, EC-8042 synergized with drugs commonly used in the therapy of TNBC in vitro, and potentiated the antitumoral effect of docetaxel in vivo. Together, these data suggest that the mithralog EC-8042 exerts an antitumoral action on TNBC cells and reinforces the action of standard of care drugs used in the therapy of this disease. These characteristics, together with a better toxicology profile of EC-8042 with respect to mithramycin, open the possibility of its clinical evaluation., The Cancer Research Institute receives support from the European Community through the regional development funding program (FEDER), and from the Fundación Ramón Areces. Ministry of Economy and Competitiveness of Spain (BFU2009-07728 and BFU2012-39151), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003) and the Scientific Foundation of the Spanish Association Against Cancer (AECC) and the CRIS Foundation to AP. JCM is a recipient of a Miguel Servet fellowship program (CP12/03073). The work carried out by A.O receives support by Instituto de Salud Carlos III; Fondo de Investigación Sanitaria (FIS), PI13/01444.

e17089 Background: Ovarian cancer is characterized by frequent mutations at TP53. These tumors also harbor germline mutations at homologous recombination repair (HR) genes, so they rely on DNA-damage checkpoint proteins, like the Checkpoint kinase 1 (CHEK1) to induce G2 arrest. In our study, by using an in silico approach, we identified a synthetic lethality interaction between CHEK1 inhibitors and mitotic Aurora Kinase A and B (AURKA/B) inhibitors. Methods: Gene expression analyses were used for the identification of relevant biological functions. OVCAR3, OVCAR8, IGROV1 and SKOV3 were used for proliferation studies. Alisertib was tested as AURKA/B inhibitor and LY2603618 as CHEK1 inhibitor. Analyses of cell cycle and intracellular mediators were performed by flow cytometry and western-blot. Stem cell properties were evaluated with immunofluorescence. Results: Gene expression analyses followed by functional annotation identified cell cycle as a deregulated function. Identified druggable kinases within this function included AURKA/B, TTK kinase and CHEK1. CHEK1 and AURKA/B inhibitors showed a synergistic interaction in different cellular models. Combination of Alisertib and LY2603618 triggered apoptosis, reduced the stem cell population and increased the effect of taxanes and platinum compounds. AURKA and CHEK1 were amplified in 8.7% and 3.9% of ovarian cancers, respectively. Expression of these genes was linked with detrimental clinical outcome. Conclusions: Amplification of AURKA and CHEK1 was observed in more than 12% of ovarian tumors. Our data describes a synthetic lethality interaction between CHEK1 and AURKA/B inhibitors with potential translation to the clinic.

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License., [Background]: The androgen receptor (AR) plays a central role in the oncogenesis of different tumors, as is the case in prostate cancer. In triple negative breast cancer (TNBC) a gene expression classification has described different subgroups including a luminal androgen subtype. The AR can be controlled by several mechanisms like the activation of membrane tyrosine kinases and downstream signaling pathways. However little is known in TNBC about how the AR is modulated by these mechanisms and the potential therapeutic strategists to inhibit its expression. [Methods]: We used human samples to evaluate the expression of AR by western-blot and phospho-proteomic kinase arrays that recognize membrane tyrosine kinase receptors and downstream mediators. Western-blots in human cell lines were carried out to analyze the expression and activation of individual proteins. Drugs against these kinases in different conditions were used to measure the expression of the androgen receptor. PCR experiments were performed to assess changes in the AR gene after therapeutic modulation of these pathways. [Results]: AR is present in a subset of TNBC and its expression correlates with activated membrane receptor kinases-EGFR and PDGFRβ in human samples and cell lines. Inhibition of the PI3K/mTOR pathway in TNBC cell lines decreased notably the expression of the AR. Concomitant administration of the anti-androgen bicalutamide with the EGFR, PDGFRβ and Erk1/2 inhibitors, decreased the amount of AR compared to each agent given alone, and had an additive anti-proliferative effect. Administration of dihydrotestosterone augmented the expression of AR that was not modified by the inhibition of the PI3K/mTOR or Erk1/2 pathways. AR expression was posttranscriptionally regulated by PI3K or Erk1/2 inhibition. [Conclusion]: Our results describe the expression of the AR in TNBC as a druggable target and further suggest the combination of bicalutamide with inhibitors of EGFR, PDGFRβ or Erk1/2 for future development., This research is supported by: Fundación Sociosanitaria de Castilla La Mancha (FISCAM) Grant Number. PI/2010/017, Ministerio de Ciencia e Innovación, Grant Number BFU2009-07728/BMC, Fondo de Investigación Sanitaria (FIS), Grant Number PI09/02144, Cancer Center Network Program from the ISCIII (RD06/0020/0041). Fundación Científica AECC.

Daily mortality displays a seasonal pattern linked to weather, air pollution, photoperiod length, influenza incidence and diet, among which temperature ranks as a leading cause. This study thus sought to assess the relationship between temperature, relative humidity, wind speed and mortality in the Madrid Autonomous Region (Spain) for the period January 1986–December 1992, controlling for the effects of air pollution and influenza incidence. Daily data on maximum, minimum and 24-hour mean temperature, relative humidity and wind speed were matched against daily mortality. Transfer function was identified using the Box–Jenkins pre-whitening method. Multivariate time series regression models were used to control for the confounding effects of air pollution and influenza incidence. Separate seasonal analyses were carried out for winter and summer periods. A J-shaped relationship between outdoor temperature, relative humidity and daily mortality was found. Mortality proved to be inversely related to cold temperature (4- to 11-day lag) and directly related to warm temperature (1-day lag). High relative humidity during summer periods was negatively related to mortality. Thermal variation ascribable to Madrid's mesothermal Mediterranean climate was strongly related to daily mortality, even where air pollution and influenza incidence were controlled for.

mTOR is a serine/threonine kinase that acts by binding different sets of proteins forming two complexes, termed mTORC1 and mTORC2. mTOR is deregulated in a substantial proportion of ovarian tumors. Despite the use of drugs directed to mTOR in ongoing clinical trials, the functional relevance of the individual mTORC branches in ovarian cancer is not known. Here, we show that mTORC1 and mTORC2 were constitutively active in ovarian cancer cell lines. Knockdown of raptor or rictor, proteins required for the function of mTORC1 or mTORC2, respectively, resulted in profound inhibition of ovarian cancer cell proliferation. The knockdown of raptor had a more important inhibitory effect than the knockdown of rictor, indicating mTORC1 had a predominant role over mTORC2 in the control of ovarian cancer cell proliferation. Rapamycin decreased the proliferation of ovarian cancer cells, and this was accompanied by inhibition of the phosphorylation of S6, a protein used as readout of mTORC1 function. However, rapamycin had only amarginal effect on the phosphorylation status of 4E-BP1, anothermTORC1 substrate. Therefore, mTORC1 probably controls p4E-BP1 along two distinct pathways, one of them sensitive to rapamycin and another insensitive. The dual PI3K/mTOR inhibitor BEZ235 was more efficient than rapamycin in its inhibitory action on ovarian cancer cell proliferation. Biochemically, BEZ235 completely inhibited pS6, p4E-BP1, and pAkt. Our results suggest that broad-spectrum mTOR inhibitors that block mTORC1 and mTORC2 are more desirable for their clinical development in ovarian cancer than agents exclusively targeting one of the mTOR branches. ©2012 AACR., This work was supported by grants from the Ministry of Science and Innovation of Spain (BFU2009-07728/BMC; A. Pandiella) and by the Consejería de Sanidad de la Junta de Castilla y León (SAN196/SA03/ 07; J.C. Montero). X. Chen is supported by the Cancer Center Network Program from the Instituto de Salud Carlos III (RD06/0020/0041). Our Cancer Research Institute and the work carried out at our laboratory received support from the European Community through the Regional Development Funding Program(FEDER) and from the Fundación Ramón Areces.

Breast cancer is the most commonly diagnosed tumor in women. While important advances in this field led to progressive increases in survival, in the metastatic setting the disease remains incurable. Because of this, important efforts are being carried out to define the molecular alterations that drive the oncogenic properties of the disease, to therapeutically act on them. In an attempt to better categorize patients to act on their disease more efficiently, several genetic studies have classified breast tumors into different subgroups, according to their gene expression profiles (Curtis et al. Nature 2012; 486:346-52; Perou et al. Nature 2000; 406:747-52). However, gene expression profiling is still not widely used to define treatment of breast cancer, which is mostly guided by anatomopathological criteria (Dawson et al. EMBO J 2013; 32:617-28). Using this approach, breast tumors can be classified into three subgroups: hormone receptor positive (HR+), HER2 positive, or triple negative breast tumors (TNBC). Hormone positive tumors express estrogen and progesterone receptors and are highly sensitive to antihormonal therapies. HER2+ tumors are characterized by overexpression of the transmembrane tyrosine kinase and receive agents against HER2 such as antibodies or small molecule kinase inhibitors. TNBC are characterized by the lack of hormone receptors, and no overexpression of HER2. In contrast to the other two subtypes, no available target is known for patients with TNBC, and their actual treatment is based on classical chemotherapeutics. This fact very much limits successful therapy of TNBC, and for this reason major research efforts aimed at identifying etiopathogenic targets are being carried out (Turner and Reis-Filho, Clin Cancer Res 2013; 19:6380-8). Genomic studies have revealed deficiencies in DNA repair mechanisms in TNBC (Shah et al. Nature 2012; 486:395-9). As a consequence, agents targeting proteins involved in such mechanisms are under clinical evaluation (Juvekar et al. Cancer Discov 2012; 2:1048-63). How can we identify additional therapeutic vulnerabilities in this type of breast cancer? Phosphokinase profiling has recently been used to analyze the activation status of several oncogenically relevant kinases in TNBC (Montero et al. Oncogene 2014; 33:148-56). Using patient samples as well as representative TNBC cell lines, these studies indicated frequent activation of components of the PI3K/mTOR pathway as well as the ERK1/2 route in this type of tumors. Genetic as well as pharmacological studies indicated an important role of the PI3K/mTOR pathway in the control of the proliferation of TNBC cells both in vitro and in vivo. In contrast, targeting of the ERK1/2 route failed to substantially affect proliferation of TNBC cells. mTOR controls cellular functions through formation of multiprotein complexes, termed mTORC1 and mTORC2. When dissecting the importance of either complex in TNBC, mTORC1 seemed to have a more relevant role than mTORC2 in the control of cellular proliferation. However, when mTOR was neutralized, by either knock down or pharmacological inhibition, the effect on cell proliferation was greater than targeting of mTORC1 or mTORC2 individually. This is therapeutically relevant, since most agents being developed with clinical purposes, i.e. rapamycin and analogues, act mainly on mTORC1. The fact that targeting mTOR was more efficient than exclusively targeting mTORC1, should stimulate an effort to clinically develop mTOR inhibitors instead of mTORC1-directed rapalogs. The precise mechanisms by which the PI3K/mTOR route is frequently active in TNBC are still unclear. Interestingly, recent massive genomic studies identified certain genetic events which may activate this pathway, including PTEN mutation/deletion in 10%, and PIK3CA mutation in 8% of the tumors. Other genomic alterations which may impinge on this route include EGFR or FGFR amplification (4%) or HER2 mutations (2%) (Shah et al. Nature 2012; 486:395-9; Curtis et al. Nature 2012; 486: 346-52). Therapeutic inhibition of the PI3K/mTOR pathway in breast cancer is currently an active area of research. Indeed, at this moment several ongoing studies are evaluating PI3K/mTOR inhibitors in TNBC alone or in combination with chemotherapy. However the incorporation of PI3K/mTOR inhibitors into the clinic raises several questions: should PI3K inhibitors be given alone? Can/should they be combined with chemotherapy or targeted agents? How can we select responsive patients? The preclinical data suggest that combinations with chemotherapy should be a preferred option (Montero et al. 2014). In fact, combination with other targeted agents including PARP inhibitors is under clinical evaluation (Juvekar et al. 2012). Preclinical evidence indicates that PI3K/mTOR targeting in TNBC and other solid tumors causes a cytostatic effect, rather than cytotoxic (Montero et al. 2014). Therefore, full exploitation of the antitumoral effect of the PI3K/mTOR inhibitors probably would require their combination with agents that cause cell death. A relevant concept is how to identify patients responsive to PI3K/mTOR inhibitors. Some studies have selected patients based on mutations at the PI3KCA gene and/or loss of PTEN. However, recent data suggest that PI3KCA mutations do not predict response to agents against this route (Dillon and Miller. Curr Drug Targets 2014; 15:65-79). This suggests that the best approach to identify a targetable route may be to evaluate its activation status, rather than an analysis of mutational or expression data. In the case of the PI3K/mTOR route, assessment of phosphorylated proteins that can be used as readouts of the activity of the pathway may represent the best method to predict sensitivity to PI3K/mTOR targeting compounds. In conclusion evaluation of cancer vulnerabilities in triple negative tumors discovered the PI3K/mTOR pathway as a key druggable element. Identification of potential responsive patients and the best partner drug to be combined will be required to optimize the efficacy of these agents in TNBCs.

[Background]: The receptor tyrosine kinase, HER2, is overexpressed in approximately 25% of patients with breast cancer and is implicated in the aggressiveness of cancer. Targeting of HER2 signaling with trastuzumab, a monoclonal antibody that inhibits HER2 activity, has demonstrated clinical benefits. [Methods]: We investigated whether the antitumor activity of trastuzumab can be potentiated by dasatinib, a small-molecule tyrosine kinase inhibitor, on breast cancer cell lines that overexpress HER2 (BT474 and SKBR3) or have normal HER2 expression (MCF7 and T47D). Functional, biochemical, and gene expression microarray studies were performed to test the effect of trastuzumab, dasatinib, or a combination of trastuzumab and dasatinib on cell proliferation; HER activation; cell cycle; DNA damage; and apoptosis. The effect of drugs on mice (n = 6 per group) bearing xenograft tumors originating from HER2-overexpressing BT474 cells was assessed, and tumors were evaluated for an effect on volume, HER signaling, and DNA damage. All statistical tests were two-sided. [Results]: Trastuzumab and dasatinib combination showed a synergistic effect on the proliferation of HER2-overexpressing breast cancer cells (combination index = 0.44, 95% confidence interval = 0.30 to 0.58). The drug combination also induced a stronger inhibitory effect on HER2 activation than the individual drugs, decreased the level of proteins involved in DNA damage response, induced DNA double-strand breaks, cell cycle arrest, and caspase-independent apoptosis. Mice (n = 6 per group) bearing xenograft tumors originating from HER2-overexpressing BT474 cells showed statistically significantly reduced tumor volume on day 28 when treated with the drug combination (control vs trastuzumab and dasatinib combination; mean volume = 2.6 vs 0.5 cm3, difference = 2.1 cm3, 95% confidence interval = 0.76 to 3.51 cm3, P = .01) and total regression of tumors by day 36 with no later relapse. [Conclusions]: Results showed that HER2 and dasatinib-sensitive tyrosine kinases act in a synergistic manner to safeguard the breast cancer cells from DNA damage. The therapeutic targeting of multikinase inhibition opens new avenues for the treatment of HER2-positive breast cancer patients., Ministry of Science and Innovation of Spain (BFU2006-01813/BMC-FEDER), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD06/0020/0041), and an Acción transversal en cancer to A.P. S.S. and J.C.M. were supported by Juan de la Cierva and Scientific Foundation of the Spanish Association Against Cancer (AECC) contracts, respectively. This work was partially sponsored by a research contract to AP from Bristol-Myers Squibb, S.A., Madrid, Spain.

Background Breast cancer is the most common neoplasia in women. Even though advances in its treatment have improved disease outcome, some patients relapse. Therefore, attempts to better define the molecular determinants that drive breast cancer cell proliferation may help in defining potential therapeutic targets. Mitogen-activated protein kinases (MAPK) play important roles in tumorigenesis. One of them, Erk5, has been linked to the proliferation of breast cancer cells in vitro. Here we have investigated the expression and prognostic value of Erk5 in human breast cancer. Methodology/Principal Findings Animal and cellular models were used to study Erk5 expression and function in breast cancer. In 84 human breast tumours the expression of Erk5 was analyzed by immunohistochemistry. Active Erk5 (pErk5) was studied by Western blotting. Correlation of Erk5 with clinicopathological parameters and with disease-free survival in early stage breast cancer patients was analyzed. Expression of Erk5 was detected in most patients, and overexpression was found in 20%. Active Erk5 was present in a substantial number of samples, as well as in tumours from an animal breast cancer model. Overexpression of Erk5 was associated with a decrease in disease-free survival time, which was independent of other clinicopathological parameters of prognosis. Transient transfection of a short hairpin RNA (shRNA) targeting Erk5, and a stable cell line expressing a dominant negative form of Erk5 (Erk5AEF), were used to investigate the influence of Erk5 on drugs used in the clinic to treat breast tumours. We found that inhibition of Erk5 decreased cancer cell proliferation and also sensitized these cells to the action of anti-HER2 therapies. Conclusions/Significance Overexpression of Erk5 is an independent predictor of disease-free survival in breast cancer, and may represent a future therapeutic target.

Background c-Kit is expressed in the plasma cells from 30% of patients with multiple myeloma. Two different isoforms of c-Kit, characterized by the presence or absence of the tetrapeptide sequence GNNK in the extracellular domain, have been described. However, their expression and function in myeloma cells are unknown.We explored the function and expression of these c-Kit isoforms in myeloma cells.Design and Methods Expression of c-Kit isoforms was investigated by reverse transcriptase polymerase chain reaction in fresh plasma cells from patients and cell lines. The function of these c-Kit isoforms was analyzed upon expression in myeloma cells. Signaling was investigated by western blotting using antibodies specific for activated forms of several signaling proteins. The impact of c-Kit on the action of drugs commonly used in the treatment of multiple myeloma was investigated by MTT proliferation assays.Results Fresh plasma cells from patients as well as myeloma cell lines expressed the two isoforms of c-Kit. Retroviral infection of myeloma cells with vectors that code for c-Kit-GNNK+ or c-Kit-GNNK− forms demonstrated differences in the kinetics of phosphorylation between these isoforms. Stem cell factor-induced activation of the GNNK− form was faster and more pronounced than that of the GNNK+ form, whose activation, however, lasted for longer. The c-Kit receptors weakly activated the Erk1/2 and Erk5 pathways. Both receptors, however, efficiently coupled to the PI3K/Akt pathway, and stimulated p70S6K activation. The latter was sensitive to the mTOR inhibitor, rapamycin. Studies of drug sensitivity indicated that cells expressing the GNNK− form were more resistant to the anti-myeloma action of bortezomib and melphalan.Conclusions Our data indicate that c-Kit expression in multiple myeloma cells is functional, and coupled to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.

The neuregulins represent the largest subclass of polypeptide factors of the epidermal growth factor family of ligands. These molecules are synthesized as membrane-bound, biologically active growth factors that act by binding to the HER/ErbB receptor tyrosine kinases. Preclinical data have indicated that increased expression and function of neuregulins may provoke cancer. Furthermore, neuregulin expression has been detected in several neoplasias, and their presence may correlate with response to treatments that target the HER receptors such as trastuzumab. In addition, the neuregulins have also been implicated in resistance to anti-HER therapies. Therefore, targeting of the neuregulins may be helpful in neoplastic diseases in which these polypeptide factors contribute to tumor generation and/or maintenance. © 2008 American Association for Cancer Research., Grant support: Ministry of Science and Education of Spain (BFU2006-01813/ BMC) and an Institutional Cancer Center Network program from the ISCIII (RD06/ 0020/0041), the Scientific Foundation of the Spanish Association for Cancer Research (J.C. Montero), a fellowship from the Ministry of Science and Education (R. Rodríguez-Barrueco), the Instituto de Salud Carlos III (A. Esparís-Ogando), and the I3PProgramfrom the CSIC (E. Díaz-Rodríguez). Our Cancer Research Institute, and the work carried out at our laboratory, receives support from the European Community through the regional development funding program (FEDER).

Purpose Human epidermal growth factor receptor 2 (HER-2) overexpression has been associated with the genesis and progression of a subset of breast cancers. The function of HER-2 may be upregulated by overexpression or by the availability of neuregulins (NRGs), a group of transmembrane growth factors. Transmembrane NRGs strongly activated HER-2 and cell proliferation in breast cancer cells that did not overexpress HER-2, and treatment with trastuzumab prevented the proliferative action of transmembrane NRG. This raised the relevant clinical question of whether patients considered as HER-2 negative, but expressing transmembrane NRG, may benefit from treatment with trastuzumab. Patients and Methods MCF7 cells expressing transmembrane NRG (MCF7-NRGα2c) were injected into mice, and their sensitivity to trastuzumab was assessed. A retrospective study of 124 patients with early-stage or metastatic breast cancer was conducted. Expression of transmembrane NRG was evaluated by immunohistochemistry. In 11 patients, Western blot for NRGs was also carried out. Statistics were performed to analyze possible correlations between NRG expression and response to trastuzumab-based therapies, event-free survival, and overall survival (OS). Results Trastuzumab inhibited tumor growth in mice injected with MCF7-NRGα2c cells. Transmembrane NRG was frequently expressed in breast cancer patients. Overexpression of transmembrane NRG significantly correlated with a longer event-free survival and OS in patients with low or normal HER-2 expression who were treated with trastuzumab-based therapies but not in patients with HER-2 overexpression. Conclusion We suggest that the spectrum of patients who may benefit from trastuzumab-based therapies may be widened to include patients with metastatic breast cancer without HER-2 amplification but who express transmembrane NRGs.

[This corrects the article on p. 115 in vol. 1, PMID: 25594005.][This corrects the article on p. 763 in vol. 1, PMID: 25621292.].

7 Figures, Multiple myeloma is characterized by the accumulation of terminally differentiated B cells in the bone marrow, due to increased proliferation and restricted apoptosis of the myelomatous clone. Here we have studied the participation of a novel mitogen-activated protein kinase (MAPK) route, the extracellular signal-regulated kinase 5 (Erk5) pathway, in the regulation of myeloma cell proliferation and apoptosis. Erk5 was expressed in cells isolated from patients and in myeloma cell lines. The myeloma growth factor interleukin 6 (IL-6) activated Erk5, and this activation was independent of Ras and Src. Expression of a dominant-negative form of Erk5 restricted the proliferation of myeloma cells and inhibited IL-6–dependent cell duplication. This dominant-negative form also sensitized myeloma cells to the proapoptotic action of dexamethasone and PS341. The latter compound caused a profound decrease in the amount of endogenous Erk5 and was less effective in inducing apoptosis when the level of Erk5 was increased by transfection of Erk5. These results place the Erk5 route as a new regulatory signaling pathway that affects multiple myeloma proliferation and apoptosis., From the Centro de Investigación del Cáncer, CSIC-Universidad de Salamanca, Spain; Hospital Universitario de Salamanca, Spain; and Centro de Investigaciones Biológicas, Madrid, Spain.

1116 Background: Receptor tyrosine kinases (RTK) and downstream signaling routes play a central role in the genesis and/or promotion of different breast cancer tumors including the triple negative ...

Background: Androgen receptor (AR) plays a central role in some tumor types like prostate cancer. Its expression and function in breast cancer is relatively unknown. Studies have linked tyrosine kinase (TK) receptors and their intracellular signaling pathways with AR status. In the present work we study the presence of AR in breast cancer human samples and how TKs can control its expression. Methods: Tumor samples from patients were used to assess the expression of AR by western-blot. Membrane and intracellular kinases were evaluated using phosphoprotein arrays. Cell lines representative of all breast cancer subtypes were used to assess the expression of the AR and other signaling proteins using western-blot and phosphoprotein arrays. Proliferation studies with several kinase inhibitors were measured by MTT assays. We also interrogated publicly available gene expression microarray data sets with annotated clinical-pathological data. Results: Across all the intrinsic subtypes of breast cancer, the expression of AR gene was found highly expressed in HER2-enriched and Luminal tumors and lowly expressed in Basal-like and/or triple-negative (TN) tumors. In TN tumors, PDGFRβ and EGFR were found highly activated, together with AKT and Erk1/2. A positive correlation was observed between activation of EGFR and PDGFRβ and the expression of AR. In vitro, AR protein expression varied across cell lines and was found more expressed in MCF7 (Luminal) and HS578T (TN), BT549 (TN), HCC70 (TN) and no expressed in HBL100 (TN) and MDA-MB231 (TN). Administration of the antiandrogen bicalutamide produced an anti-proliferative effect that was increased by association of an Erk1/2 inhibitor. Inhibition of the PI3K-mTOR pathway in BT549 and HS578T reduced the expression of AR but did not augment the action of bicalutamide. In TN tumors, AR gene and protein was found more expressed in older compared to younger patients. Conclusions: In breast cancer, AR is expressed and is controlled by receptor TKs through the PI3K-mTOR pathway. An increased anti-proliferative effect is observed with the combination of bicalutamide and Erk1/2 inhibitors. AR expression correlates with age at diagnosis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-04.

Breast cancer represents one of the most common tumoral pathologies in women. Although continuous advances in its treatment have improved its outcome, in the metastatic setting the disease still represents an important medical problem. For this reason, the incorporation of novel therapies is required. Immunohistochemical characterization of breast cancers led to their division into three main subtypes: hormone receptor positive, HER2 positive, or triple negative breast cancers. Triple negative breast cancers (TNBC) are characterized by the absence of hormonal and HER2 receptors, and represents around 15% of all breast tumors. TNBC are aggressive from the clinical perspective, and their therapy is mainly based on classical chemotherapeutic agents, with limited success in the metastatic setting. Here we have explored the action of a novel drug, named EC8042, in TNBC. The drug, which is a mithramycin derivative, decreased MTT uptake of several TNBC cell lines. The IC50 values obtained were in the nM range, indicating that the drug had a potent effect on these cell lines. Mechanistically, the drug caused accumulation of cells in the S phase of the cell cycle at doses of 100 and 500 nM in the cell lines HBL100 and MDA-MB231. Biochemically, EC8042 caused an increase in cyclin E, cyclin A, cyclin D3, cyclin B, and p27, and decreased cyclin D1 and Rb phosphorylation at serine 780. These data are consistent with an action of EC8042 on the cell cycle. Moreover, the increase in wee1 and pY15-CDK1 are in line with an effect of the drug blocking cell cycle progression through the S phase. Cell cycle analyses using propidium iodide staining also revealed an increase in the subG0 proportion of cells, especially in HBL100, suggestive of a potential indiction of cell death by EC8042. Annexin V staining indicated that treatment of HBL100 with 500 nM of EC8042 provoked apoptosis, which was also revealed by PARP processing. The treatment reduced the amounts of MCL1, BCLXL, and XIAP, but did not affect BCL2 levels. Together, our results indicate that EC8042 has a strong antitumoral action on TNBC cells, which is caused by increased apoptosis and a decrease in cell cycle progression, and opens the possibility for further development of this drug in TNBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B39.

e15523 Background: Although improvements in the treatment of ovarian cancer have increased the global prognosis, the metastatic disease is still an incurable situation. Therefore, it is necessary t...

1084 Background: Breast cancer is the most common neoplasia in women. Although important advances in its treatment have been made along the last years, in the metastatic setting this disease remains incurable. One group of molecules involved in breast cancer genesis/progression are tyrosine kinases. HER-2 is a receptor tyrosine kinase that is overexpressed in a subset of patients with breast cancer. Targeting of HER-2 with trastuzumab a monoclonal antibody that acts on HER-2 has demonstrated clinical benefit. However, as a single agent its antitumoral activity is limited. Methods: Here we have investigated whether the action of trastuzumab, can be potentiated by dasatinib, a small molecule inhibitor that acts on several receptor and non-receptor tyrosine kinases. To this end, we have used cancer cell lines and in vivo xenografted mouse models. MTT proliferation and other apoptosis assays, biochemical and microarray experiments were performed to investigate the molecular bases of the action of the combined trastuzumab plus dasatinib treatment. Results: Combination of trastuzumab and dasatinib was synergic on breast cancer cells overexpressing HER-2, but not on cells with a normal complement of its receptor. In vivo xenograft studies confirmed the antitumoral action of this drug combination. Studies on the mechanism of action showed that this drug combination triggered apoptosis in HER-2 overexpressing cells. Apoptosis was evidenced by increased Annexin V staining, and was found to occur in a caspase-independent manner. In addition, an action of the drug combination on cell cycle could be identified, and the drugs decreased cyclins E1/2, B, and Cdk2, in addition to increasing p27. Microarray as well as biochemical analyses showed that the drug combination provoked a decrease in HER receptor phosphorylation, followed by interruption of downstream signalling. These changes resulted in alterations in the levels of proteins involved in DNA repair. Moreover, treatment with the drug combination caused a DNA damage response as evidenced by increased γH2AX. Conclusions: The effective antitumoral action of the drug combination opens new avenues for the clinical exploration of this drug combination in breast cancer patients overexpressing HER-2. No significant financial relationships to disclose.

Background and Aims: Although recent therapeutic advances have led to an improvement in the outcome of Multiple Myeloma (MM), it still remains an incurable disease, and therefore, new drugs with novel mechanisms of action are needed for myeloma patients. Zalypsis is a new synthetic alkaloid derived from certain marine compounds which has demonstrated significant in vitro and in vivo antitumor activity in different malignancies. It is currently under late Phase I development in solid tumours, with preliminary evidence of activity. In this study, we have analysed the preclinical activity and mechanism of action of Zalypsis in MM. Material and methods: Nine different MM cell lines and BM samples from MM patients and normal donors were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and gene expression profile analysis. The in vivo activity was explored in a human subcutaneous plasmocytoma model and immunohistochemistry was performed in selected tumours. Results: Zalypsis turned out to be the most potent antimyeloma agent we have tested so far in our laboratory, with IC50s in picomolar or low nanomolar ranges depending on the cell lines studied. Interestingly, the sensitivity to Zalypsis was independent of the pattern of resistance of the cell lines to conventional antimyeloma agents such as Dexamethasone or Melphalan. It also showed remarkable ex vivo potency in freshly isolated plasma cells from six patients (including two with plasma cell leukemia) and synergized with many other antimyeloma compounds, being the combination of Zalypsis + Lenalidomide + Dexamethasone particularly attractive. Regarding toxicity, Zalypsis preserved the CD34+ hematopoietic progenitor cells from MM and normal donor BM samples. This remarkable activity prompted us to investigate the mechanism of action of the drug. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double strand breaks, which were evidenced by an increase in phospho Histone H2AX and phospho CHK2, followed by a striking overexpression of p53 in MM cell lines bearing wild type forms of this protein. Of note, no other compound currently used in the MM clinic induced such an increase in p53 protein levels. In addition, in a subset of MM cell lines in which p53 was mutated, Zalypsis also provoked DNA double strand breaks and induced cell death, although higher concentrations were required. Changes in the gene expression profile of MM cells treated with Zalypsis were concordant with these results, since important genes involved in DNA damage response were deregulated. This include genes implicated in the ATM repair pathway, such as TLK2, ATR, CHEK2, RAD5 and BRIP1 and other mRNAs related to DNA repair, such as RAD23B, XPC, XRCC1, XRCC5 and GADD45A. These results were confirmed in vivo in a model of human subcutaneous plasmocytoma in SCID mice. Zalypsis (0.8 and 1 mg/Kg) decreased tumour growth and improved survival of mice implanted with MM1S (wild type p53) and OPM-1 (mutated p53) plasmocytomas. Immunohistochemical studies in tumours from treated animals also demonstrated DNA damage with H2AX phosphorylation and p53 overexpression. Conclusion: The potent in vitro and in vivo antimyeloma activity and the singular mechanism of action of Zalypsis uncovers the high sensitivity of tumour plasma cells to double strand breaks, and strongly supports the potential use of this compound in multiple myeloma patients.

The development of novel targeted therapies in Multiple Myeloma (MM) has opened promising expectations for the treatment of this incurable hematological malignancy. However, the molecular mechanisms of both novel biologically based therapies and conventional treatments are still unclear. The purpose of the present study was to evaluate the changes in the gene expression profile of the human multiple myeloma cell line (MM.1S) following exposure to Doxorubicin, Melphalan, Bortezomib, Aplidin and Arsenic Trioxide. We have focused on the analysis of the early steps of activation of molecular mechanisms that lead to MM cell death. For this purpose we investigated with a time-course the onset of the apoptosis for every drug, according to the flow cytometric analysis with Annexin V- FITC Apoptosis Detection Reagent Kit. Based on these results the optimal concentration and time of exposure for each of the drugs were as follows Melphalan 50 μM, 9hours; Doxorubicin 1 μM, 17hours; Bortezomib 10 nM, 6 hours; Aplidin 50 nM, 4 hours and Arsenic Trioxide 5 mM, 3 hours. Affymetrix HG-U133A array containing around 15,000 full-length genes was used for mRNA expression profiling. All the experiments were performed in duplicate. The DNA- Chip Analyzer (DChip) was used to normalize and compare samples. Genes with expression changes greater than twofold in either direction were considered significant. A total of 269, 74, 74, 808 and 525 genes showed a significantly altered expression pattern, in response to Melphalan, Doxorubicin, Bortezomib, Aplidin and Arsenic Trioxide, respectively. Our results demonstrate that treatment with Melphalan inhibits DNA replication and transcription (underexpression of POLA, RFC1) as well as proliferation and survival (underexpression of IGF-1); it blocks the cell cycle (overexpression of CDKN1A and PA26) and induces apoptosis (overexpression of GADD45B). Doxorubicin deregulates many genes involved in cell cycle arrest (high expression of CDKN1A, PA26, GADD45A and low expression of CCND2 y CDC20) as well as up-regulation of several members of the TNF family (CD95, TRAILR2 and CD27). Bortezomib increases the expression of many “heat shock proteins” (HSP 110, HSP 70B, HSP 70B′) and decreases the level expression of IGF-1. Aplidin triggers early induction of many genes involved in apoptosis (overexpression of MAP4K3 and EGR2) and down-regulation of genes that play and important role in G2/M phase transition (NEK2, CENPF, BUB1). Arsenic Trioxide induces underexpression of essential genes for G1/S phase transition and cell cycle progression (CDC7L1, CDC25A) as well as underexpression of genes that mediate spindle formation and cromosome segregation (STK6, PRC1, HCAP-G, ZWINT, KIF4A, BUB1). Moreover, Arsenic Trioxide alike Bortezomib treatment up-regulates several HSP (HSP 40, HSP70B, HSP27). TNFSF9 which inhibits proliferation was the only gene up-regulated by all the drugs. Microarray technology demonstrates that treatment with novel targeted therapies (Bortezomib, Aplidin and Arsenic Trioxide) induces deregulation of molecular mechanisms which are not involved in anti-myeloma activity of conventional treatments (Melphalan, Doxorubicin). In addition it is an efficient tool to understand the differences in mechanisms of action of novel drugs.

This is an open-access article distributed under the terms of the Creative Commons Attribution License., Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease., This work was supported by grants from the Spanish MINECO-ISCIII (PI12/02591, PI12/00624) and FEDER (European Funds for Regional Development); the Centro en Red for Regenerative Medicine and Cellular Therapy from Castilla y León; the Spanish Health Thematic Network of Cooperative Research in Cancer (RTICC RD12/0056/0058 and RD12/0036/0003), and Spanish FIS (PS09/01897 and PS09/00843). AG-G received support from the Centro en Red for Regenerative Medicine and Cellular Therapy from Castilla y León and from the Spanish Society of Hematology and Hemotherapy (SEHH), and EDR from the Spanish Association for Cancer Research (AECC).