Latent-Transforming Growth Factor ß-Binding Protein 1/Transforming Growth Factor ß1 Complex Drives Antitumoral Effects upon ERK5 Targeting in Melanoma.

Melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. Available treatments have improved survival, although long-term benefits still are unsatisfactory. The mitogen-activated protein kinase extracellular signal-regulated kinase 5 (ERK5) promotes melanoma growth, and ERK5 inhibition determines cellular senescence and the senescence-associated secretory phenotype. Here, latent-transforming growth factor ß-binding protein 1 (LTBP1) mRNA was found to be up-regulated in A375 and SK-Mel-5 BRAFV600E melanoma cells after ERK5 inhibition. In keeping with a key role of LTBP1 in regulating transforming growth factor ß (TGF-ß), TGF-ß1 protein levels were increased in lysates and conditioned media of ERK5-knockdown (KD) cells, and were reduced upon LTBP1 KD. Both LTBP1 and TGF-ß1 proteins were increased in melanoma xenografts in mice treated with the ERK5 inhibitor XMD8-92. Moreover, treatment with conditioned media from ERK5-KD melanoma cells reduced cell proliferation and invasiveness, and TGF-ß1-neutralizing antibodies impaired these effects. In silico data sets revealed that higher expression levels of both LTBP1 and TGFB1 mRNA are associated with better overall survival of melanoma patients, and that increased LTBP1 or TGF-ß1 expression proved a beneficial role in patients treated with anti-PD1 immunotherapy, making a possible immunosuppressive role of LTBP1/TGF-ß1 unlikely upon ERK5 inhibition. This study, therefore, identifies additional desirable effects of ERK5 targeting, providing evidence of an ERK5-dependent tumor-suppressive role of TGF-ß in melanoma.

ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression.

Many of the biological processes of the cell, from its structure to signal transduction, involve protein-protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis.

Etiopathogenic role of ERK5 signaling in sarcoma: prognostic and therapeutic implications.

Sarcomas constitute a heterogeneous group of rare and difficult-to-treat tumors that can affect people of all ages, representing one of the most common forms of cancer in childhood and adolescence. Little is known about the molecular entities involved in sarcomagenesis. Therefore, the identification of processes that lead to the development of the disease may uncover novel therapeutic opportunities. Here, we show that the MEK5/ERK5 signaling pathway plays a critical role in the pathogenesis of sarcomas. By developing a mouse model engineered to express a constitutively active form of MEK5, we demonstrate that the exclusive activation of the MEK5/ERK5 pathway can promote sarcomagenesis. Histopathological analyses identified these tumors as undifferentiated pleomorphic sarcomas. Bioinformatic studies revealed that sarcomas are the tumors in which ERK5 is most frequently amplified and overexpressed. Moreover, analysis of the impact of ERK5 protein expression on overall survival in patients diagnosed with different sarcoma types in our local hospital showed a 5-fold decrease in median survival in patients with elevated ERK5 expression compared with those with low expression. Pharmacological and genetic studies revealed that targeting the MEK5/ERK5 pathway drastically affects the proliferation of human sarcoma cells and tumor growth. Interestingly, sarcoma cells with knockout of ERK5 or MEK5 were unable to form tumors when engrafted into mice. Taken together, our results reveal a role of the MEK5/ERK5 pathway in sarcomagenesis and open a new scenario to be considered in the treatment of patients with sarcoma in which the ERK5 pathway is pathophysiologically involved.

The WNK1-ERK5 route plays a pathophysiological role in ovarian cancer and limits therapeutic efficacy of trametinib.

BACKGROUND: The dismal prognosis of advanced ovarian cancer calls for the development of novel therapies to improve disease outcome. In this regard, we set out to discover new molecular entities and to assess the preclinical effectiveness of their targeting. METHODS: Cell lines, mice and human ovarian cancer samples were used. Proteome profiling of human phosphokinases, in silico genomic analyses, genetic (shRNA and CRISPR/Cas9) and pharmacological strategies as well as an ex vivo human preclinical model were performed. RESULTS: We identified WNK1 as a highly phosphorylated protein in ovarian cancer and found that its activation or high expression had a negative impact on patients' survival. Genomic analyses showed amplification of WNK1 in human ovarian tumours. Mechanistically, we demonstrate that WNK1 exerted its action through the MEK5-ERK5 signalling module in ovarian cancer. Loss of function, genetic or pharmacological experiments, demonstrated anti-proliferative and anti-tumoural effects of the targeting of the WNK1-MEK5-ERK5 route. Additional studies showed that this pathway modulated the anti-tumoural properties of the MEK1/2 inhibitor trametinib. Thus, treatment with trametinib activated the WNK1-MEK5-ERK5 route, raising the possibility that this effect may limit the therapeutic benefit of ERK1/2 targeting in ovarian cancer. Moreover, in different experimental settings, including an ex vivo patient-derived model consisting of ovarian cancer cells cultured with autologous patient sera, we show that inhibition of WNK1 or MEK5 increased the anti-proliferative and anti-tumour efficacy of trametinib. CONCLUSIONS: The present study uncovers the participation of WNK1-MEK5-ERK5 axis in ovarian cancer pathophysiology, opening the possibility of acting on this pathway with therapeutic purposes. Another important finding of the present study was the activation of that signalling axis by trametinib, bypassing the anti-tumoural efficacy of this drug. That fact should be considered in the context of the use of trametinib in ovarian cancer.

ERK5 Is a Major Determinant of Chemical Sarcomagenesis: Implications in Human Pathology.

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.

Inhibition of ERK5 Elicits Cellular Senescence in Melanoma via the Cyclin-Dependent Kinase Inhibitor p21.

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 ß-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. SIGNIFICANCE: This study shows that targeting ERK5 induces p21-mediated cellular senescence in melanoma, identifying a prosenescence effect of ERK5 inhibitors that may be exploited for melanoma treatment.

Clinical, genetic and pharmacological data support targeting the MEK5/ERK5 module in lung cancer.

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.

Inhibition of the mitotic kinase PLK1 overcomes therapeutic resistance to BET inhibitors in triple negative breast cancer.

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.

Activity of BET-proteolysis targeting chimeric (PROTAC) compounds in triple negative breast cancer.

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.

Resistance to MAPK Inhibitors in Melanoma Involves Activation of the IGF1R-MEK5-Erk5 Pathway.

Combined treatment of metastatic melanoma with BRAF and MEK inhibitors has improved survival, but the emergence of resistance represents an important clinical challenge. Targeting ERK is a suitable strategy currently being investigated in melanoma and other cancers. To anticipate possible resistance to ERK inhibitors (ERKi), we used SCH772984 (SCH) as a model ERKi to characterize resistance mechanisms in two BRAF V600E melanoma cell lines. The ERKi-resistant cells were also resistant to vemurafenib (VMF), trametinib (TMT), and combined treatment with either VMF and SCH or TMT and SCH. Resistance to SCH involved stimulation of the IGF1R-MEK5-Erk5 signaling pathway, which counteracted inhibition of Erk1/2 activation and cell growth. Inhibition of IGF1R with linsitinib blocked Erk5 activation in SCH-resistant cells and decreased their growth in 3D spheroid growth assays as well as in NOD scid gamma (NSG) mice. Cells doubly resistant to VMF and TMT or to VMF and SCH also exhibited downregulated Erk1/2 activation linked to stimulation of the IGF1R-MEK5-Erk5 pathway, which accounted for resistance. In addition, we found that the decreased Erk1/2 activation in SCH-resistant cells involved reduced expression and function of TGFα. These data reveal an escape signaling route that melanoma cells use to bypass Erk1/2 blockade during targeted melanoma treatment and offer several possible targets whose disruption may circumvent resistance. SIGNIFICANCE: Activation of the IGF1R-MEK5-Erk5 signaling pathway opposes pharmacologic inhibition of Erk1/2 in melanoma, leading to the reactivation of cell proliferation and acquired resistance.

A Transcriptomic Immunologic Signature Predicts Favorable Outcome in Neoadjuvant Chemotherapy Treated Triple Negative Breast Tumors.

Limited therapeutic options exist for the treatment of patients with triple negative breast cancer (TNBC). Neoadjuvant chemotherapy is currently the standard of care treatment in the early stages of the disease, although reliable biomarkers of response have been scarcely described. In our study we explored whether immunologic signatures associated with inflamed tumors or hot tumors could predict the outcome to neoadjuvant chemotherapy. Publicly available transcriptomic data of more than 2,000 patients were evaluated. ROC plots were generated to assess the response to therapy. Cox proportional hazards regression was computed. Kaplan-Meier plots were drawn to visualize the survival differences. Higher expression of IDO1, CXCL9, CXCL10, HLA-DRA, HLA-E, STAT1, and GZMB were associated with a higher proportion without relapse in the first 5 y after chemotherapy in TNBC. The expression of these genes was associated with a high presence of CD8 T cells in responder patients using the EPIC bioinformatic tool. The strongest effect was observed for STAT1 (p = 1.8e-05 and AUC 0.69, p = 2.7e-06). The best gene-set signature to predict favorable RFS was the combination of IDO1, LAG3, STAT1, and GZMB (HR = 0.28, CI = 0.17-0.46, p = 9.8 E-08, FDR = 1%). However, no influence on pathological complete response (pCR) was observed. Similarly, no benefit was identified in any other tumor subtype: HER2 or estrogen receptor positive. In conclusion, we describe a set of immunologic genes that predict the outcome to neoadjuvant chemotherapy in TNBC, but not pCR, suggesting that this non-time to event endpoint is not a good surrogate marker to detect the long term outcome for immune activated tumors.

Targeting the EGF/HER Ligand-Receptor System in Cancer.

Receptor tyrosine kinases (RTKs) are a superfamily of transmembrane proteins that mediate intracellular signaling by phosphorylating substrate proteins involved in cell proliferation, survival, differentiation or migration. The Human Epidermal growth factor Receptor (HER) family belongs to the RTKs superfamily, and comprises four members: EGFR (epidermal growth factor receptor), HER2, HER3 and HER4. Physiologically, these receptors are activated by the ligands of the EGF family. In solid tumors other mechanisms of activation, such as overexpression or molecular alterations have been reported, and have been linked to tumour initiation/progression. Because of that, several strategies have been developed to target HER receptors and include i) antibody-based therapies using monoclonal antibodies against the extracellular domain of these receptors, and ii) small molecule tyrosine kinase inhibitors (TKIs) against the intracellular kinase domain. In this review we will provide basic information about biological aspects of HER receptors and their ligands as well as the therapeutic strategies to target them. We also summarize general mechanisms of resistance generated in patients to such anti-HER therapies.

Neuregulin expression in solid tumors: prognostic value and predictive role to anti-HER3 therapies.

BACKGROUND: Neuregulins (NRG) are a family of epidermal growth factor ligands which act through binding to HER3 and HER4 receptors. NRGs are widely expressed in solid tumors. Their prognostic significance or their role as predictors of benefit from anti-HER3 therapy is not known. RESULTS: Of 29 included studies, 7 studies reported the association between NRG and outcome. NRG was most commonly expressed in breast, prostate, colon and bladder cancers. NRG expression was not associated with either OS or PFS (HR: 3.47, 95% CI 0.78-15.47, p = 0.10 and HR: 1.64, 95% CI 0.94-2.86, p = 0.08, respectively). In 4 placebo controlled trials of anti-HER3 therapy, the addition of anti-HER3 antibodies to control therapy in unselected patients was not associated with improved PFS (HR: 0.88, 95% CI 0.75-1.04. p = 0.14). However, in patients with high NRG expression, there was significantly delayed progression (HR: 0.35, 95% CI 0.23-0.52, p < 0.001). Anti-HER3 antibodies were associated with increased risk of diarrhea, nausea and rash. METHODS: A search of electronically available databases identified studies exploring clinical outcomes based on NRG expression, as well as placebo-controlled trials of HER3-directed therapy reporting results based on NRG expression status. Data were combined in a meta-analysis using generic inverse variance and random effects modeling for studies reporting the hazard ratio (HR) for overall (OS) or progression-free survival (PFS). Mantel-Haenszel random-effect modeling was used for odds ratio (OR) for 3-year and 5-year OS and PFS. CONCLUSIONS: NRG expression is not associated with either OS or PFS, but is a predictor of benefit from anti-HER3 antibodies.

The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma.

OBJECTIVE: The extracellular signal-regulated kinase 5 (ERK5 or BMK1) is involved in tumour development. The ERK5 gene may be amplified in hepatocellular carcinoma (HCC), but its biological role has not been clarified. In this study, we explored the role of ERK5 expression and activity in HCC in vitro and in vivo. DESIGN: ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacological inhibitor, XMD8-92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts. RESULTS: In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue compared with normal liver tissue. Inhibition of ERK5 decreased HCC cell proliferation and increased the proportion of cells in G0/G1 phase. These effects were associated with increased expression of p27 and p15 and decreased CCND1. Treatment with XMD8-92 or ERK5 silencing prevented cell migration induced by epidermal growth factor or hypoxia and caused cytoskeletal remodelling. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 was silenced for ERK5. Moreover, systemic treatment with XMD8-92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel. CONCLUSIONS: ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC.

Therapeutic potential of ERK5 targeting in triple negative breast cancer.

Triple negative breast cancers (TNBCs) account for 15% of all breast cancers, and represent one of the most aggressive forms of the disease, exhibiting short relapse-free survival. In contrast to other breast cancer subtypes, the absence of knowledge about the etiopathogenic alterations that cause TNBCs force the use of chemotherapeutics to treat these tumors. Because of this, efforts have been devoted with the aim of incorporating novel therapies into the clinical setting. Kinases play important roles in the pathophysiology of several tumors, including TNBC. Since expression of the MAP kinase ERK5 has been linked to patient outcome in breast cancer, we analyzed the potential value of its targeting in TNBC. ERK5 was frequently overexpressed and active in samples from patients with TNBC, as well as in explants from mice carrying genetically-defined TNBC tumors. Moreover, expression of ERK5 was linked to a worse prognosis in TNBC patients. Knockdown experiments demonstrated that ERK5 supported proliferation of TNBC cells. Pharmacological inhibition of ERK5 with TG02, a clinical stage inhibitor which targets ERK5 and other kinases, inhibited cell proliferation by blocking passage of cells through G1 and G2, and also triggered apoptosis in certain TNBC cell lines. TG02 had significant antitumor activity in a TNBC xenograft model in vivo, and also augmented the activity of chemotherapeutic agents commonly used to treat TNBC. Together, these data indicate that ERK5 targeting may represent a valid strategy against TNBC, and support the development of trials aimed at evaluating the clinical effectiveness of drugs that block this kinase.

ERK5/BMK1 is a novel target of the tumor suppressor VHL: implication in clear cell renal carcinoma.

Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas.

Potent antimyeloma activity of a novel ERK5/CDK inhibitor.

PURPOSE: To analyze the antimyeloma potential of TG02, an ERK5/CDK inhibitory drug. EXPERIMENTAL DESIGN: Utilizing different multiple myeloma cell lines we determined the effect of TG02 over viability by MTT assays. The apoptotic effect over multiple myeloma patient samples was studied ex vivo by cytometry. The mechanism of action of TG02 was analyzed in the cell line MM1S, studying its effect on the cell cycle, the induction of apoptosis, and the loss of mitochondrial membrane potential by cytometry and Western blot. Two models of multiple myeloma xenograft were utilized to study the in vivo action of TG02. RESULTS: TG02 potently inhibited proliferation and survival of multiple myeloma cell lines, even under protective bone marrow niche conditions, and selectively induced apoptosis of primary patient-derived malignant plasma cells. TG02 displayed significant single-agent activity in two multiple myeloma xenograft models, and enhanced the in vivo activity of bortezomib and lenalidomide. Signaling analyses revealed that the drug simultaneously blocked the activity of CDKs 1, 2, and 9 as well as the MAP kinase ERK5 in MM1S cells, leading to cell-cycle arrest and rapid commitment to apoptosis. TG02 induced robust activation of both the intrinsic and extrinsic pathways of apoptosis, and depletion of XIAP and the key multiple myeloma survival protein Mcl-1. CONCLUSIONS: TG02 is a promising new antimyeloma agent that is currently in phase I clinical trials in leukemia and multiple myeloma patients.

Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue.

Glioblastoma (GBM) is associated with infiltration of peritumoral (PT) parenchyma by isolated tumor cells that leads to tumor regrowth. Recently, GBM stem-like or initiating cells (GICs) have been identified in the PT area, but whether these GICs have enhanced migratory and invasive capabilities compared with GICs from the tumor mass (TM) is presently unknown. We isolated GICs from the infiltrated PT tissue and the TM of three patients and found that PT cells have an advantage over TM cells in two-dimensional and three-dimensional migration and invasion assays. Interestingly, PT cells display a high plasticity in protrusion formation and cell shape and their migration is insensitive to substrate stiffness, which represent advantages to infiltrate microenvironments of different rigidity. Furthermore, mouse and chicken embryo xenografts revealed that only PT cells showed a dispersed distribution pattern, closely associated to blood vessels. Consistent with cellular plasticity, simultaneous Rac and RhoA activation are required for the enhanced invasive capacity of PT cells. Moreover, Rho GTPase signaling modulators αVß3 and p27 play key roles in GIC invasiveness. Of note, p27 is upregulated in TM cells and inhibits RhoA activity. Gene silencing of p27 increased the invasive capacity of TM GICs. Additionally, ß3 integrin is upregulated in PT cells. Blockade of dimeric integrin αVß3, a Rac activator, reduced the invasive capacity of PT GICs in vitro and abrogated the spreading of PT cells into chicken embryos. Thus, our results describe the invasive features acquired by a unique subpopulation of GICs that infiltrate neighboring tissue.

ERK2, but not ERK1, mediates acquired and "de novo" resistance to imatinib mesylate: implication for CML therapy.

Resistance to Imatinib Mesylate (IM) is a major problem in Chronic Myelogenous Leukaemia management. Most of the studies about resistance have focused on point mutations on BCR/ABL. However, other types of resistance that do not imply mutations in BCR/ABL have been also described. In the present report we aim to study the role of several MAPK in IM resistance not associate to BCR/ABL mutations. Therefore we used an experimental system of resistant cell lines generated by co-culturing with IM (K562, Lama 84) as well as primary material from resistant and responder patient without BCR/ABL mutations. Here we demonstrate that Erk5 and p38MAPK signaling pathways are not implicated in the acquired resistance phenotype. However, Erk2, but not Erk1, is critical for the acquired resistance to IM. In fact, Bcr/Abl activates preferentially Erk2 in transient transfection in a dose dependent fashion through the c-Abl part of the chimeric protein. Finally, we present evidences demonstrating how constitutive activation of Erk2 is a de novo mechanism of resistance to IM. In summary our data support the use of therapeutic approaches based on Erk2 inhibition, which could be added to the therapeutic armamentarium to fight CML, especially when IM resistance develops secondary to Erk2 activation.