Clinical Outcomes in Patients with Multi-Hit TP53 Chronic Lymphocytic Leukemia Treated with Ibrutinib.

PURPOSE: TP53 aberration (TP53 mutation and/or 17p deletion) is the most important predictive marker in chronic lymphocytic leukemia (CLL). Although each TP53 aberration is considered an equal prognosticator, the prognostic value of carrying isolated (single-hit) or multiple (multi-hit) TP53 aberrations remains unclear, particularly in the context of targeted agents. PATIENTS AND METHODS: We performed deep sequencing of TP53 using baseline samples collected from 51 TP53 aberrant patients treated with ibrutinib in a phase II study (NCT01500733). RESULTS: We identified TP53 mutations in 43 patients (84%) and del(17p) in 47 (92%); 9 and 42 patients carried single-hit and multi-hit TP53, respectively. The multi-hit TP53 subgroup was enriched with younger patients who had prior treatments and unmutated immunoglobulin heavy-chain variable region gene status. We observed significantly shorter overall survival, progression-free survival (PFS), and time-to-progression (TTP) in patients with multi-hit TP53 compared with those with single-hit TP53. Clinical outcomes were similar in patient subgroups stratified by 2 or >2 TP53 aberrations. In multivariable analyses, multi-hit TP53 CLL was independently associated with inferior PFS and TTP. In sensitivity analyses, excluding mutations below 1% VAF demonstrated similar outcome. Results were validated in an independent population-based cohort of 112 patients with CLL treated with ibrutinib. CONCLUSIONS: In this study, single-hit TP53 defines a distinct subgroup of patients with an excellent long-term response to single-agent ibrutinib, whereas multi-hit TP53 is independently associated with shorter PFS. These results warrant further investigations on prognostication and management of multi-hit TP53 CLL.See related commentary by Bomben et al., p. 4462.

Aurora A and Mcl-1: new potential treatment targets in antiestrogen-resistant breast cancer.

Antiestrogen resistance is a major clinical limitation in treatment of breast cancer. We have recently reported that Aurora A and Mcl-1 (myeloid cell leukemia 1) are potential novel treatment targets in antiestrogen-resistant breast cancer cells and that Aurora A expression is a biomarker for tamoxifen resistance in breast cancer patients. Abbreviations: Bcl-2, B-cell lymphoma 2; EGF, epidermal growth factor; ERα, estrogen receptor α; Mcl-1, myeloid cell leukemia 1; VEGF, vascular endothelial growth factor.

Genomic profiling of newly diagnosed glioblastoma patients and its potential for clinical utility - a prospective, translational study.

Glioblastoma (GBM) is an incurable brain tumor for which new treatment strategies are urgently needed. Next-generation sequencing of GBM has most often been performed retrospectively and on archival tissue from both diagnostic and relapse surgeries with limited knowledge of clinical information, including treatment given. We sought to investigate the genomic composition prospectively in treatment-naïve patients, searched for possible targetable aberrations, and investigated for prognostic and/or predictive factors. A total of 108 newly diagnosed GBM patients were included. Clinical information, progression-free survival, and overall survival (OS) were noted. Tissues were analyzed by whole-exome sequencing, single nucleotide polymorphism (SNP) and transcriptome arrays, and RNA sequencing; assessed for mutations, fusions, tumor mutational burden (TMB), and chromosomal instability (CI); and classified into GBM subgroups. Each genomic report was discussed at a multidisciplinary tumor board meeting to evaluate for matching trials. From 111 consecutive patients, 97.3% accepted inclusion in this study. Eighty-six (77%) were treated with radiation therapy/temozolomide (TMZ) and adjuvant TMZ. One NTRK2 and three FGFR3-TACC3 fusions were identified. Copy number alterations in GRB2 and SMYD4 were significantly correlated with worse median OS together with known clinical variables like age, performance status, steroid dose, and O6-methyl-guanine-DNA-methyl-transferase status. Patients with CI-median or TMB-high had significantly worse median OS compared to CI-low/high or TMB-low/median. In conclusion, performing genomic profiling at diagnosis enables evaluation of genomic-driven therapy at the first progression. Furthermore, TMB-high or CI-median patients had worse median OS, which can support the possibility of offering experimental treatment already at the first line for this group.

Application of cell-free DNA for genomic tumor profiling: a feasibility study.

PURPOSE: Access to genomic tumor material is required to select patients for targeted therapies. However, tissue biopsies are not always feasible and therefore circulating cell-free DNA (cfDNA) has emerged as an alternative. Here we investigate the utility of cfDNA for genomic tumor profiling in the phase I setting. STUDY DESIGN: Peripheral blood was collected from patients with advanced solid cancers eligible for phase I treatment. Patients failing the initial tissue biopsy due to inaccessible lesions or insufficient tumor cellularity (<10%) were included in the study. Genomic profiling of cfDNA including whole exome sequencing (WES) and somatic copy number alterations (SCNAs) analysis (OncoScan). RESULTS: Plasma cfDNA was pro- and retrospectively profiled from 24 and 20 patients, respectively. The median turnaround time was 29 days (N= 24, range 13-87 days) compared to tissue re-analyses of median 60 days (N= 6, range 29-98). Selected cancer-associated alterations (SCAAs) were identified in 70% (31/44) of patients, predominantly by WES due to the low sensitivity of OncoScan on cfDNA. Primarily, inaccessible cases of prostate and lung cancers could benefit from cfDNA profiling. In contrast, breast cancer patients showed a low level of tumor-specific cfDNA which might be due to cancer type and/or active treatment at the time of plasma collection. CONCLUSION: Plasma cfDNA profiling using WES is feasible within a clinically relevant timeframe and represents an alternative to invasive tissue biopsies to identify possible treatment targets. Especially, difficult-to-biopsy cancers can benefit from cfDNA profiling, but tumor tissue remains the gold standard for molecular analyses.

Deep targeted sequencing of TP53 in chronic lymphocytic leukemia: clinical impact at diagnosis and at time of treatment.

In chronic lymphocytic leukemia, TP53 mutations and deletion of chromosome 17p are well-characterized biomarkers associated with poor progression-free and overall survival following chemoimmunotherapy. Patients harboring low burden TP53 mutations with variant allele frequencies of 0.3-15% have been shown to have similar dismal outcome as those with high burden mutations. We here describe a highly sensitive deep targeted next-generation sequencing assay allowing for the detection of TP53 mutations as low as 0.2% variant allele frequency. Within a consecutive, single center cohort of 290 newly diagnosed patients with chronic lymphocytic leukemia, deletion of chromosome 17p was the only TP53 aberration significantly associated with shorter overall survival and treatment-free survival. We were unable to demonstrate any impact of TP53 mutations, whether high burden (variant allele frequency >10%) or low burden (variant allele frequency ≤10%), in the absence of deletion of chromosome 17p. In addition, the impact of high burden TP53 aberration (deletion of chromosome 17p and/or TP53 mutation with variant allele frequency >10%) was only evident for patients with IGHV unmutated status; no impact of TP53 aberrations on outcome was seen for patients with IGHV mutated status. In 61 patients at time of treatment, the prognostic impact of TP53 mutations over 1% variant allele frequency could be confirmed. This study furthers the identification of a clinical significant limit of detection for robust TP53 mutation analysis in chronic lymphocytic leukemia. Multicenter studies are needed for validation of ultra-sensitive TP53 mutation assays in order to define and implement a technical as well as a clinical lower limit of detection.

Concordance of Mutation Detection in Circulating Tumor DNA in Early Clinical Trials Using Different Blood Collection Protocols.

BACKGROUND: Small fragments of tumor DNA can be found in the circulation of cancer patients, providing a noninvasive access to tumor material (liquid biopsy). Analysis of circulating tumor DNA (ctDNA) has been used for diagnosis, treatment decisions, and detection of therapy resistance, including in patients with tumors inaccessible for biopsy, making ctDNA an important alternative source of tumor material. Immediate separation of plasma is widely used in standard isolation of cell-free DNA to ensure high quality plasma DNA. However, these procedures are labor intensive and logistically challenging in a clinical setting. Here we investigate the concordance between standard blood collection for molecular analysis using immediate separation of plasma, compared to the use of collection tubes allowing for delayed processing. METHODS: In this study, we measured the fractional abundance of tumor specific mutations (BRAF p.V600E and PIK3CA p.H1047R) in ctDNA isolated from blood samples collected in either cell-stabilizing Cell-Free DNA BCT tubes (delayed processing within 72 hours) or standard K3EDTA tubes (immediate processing within 15 minutes). Twenty-five blood sample pairs (EDTA/BCT) were collected from patients with advanced solid cancers enrolled in early clinical trials. RESULTS: Concordance in the fractional abundance of mutations in ctDNA isolated from blood collected in either K3EDTA or BCT tubes from patients with different solid cancers was observed. CONCLUSIONS: This study indicates that BCT tubes are preferable for collection of circulating DNA in a clinical setting due to the favorable storage and shipping conditions.

Next-Generation Sequencing-Based Detection of Germline Copy Number Variations in BRCA1/BRCA2: Validation of a One-Step Diagnostic Workflow.

Genetic testing of BRCA1/2 includes screening for single nucleotide variants and small insertions/deletions and for larger copy number variations (CNVs), primarily by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). With the advent of next-generation sequencing (NGS), it has become feasible to provide CNV information and sequence data using a single platform. We report the use of NGS gene panel sequencing on the Illumina MiSeq platform and JSI SeqPilot SeqNext software to call germline CNVs in BRCA1 and BRCA2. For validation 18 different BRCA1/BRCA2 CNVs previously identified by MLPA in 48 Danish breast and/or ovarian cancer families were analyzed. Moreover, 120 patient samples previously determined as negative for BRCA1/BRCA2 CNVs by MLPA were included in the analysis. Comparison of the NGS data with the data from MLPA revealed that the sensitivity was 100%, whereas the specificity was 95%. Taken together, this study validates a one-step bioinformatics work-flow to call germline BRCA1/2 CNVs using data obtained by NGS of a breast cancer gene panel. The work-flow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing and adapted to genes other than BRCA1/2.

Aurora kinase B is important for antiestrogen resistant cell growth and a potential biomarker for tamoxifen resistant breast cancer.

BACKGROUND: Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets. METHODS: Antiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose-response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen. RESULTS: The selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment. CONCLUSIONS: Our results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.

Aurora kinase A and B as new treatment targets in aromatase inhibitor-resistant breast cancer cells.

Aromatase inhibitors (AIs) are used for treatment of estrogen receptor α (ER)-positive breast cancer; however, resistance is a major obstacle for optimal outcome. This preclinical study aimed at identifying potential new treatment targets in AI-resistant breast cancer cells. Parental MCF-7 breast cancer cells and four newly established cell lines, resistant to the AIs exemestane or letrozole, were used for a functional kinase inhibitor screen. A library comprising 195 different compounds was tested for preferential growth inhibition of AI-resistant cell lines. Selected targets were validated by analysis of cell growth, cell cycle phase distribution, protein expression, and subcellular localization. We identified 24 compounds, including several inhibitors of Aurora kinases e.g., JNJ-7706621 and barasertib. Protein expression of Aurora kinase A and B was found upregulated in AI-resistant cells compared with MCF-7, and knockdown studies showed that Aurora kinase A was essential for AI-resistant cell growth. In AI-resistant cell lines, the clinically relevant Aurora kinase inhibitors alisertib and danusertib blocked cell cycle progression at the G2/M phase, interfered with chromosome alignment and spindle pole formation, and resulted in preferential growth inhibition compared with parental MCF-7 cells. Even further growth inhibition was obtained when combining the Aurora kinase inhibitors with the antiestrogen fulvestrant. Our study is the first to demonstrate that Aurora kinase A and B may be treatment targets in AI-resistant cells, and our data suggest that therapy targeting both ER and Aurora kinases may be a potent treatment strategy for overcoming AI resistance in breast cancer.

New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

Sorafenib and nilotinib resensitize tamoxifen resistant breast cancer cells to tamoxifen treatment via estrogen receptor α.

Tamoxifen­resistant breast cancer is a major clinical problem and new treatment strategies are highly warranted. In this study, the multitargeting kinase inhibitors sorafenib and nilotinib were investigated as potential new treatment options for tamoxifen­resistant breast cancer. The two compounds inhibited cell growth, reduced expression of total estrogen receptor α (ER), Ser118-phosphorylated ER, FOXA1 and AIB1 and resensitized tamoxifen­resistant cells to tamoxifen. The ER downmodulator fulvestrant exerted strong growth inhibition of tamoxifen­resistant cells and addition of sorafenib and nilotinib could not further suppress growth, showing that sorafenib and nilotinib exerted growth inhibition via ER. In support of this, estradiol prevented sorafenib and nilotinib mediated growth inhibition. These results demonstrate that sorafenib and nilotinib act via ER and ER-associated proteins, indicating that these kinase inhibitors in combination with tamoxifen may be potential new treatments for tamoxifen­resistant breast cancer.

The broad-spectrum metalloproteinase inhibitor BB-94 inhibits growth, HER3 and Erk activation in fulvestrant-resistant breast cancer cell lines.

Breast cancer cells can switch from estrogen receptor α (ER)- to human epidermal growth factor receptor (HER)-driven cell growth upon acquiring antiestrogen resistance. HER ligands are cleaved by metalloproteinases leading to release of active HER ligands, activation of HER receptors and consequently increased cell growth. In this study, we investigated the importance of HER receptors, in particular HER3, and HER ligand shedding for growth and signaling in human MCF-7 breast cancer cells and MCF-7-derived sublines resistant to the antiestrogen fulvestrant. The HER3/HER4 ligand heregulin 1ß induced phosphorylation of HER3, Akt and Erk, and partly rescued fulvestrant-inhibited growth of MCF-7 cells. HER3 ligands were found to be produced and shed from the fulvestrant-resistant cells as conditioned medium from fulvestrant-resistant MCF-7 cells induced phosphorylation of HER3 and Akt in MCF-7 cells. This was prevented by treatment of resistant cells with the metalloproteinase inhibitor TAPI-2. Only the broad-spectrum metalloproteinase inhibitor BB-94, and not the more selective inhibitors GM6001 or TAPI-2, which inhibited shedding of the HER ligands produced by the fulvestrant-resistant cells, was able to inhibit growth and activation of HER3 and Erk in resistant cells. Compared to MCF-7, fulvestrant-resistant cells have increased HER3 phosphorylation, but knockdown of HER3 had no inhibitory effect on resistant cell growth. The EGFR inhibitor gefitinib exhibited only a minor growth inhibition, whereas the pan-HER inhibitor CI-1033 exerted growth arrest. Thus, neither HER3 nor EGFR alone are the main driver of fulvestrant-resistant cell growth and treatment should target both receptors. Ligand shedding is not a treatment target, as receptor activation occurred, independent of release of ligands. Only the broad-spectrum metalloproteinase inhibitor BB-94 could abrogate HER3 and Erk activation in the resistant cells, which stresses the complexity of the resistance mechanisms and the requirement of targeting signaling from HER receptors by multiple strategies.

1α,25-dihydroxyvitamin D3 inhibits cell growth and NFκB signaling in tamoxifen-resistant breast cancer cells.

Resistance to antiestrogens is a major clinical problem in current breast cancer treatment and development of new treatment strategies for these tumors is highly prioritized. In this study, we have investigated the effects of 1α,25-dihydroxyvitamin D3 on the proliferation of tamoxifen-resistant cells. Further, we have investigated on a molecular level the effects of vitamin D on NFkB signaling in tamoxifen-resistant breast cancer cells. Parental human breast cancer MCF-7 cells and four tamoxifen-resistant sublines have been used to investigate the effects of 1α,25-dihydroxyvitamin D3 on cell proliferation using a colorimetric method, gene expression using quantitative PCR, protein phosphorylation using Western blot analysis and cellular localization of proteins using immunofluorescence microscopy. We found that 1α,25-dihydroxyvitamin D3 is able to strongly decrease the growth of both tamoxifen-sensitive and -resistant breast cancer cells and that this antiproliferative effect of 1α,25-dihydroxyvitamin D3 might be mediated via inhibition of the NFκB pathway. We found that 1α,25-dihydroxyvitamin D3 stimulates the gene expression of IkB, an NFκB-inhibiting protein, and that cells pretreated with 1α,25-dihydroxyvitamin D3 have a decreased sensitivity to TNFα stimulation. Further, we show that 1α,25-dihydroxyvitamin D3 treatment strongly decreases the TNFα-induced translocation of p65 into the nucleus. This manuscript reports novel findings regarding the effects of 1α,25-dihydroxyvitamin D3 on NFκB signaling in tamoxifen-resistant breast cancer cells and suggests that vitamin D might be interesting for further evaluation as a new strategy to treat antiestrogen-resistant breast cancers.

Estrogen receptor α is the major driving factor for growth in tamoxifen-resistant breast cancer and supported by HER/ERK signaling.

Resistance to tamoxifen is a major clinical challenge in the treatment of breast cancer; however, it is still unclear which signaling pathways are the major drivers of tamoxifen-resistant growth. To characterize resistance mechanisms, we have generated different tamoxifen-resistant breast cancer cell lines from MCF-7. In this model, we investigated whether signaling from human epidermal growth factor receptors (HERs), their downstream kinases, or from the estrogen receptor α (ERα) was driving tamoxifen-resistant cell growth. Increased expression of EGFR and increased phosphorylation of HER3 were observed upon acquisition of tamoxifen resistance, and the extracellular activated kinase (ERK) signaling pathway was highly activated in the resistant cells. The EGFR inhibitor gefitinib and the ERK pathway inhibitor U0126 resulted in partial and preferential growth inhibition of tamoxifen-resistant cells. All the tamoxifen-resistant cell lines retained ERα expression but at a lower level compared to that in MCF-7. Importantly, we showed via ERα knockdown that the tamoxifen-resistant cells were dependent on functional ERα for growth and we observed a clear growth stimulation of resistant cell lines with clinically relevant concentrations of tamoxifen and 4-OH-tamoxifen, indicating that tamoxifen-resistant cells utilize agonistic ERα stimulation by tamoxifen for growth. The tamoxifen-resistant cells displayed high phosphorylation of ERα at Ser118 in the presence of tamoxifen; however, treatment with U0126 neither affected the level of Ser118 phosphorylation nor expression of the ERα target Bcl-2, suggesting that ERK contributes to cell growth independently of ERα in our cell model. In support of this, combined treatment against ERα and ERK signaling in resistant cells was superior to single-agent treatment and as effective as fulvestrant treatment of MCF-7 cells. Together, these findings demonstrate that ERα is a major driver of growth in tamoxifen-resistant cells supported by HER/ERK growth signaling, implying that combined targeting of these pathways may have a clinical potential for overcoming tamoxifen resistance.

Carboplatin treatment of antiestrogen-resistant breast cancer cells.

Antiestrogen resistance is a major clinical problem in current breast cancer treatment. Therefore, biomarkers and new treatment options for antiestrogen-resistant breast cancer are needed. In this study, we investigated whether antiestrogen­resistant breast cancer cell lines have increased sensitivity to carboplatin, as it was previously shown with cisplatin, and whether low Bcl-2 expression levels have a potential value as marker for increased carboplatin sensitivity. Breast cancer cells resistant to the pure antiestrogen fulvestrant, and two out of four cell lines resistant to the antiestrogen tamoxifen, were more sensitive to carboplatin treatment compared to the parental MCF-7 cell line. This indicates that carboplatin may be an advantageous treatment in antiestrogen­resistant breast cancer; however, a marker for increased sensitivity would be needed. Low Bcl-2 expression was correlated with increased carboplatin response in the tamoxifen­resistant cell line MCF-7/TAMR-1 and overexpression of Bcl-2 in this cell line resulted in significantly reduced carboplatin sensitivity, confirming the anti-apoptotic role of Bcl-2. However, neither Bcl-2 expression alone, nor Bcl-2 in combination with Bcl-xL and Bax, could explain the observed responses to carboplatin in all tamoxifen­resistant cell lines, indicating that more markers are needed to predict the response to carboplatin in tamoxifen­resistant breast cancer.

NFκB signaling is important for growth of antiestrogen resistant breast cancer cells.

Resistance to endocrine therapy is a major clinical challenge in current treatment of estrogen receptor-positive breast cancer. The molecular mechanisms underlying resistance are yet not fully clarified. In this study, we investigated whether NFκB signaling is causally involved in antiestrogen resistant cell growth and a potential target for re-sensitizing resistant cells to endocrine therapy. We used an MCF-7-derived cell model for antiestrogen resistant breast cancer to investigate dependence on NFκB signaling for antiestrogen resistant cell growth. We found that targeting NFκB preferentially inhibited resistant cell growth. Antiestrogen resistant cells expressed increased p50 and RelB, and displayed increased phosphorylation of p65 at Ser529 and Ser536. Moreover, transcriptional activity of NFκB after stimulation with tumor necrosis factor α was enhanced in antiestrogen resistant cell lines compared to the parental cell line. Inhibition of NFκB signaling sensitized tamoxifen resistant cells to the growth inhibitory effects of tamoxifen but was not sufficient to fully restore sensitivity of fulvestrant resistant cells to fulvestrant. In support of this, depletion of p65 with siRNA in tamoxifen resistant cells increased sensitivity to tamoxifen treatment. Our data provide evidence that NFκB signaling is enhanced in antiestrogen resistant breast cancer cells and plays an important role for antiestrogen resistant cell growth and for sensitivity to tamoxifen treatment in resistant cells. Our results imply that targeting NFκB might serve as a potential novel treatment strategy for breast cancer patients with resistance toward antiestrogen.

The antipsychotic drug chlorpromazine enhances the cytotoxic effect of tamoxifen in tamoxifen-sensitive and tamoxifen-resistant human breast cancer cells.

Tamoxifen resistance is a major clinical problem in the treatment of estrogen receptor alpha-positive breast tumors. It is, at present, unclear what exactly causes tamoxifen resistance. For decades, chlorpromazine has been used for treating psychotic diseases, such as schizophrenia. However, the compound is now also recognized as a multitargeting drug with diverse potential applications, for example, it has antiproliferative properties and it can reverse resistance toward antibiotics in bacteria. Furthermore, chlorpromazine can reverse multidrug resistance caused by overexpression of P-glycoprotein in cancer cells. In this study, we have investigated the effect of chlorpromazine on tamoxifen response of human breast cancer cells. We found that chlorpromazine worked synergistically together with tamoxifen with respect to reduction of cell growth and metabolic activity, both in the antiestrogen-sensitive breast cancer cell line, MCF-7, and in a tamoxifen-resistant cell line, established from the MCF-7 cells. Tamoxifen-sensitive and tamoxifen-resistant cells were killed equally well by combined treatment with chlorpromazine and tamoxifen. This synergistic effect could be prevented by addition of estrogen, suggesting that chlorpromazine enhances the effect of tamoxifen through an estrogen receptor-mediated mechanism. To investigate this putative mechanism, we applied biophysical techniques to simple model membranes in the form of unilamellar liposomes of well-defined composition and found that chlorpromazine interacts strongly with lipid bilayers of different composition leading to increased permeability. This implies that chlorpromazine can change influx properties of membranes hence suggesting that chlorpromazine may be a promising chemosensitizing compound for enhancing the cytotoxic effect of tamoxifen.

Pyrazole carboxamides and carboxylic acids as protein kinase inhibitors in aberrant eukaryotic signal transduction: induction of growth arrest in MCF-7 cancer cells.

Densely functionalised pyrazole carboxamides and carboxylic acids were synthesised in an expedient manner through saponification and transamidation, respectively, of ester-functionalised pyrazoles. This synthetic protocol allowed for three diversifying steps in which appendages on the pyrazole scaffold were adjusted to optimise inhibition of protein kinases. Thirty-five analogues were tested in CK2, AKT1, PKA, PKCalpha, and SAPK2a (p38) kinase inhibition bioassays. Blocking of these kinases may lead to effective therapies for treating inflammatory diseases and cancer. In order to investigate potential biological activity, MCF-7 human breast cancer cells were incubated with the most promising derivatives. Two analogues caused changes in MCF-7 cell growth, one of them through cell cycle arrest demonstrated by cell cycle analysis.

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases.

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.

Inclining the purine base binding plane in protein kinase CK2 by exchanging the flanking side-chains generates a preference for ATP as a cosubstrate.

Protein kinase CK2 (casein kinase 2) is a highly conserved and ubiquitously found eukaryotic serine/threonine kinase that plays a role in various cellular key processes like proliferation, apoptosis and circadian rhythm. One of its prominent biochemical properties is its ability to use GTP as well as ATP as a cosubstrate (dual-cosubstrate specificity). This feature is exceptional among eukaryotic protein kinases, and its biological significance is unknown. We describe here a mutant of the catalytic subunit of protein kinase CK2 (CK2alpha) from Homo sapiens (hsCK2alpha) with a clear and CK2-atypical preference for ATP compared to GTP. This mutant was designed on the basis of several structures of CK2alpha from Zea mays (zmCK2alpha) in complex with various ATP-competitive ligands. A structural overlay revealed the existence of a "purine base binding plane" harbouring the planar moiety of the respective ligand like the purine base of ATP and GTP. This purine base binding plane is sandwiched between the side-chains of Ile66 (Val66 in hsCK2alpha) and Met163, and it adopts a significantly different orientation than in prominent homologues like cAMP-dependent protein kinase (CAPK). By exchanging these two flanking amino acids (Val66Ala, Met163Leu) in hsCK2alpha(1-335), a C-terminally truncated variant of hsCK2alpha, the cosubstrate specificity shifted in the expected direction so that the mutant strongly favours ATP. A structure determination of the mutant in complex with an ATP-analogue confirmed the predicted change of the purine base binding plane orientation. An unexpected but in retrospect plausible consequence of the mutagenesis was, that the helix alpha D region, which is in the direct neighbourhood of the ATP-binding site, has adopted a conformation that is more similar to CAPK and less favourable for binding of GTP. These findings demonstrate that CK2alpha possesses sophisticated structural adaptations in favour of dual-cosubstrate specificity, suggesting that this property could be of biological significance.