XRCC5 Plays an Important Role in Homologous Recombination, Genome Stability and Survival of Myeloma Cells

Abstract Understanding mechanisms underlying genomic instability is critical in delineating pathogenesis and development of new treatments for prevention and treatment of cancer. We have previously shown that dysregulated homologous recombination (HR) significantly contributes to genomic instability and progression in multiple myeloma (MM). To identify the regulators of HR and genome stability in MM, we conducted a functional shRNA screen and identified XRCC5 (Ku80) as a novel regulator of HR in MM cells. XRCC5 has been known to work as part of DNA ligase IV-XRCC4 complex in the repair of DNA breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events. Evaluation by Western blotting showed that all myeloma cell lines tested (RPMI, MM1S, OPM2, MM1R, U266, ARP, H929) had elevated expression of XRCC5, ranging from 3- to 10-fold elevation relative to average expression in two normal PBMC samples. Expression profiling showed a wide range of XRCC5 expression in myeloma patients, with a subset of patients with very high expression. To investigate the role of XRCC5 in ongoing acquisition of genomic changes, we investigated the association of XRCC5 with genomic instability using two different patient datasets (gse26863, n=246 and IFM 170 pt dataset) in which both the gene expression and genomic copy number information for each patient was available. Copy events were defined as changes observed in ≥ 3 and/or 5 consecutive SNPs. Higher XRCC5 expression significantly correlated with increase in the number of copy number change events in both the 170 dataset (p ≤ 0.005 for amplifications and p = 0.0001 for deletions) as well as in gse26863 dataset (p ≤ 0.004 for amplifications and p ≤ 0.00003 for deletions). To understand mechanisms by which XRCC5 regulates HR in myeloma cells, we investigatedprotein-protein interactions using a custom protein array coated with antibodies against major DNA repair and cell cycle proteins. Array was sequentially incubated with MM cell lysate and HRP-conjugated anti-XRCC5 antibody, and interacting partners were then identified by their address on the array. Investigation in two different cell lines (RPMI and U266) showed that XRCC5 in myeloma interacts with XRCC4 (an NHEJ protein), a panel of major HR regulators (RAD51, RAD52, BRCA2, BRCA1, BARD1, P73, P53, C-ABL) and with components of cell cycle including CDC42, CDK1 (which controls entry from G2 to mitosis), CDK4, CDK6, CHK, CDC36, CDC34, and cyclins E and H. Consistent with these data, knockdown (KD) of XRCC5 was associated with reduced HR as well as reduced proliferation rate followed by a complete cell death over a period of two to three weeks in different experiments, in all 3 myeloma cell lines tested. Moreover, the investigation in U266 cells showed that XRCC5-KD is associated with 3-fold increase in the fraction of cells in G2 phase of cell cycle. Importantly, the elevated expression of XRCC5 was associated with shorter event free (p < 0.013) as well as poor overall survival (p < 0.008) in 170 patient dataset. We evaluted the expression and clinical correlation of XRCC5 in RNA-seq data from 311 newly-diagnosed MM patients and observed that the elevated expression of XRCC5 also correlated with event free survival (p = 0.03). In summary, we report that XRCC5, besides its known role in NHEJ, has important roles in HR, cell cycle and may be involved in the crosstalk among these DNA repair pathways. Elevated XRCC5 expression is associated with dysregulation of HR with consequent impact on survival of myeloma patients. Elevated XRCC5 is, therefore, a promising new target to inhibit/reduce genomic evolution as well as MM cell growth. Disclosures Avet-Loiseau: celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.

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Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.803.803 ◽

2016 ◽

Vol 128 (22) ◽

pp. 803-803

Author(s):

Srikanth Talluri ◽

Mehmet Kemal Samur ◽

Leutz Buon ◽

Stekla A Megan ◽

Purushothama Nanjappa ◽

...

Keyword(s):

Genomic Instability ◽

Cell Lines ◽

Copy Number ◽

Cytidine Deaminase ◽

Myeloma Cell ◽

Mrna Editing ◽

Genomic Changes ◽

Elevated Expression ◽

Apobec Family

Abstract The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutations In order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%. We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045). In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. Disclosures Munshi: Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.

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Critical Role of Split Hand/Foot Malformation Type 1 (SHFM1) in Homologous Recombination and Cell Survival in Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v126.23.3625.3625 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3625-3625

Author(s):

Purushothama Nanjappa ◽

Subodh Kumar ◽

Srikanth Talluri ◽

Humza Ahmad ◽

Ahsun Bajwa ◽

...

Keyword(s):

Homologous Recombination ◽

Board Of Directors ◽

Genomic Instability ◽

Copy Number ◽

Critical Role ◽

Genomic Evolution ◽

Advisory Committees ◽

Myeloma Cells ◽

Equity Ownership

Abstract Genomic instability is associated with development and progression of cancer. Our previous studies have demonstrated that myeloma cells display a marked genomic instability and the number of mutations correlates with clinical outcome. Based on published observation that dysregulated homologous recombination (HR) contributes to genomic instability in multiple myeloma (MM) we have investigated mechanisms underlying dysregulated HR. We conducted a functional high-throughput shRNA screen using HR assay and identified Split hand/foot malformation type 1 (SHFM1) as a novel regulator of HR in MM. SHFM1 has been shown to directly interact with BRCA2 and function in DNA binding and recombination. Based on these we have here investigated the role/s of SHFM1 in genomic evolution and impact on other oncogenic pathways/activities in MM. Using 2 different datasets (gse26863, n=246 and IFM n=170) with both expression data and copy number/SNP information, we analyzed the association of SHFM1 with genomic instability in vivo, Genomic instability in each patient was determined by counting the total number of copy number change events (deletions and amplifications) defined as changes in ≥ 5 consecutive SNPs. We observed that the higher SHFM1 expression correlated with increase in both the amplifications (p=0.0015) and deletions (p=0.0188) in gse26863 dataset, and correlated with increase in amplifications in 170 dataset (p=0.0006). When events were defined as changes in ≥ 3 consecutive SNPs, the increased SHFM1 correlated with increase in amplifications (p ≤ 0.0004) in both datasets. The increased SHFM1 expression also correlated with hyperdiploidy (P=0.0008) in MM patients. These observations suggest that elevated SHFM1 is associated with evolution, including hyperdiploidy, in myeloma. In a loss of function study, we suppressed SHFM1 in three different myeloma cell lines (ARP, RPMI, U266) using shRNA. In all three cell lines the SHFM1-KD was associated with reduction in live cell number (~50% of control non-targeting shRNA) at day 1 after selection, to complete cell death over a period of 1 - 3 weeks, in 3 different experiments. Both cell cycle and annexin V-labeling showed that mechanism of cell death was apoptotic. These data suggest that elevated SHFM1 has a critical role in survival and prevention of apoptosis in myeloma cells. To further understand the roles of SHFM1 in myeloma, we identified the genes whose expression correlated with SHFM1. In two different datasets, the expression of SHFM1 positively correlated with genes involved in DNA synthesis, homologous recombination (ATR, TOPBP1, RAD1, RAD50, POLD2, NBN, SSBP1, MRE11A), cell cycle progression and telomere maintenance, whereas negatively correlated with several apoptosis related genes (including TNFRSF12A, TNFRSF9 and DAPK2) and SUV420H2 (involved in epigenetic transcriptional repression). Investigation by real time PCR confirmed that SHFM1-KD was associated with ~2.0-fold increase in the expression of TNFRSF9; these and other associations are being further investigated by mass spectrometry. In summary, our data show that SHFM1 has critical roles in genomic evolution as well as prevention of apoptosis in myeloma cells, and can be targeted to make myeloma cells static. Disclosures Anderson: acetylon pharmaceuticals: Equity Ownership; BMS: Consultancy; Oncocorp: Equity Ownership; Celgene Corporation: Consultancy; Gilead: Consultancy; Millennium: Consultancy. Avet-Loiseau:onyx: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

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Sensitizing shRNA Screen for Molecular Targets in CDK4/CDK6-Based Combination Therapy in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.3440.3440 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3440-3440 ◽

Cited By ~ 1

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

David Chiron ◽

Ruben Niesvizky ◽

Anna C. Schinzel ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Clinical Trial ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Proteasome Inhibitors ◽

Advisory Committees ◽

Myeloma Cells ◽

Shrna Screen

Abstract CDK4 and CDK6 are rarely mutated but are overexpressed or hyperactivated at a very high frequency in human cancers. By inhibiting CDK4/CDK6 with an exceptionally selective and reversible inhibitor, palbociclib (PD 0332991), we have developed a novel strategy to reprogram cancer cells for cytotoxic killing through induction of prolonged early G1 arrest (pG1). We have demonstrated that pG1 sensitizes cancer cells expressing Rb, the substrate of CDK4 and CDK6, to cytotoxic killing by forcing an imbalance in gene expression because only genes scheduled for early G1 are expressed. This sensitization is exacerbated after palbociclib withdrawal due to incomplete restoration of gene expression despite S phase synchronization (pG1-S). This study aims to identify genes that mediate pG1-S sensitization to two clinically-relevant agents for myeloma, the proteasome inhibitors carfilzomib and bortezomib, in model cell lines by a sensitizing pool genome-wide shRNA screen, and by validating the hits in a clinical trial of palbociclib in combination with bortezomib and dexamethasone. We ranked the hits based on the enrichment of target shRNAs, and representation in replica of each cell lines and among different human myeloma cell lines (HMCLs) as well as functional analyses. In myeloma cells, cell cycle control by palbociclib was intact in all hits, demonstrating that CDK4 and CDK6 are indispensable for myeloma replication. Among the top ranking 20 candidates, we found that NEDD4L was essential for proteasome inhibitor killing, FTH1 modulated the threshold of killing by diverse agents especially in pG1-S, and IL10RAappeared to be required for pG1-S sensitization to proteasome inhibitors. Moreover, RNA-sequencing analysis of primary myeloma cells from a phase II clinical trial targeting CDK4/CDK6 with palbociclib in combination with bortezomib in myeloma revealed that a higher level of FTH1 expression in myeloma cells in vivo correlated with sensitivity to this therapy, suggesting a role for FTH1 in differential sensitivity to this CDK4/CDK6-based therapy in myeloma. Selective inhibition of CDK4/CDK6 with palbociclib, or another specific inhibitor such as LY2835219 or LEE011, in combination therapy has now achieved unprecedented clinical efficacy in diverse human cancers. Most notably, palbociclib more than doubled the progression free survival of metastatic breast cancer patients when it was combined with letrozole, and has been designated a “breakthrough therapy” by the FDA for breast cancer. Our work provides the first insight into genes that mediate cell cycle sensitization to cytotoxic killing through selective CDK4/CDK6 inhibition. It provides an exciting potential for further investigation in a clinical context, such as the ongoing phase I clinical trial combining palbociclib with the immunomodulatory drug lenalidomide in patients with relapsed/refractory myeloma. Disclosures Huang: Celgene: Research Funding. Off Label Use: PD 0332991 (palbociclib) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Niesvizky:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Chen-Kiang:Celgene: Consultancy, Research Funding.

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Mafb Protein Confers Primary Resistance of Myeloma to Proteasome Inhibitors

Blood ◽

10.1182/blood.v124.21.2091.2091 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2091-2091

Author(s):

Ya-Wei Qiang ◽

Shiqiao Ye ◽

Faith E Davies ◽

Bart Barlogie ◽

Joshua Epstein ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Proteasome Inhibitors ◽

Myeloma Cell ◽

Molecular Subgroups ◽

Advisory Committees ◽

Primary Resistance ◽

Molecular Subgroup ◽

Myeloma Cells ◽

20 Nm

Abstract Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that both the MS and MF subgroups associated with inferior survival (Zhan et al, Blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup while patients in the MF subgroups (including C-MAF and MAFb) did not benefit from Bzb (Nair, Blood 2010). We have previously demonstrated that Bzb prevents c-MAF protein degradation leading to primary drug resistance (Abstract # 281, ASH 2013). In the present study, we assessed the ability of MAFb, another MAF family member, to influence the innate resistance to proteasome inhibitors (PI) and identify the molecular mechanism underlying the resistance of proteasome inhibitors in high MAFb-expressing patients. To investigate the association of the limited therapeutic effect of proteasome inhibitors in the different molecular subgroup of myeloma, we compared the IC50 of Bzb and carfilzomib (CFZ) in 29 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentrations of Bzb were higher (>25 nM) in all 4 MAFb MMCL and >60 nM in one MAFb MMCL, which expressed the highest level of MAFb protein, as determined by immunoblot analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. For CFZ, IC50 concentrations were higher (>30nM) in all 11 c-MAF cell lines, while the IC50 levels were lower (2-20 nM) for the MMCL belonging to other molecular subgroup. One MMCL harboring t (14; 20) with high IC50 of Bzb (35 nM) showed low IC50 (10 nM) for CFZ. These results indicate that high MAFb expression in myeloma cells may contribute to primary resistance to Bzb and MM cells that express high MAFb protein although resistant to Bzb are sensitive to CFZ. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAFb protein levels in a dose-dependent manner, suggesting that Bzb prevents the degradation of MAFb protein in myeloma cells. To further confirm that drug-induced stabilization of MAFb protein confers resistance to Bzb and partially to CFZ, we generated loss of functional MAFb cells by silencing MAFb expression in a t (14;20) positive myeloma cell line using lentiviral shRNA specific to MAFb mRNA (shMAFb). shMAFb infected myeloma cells had 85% lower levels of MAFb mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased ITGB (9.1 fold), E-cadherin (2.5-fold), CCND2 (5.5-fold), and CCR1 (25-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAFb expression significantly decreased proliferation of myeloma cells (65.1% decrease, p=2.1E-6). Moreover, Bzb treatment of the cells infected with shMAFb led to 50.2%, inhibition (P=2.9E-8) of proliferation compared with control cells. Similarly, CFZ treatment of cells with silenced MAFb resulted in 54.3% (P=3.5E-8) inhibition of proliferation compared with control cells. Taken together, our results indicate that high expression of MAFb protein, similar to c-MAF, confers primary resistance to Bzb as well as to CFZ. In addition Bzb induces stabilization of MAFb protein, further increasing resistance to Bzb. These data provide the molecular rational for adopting an alternative therapeutic strategy for high-MAFb expressing myeloma patients. Disclosures Morgan: Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

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Mapping of the Multiple Myeloma Transcriptional Core Regulatory Circuitry Reveals TCF3 As a Novel Dependency and an Oncogenic Collaborator of MYC

Blood ◽

10.1182/blood.v130.suppl_1.64.64 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 64-64

Author(s):

Charles Y Lin ◽

Mariateresa Fulciniti ◽

Michael A Lopez ◽

Mehmet Kemal Samur ◽

Raphael Szalat ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

Transcription Factors ◽

Board Of Directors ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Advisory Committees ◽

Myeloma Cells ◽

Over Time

Abstract Multiple Myeloma (MM) is a complex plasma cell malignancy driven by numerous genetic and epigenetic alterations that are acquired over time. The events controlling and modifying transcriptomic changes that drive MM cell growth and progression remains undefined. To reveal the epigenetic circuitry governing myeloma cells, we performed a comprehensive analysis integrating data obtained from Multiplexed Indexed T7 Chromatin IP (Mint-ChIP), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), and RNA-seq in 10 primary MM cells as well as 3 MM cell lines to identify genome-wide the master transcription factors (TFs), the enhancer elements they occupy, and the genes they regulate. Using these data, we have identified myeloma-specific core regulatory circuitry which includes several well-established regulators of MM such as IKZF, E2F, MYC and IRF family of genes. For example, our data show elevated MYC at numerous tissue specific enhancers in myeloma cells, including those that regulate lineage specifying transcription factors such as IRF4 and TCF3 (aka E2A). When translocated to the immunoglobulin enhancer, MYC in turn is regulated by these lineage transcription factors thus integrating MYC into the interconnected transcriptional core regulatory circuitry of MM (Figure 1a,b). We propose that this oncogenic "re-wiring" accounts for the observed addiction of MM cells to lineage factors such as IRF4 and in this work, we implicate the B-cell factor TCF3 as a novel multiple myeloma dependency. Using myeloma cell lines and primary samples, we observed elevated enhancer activity at TCF3 in primary CD138+ cells from myeloma patients compared to normal plasma cells (NPCs) (Figure 1c). As a result, TCF3 expression is significantly upregulated in our large cohort of MM patients (n=370) compared to normal bone marrow plasma cells (n=18). As MYC proteins can only bind pre-established and acetylated regions of active chromatin, we hypothesize that enhancer specifying lineage transcription factors such as TCF3 may cooperate with MYC to alter tissue specific gene expression programs. We show that TCF3 is regulated by a large proximal enhancer that is bound by MYC, and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. As a helix-loop-helix transcription factor that similar to MYC binds short (CANNTG) E-box sequences, we computationally predict co-occupancy of MYC and TCF3 at ~80% of all enhancers that form the multiple myeloma transcriptional core regulatory circuitry. To evaluate the functional role of TCF3 in myeloma cells, we established TCF3 knock down myeloma cell lines and followed the cell growth over time. Stable knockdown of TCF3 preferentially blocks proliferation of IgH MYC translocated cell lines (such as MM1.S cells) versus non-translocated lines (such as U266 cells). Finally, high expression of TCF3 correlates with poor clinical outcome in myeloma patients. Together these data suggest TCF3 acts as an oncogenic collaborator with deregulated MYC and implicates transcriptional control of lineage as a dependency in multiple myeloma. Figure 1: Transcriptional core regulatory circuitry of multiple myeloma: A) ChIP-Seq tracks of IRF4, MYC, BRD4, and H3K27ac occupancy at the IRF4, IgH enhancer, and TCF3 loci respectively. B) Schematic of transcription factor to enhancer connectivity of the partial multiple myeloma transcriptional core regulatory circuitry highlighting interactions between IRF4, MYC, and TCF3 (computationally predicted based on TCF3 motif data). C) ChIP-Seq tracks of H3K27ac occupancy at the TCF3 locus in patient multiple myeloma (top, n=3) or normal plasma cells (bottom, n=2). Figure 1 Figure 1. Disclosures Bradner: Acetylon: Other: Scientific Founder; Novartis: Employment. Anderson: Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

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Targeting PI3K-AKT-mTOR Signaling in Multiple Myeloma Mesenchymal Stem Cells Mediates Antiproliferative Effect on Myeloma Cells

Blood ◽

10.1182/blood-2021-150413 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1600-1600

Author(s):

Luca Heinmann ◽

Helal Mohammed Mohammed Ahmed Noman ◽

Klara Möllers ◽

Subbaiah Chary Nimmagadda ◽

Kaiyan Sun ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Therapeutic Strategies ◽

Advisory Committees ◽

Myeloma Cells ◽

Bm Niche ◽

Novel Therapeutic Strategies ◽

Novel Therapeutic

Abstract Introduction: Multiple myeloma (MM) is a B-cell malignancy characterized by an abnormal proliferation and infiltration of malignant plasma cells in the bone marrow (BM). Mesenchymal stromal cells (MSCs) represent a crucial component of the BM niche and mediate essential signalling via cytokines and cell-cell interactions. The interplay of MM cells and BM-MSC is complex and relies on multiple signaling pathways leading to MM progression and therapeutic resistance. Objectives: MM remains an incurable disease so far. Distinctive for this disease is a long-lasting polarization of the BM niche influencing MM progression and prognosis. We, therefore, focussed on MSCs to identify enrichment for different hallmark gene sets and their aberrant signaling contributing to the pathogenesis of the disease, therapy response and to further identify novel therapeutic strategies. Methods: BM-MSCs were isolated from patients with MM at diagnosis (MM-D-MSC) and in remission (MM-R-MSC) as well as from donors with other malignant diseases (CTR-MSC). RNA sequencing and Western Blot were used for examination of enriched pathways. Various functional assays for proliferation, apoptosis and cell cycle were performed either using a mono-culture or co-culture protocol of MSC and the MM-cell lines MM.1S and SKMM2 treating the cells with the pan-PI3K-inhibitor GDC-0941. Results: MM-D-MSCs supported the growth of myeloma cell lines better (3 fold, p<0.01) than MM-R- and CTR-MSCs. Our results demonstrate that MM-D-MSCs have a distinct gene expression profile compared to CTR-MSC indicating potential nodes of crosstalk and therapeutic importance. Amongst others, the PI3K-AKT-mTOR hallmark gene set was significantly enriched in MM-D-MSCs as compared to CTR-MSCs (p<0.001). We confirmed these findings on a proteomic level. We found evidence for the upregulation of PI3Kα, AKT, pAKT and mTOR in MM-D-MSC comparing to the other MM-R- and CTR-MSCs (p<0.05). We treated these MSC and the MM-cell lines MM.1S and SKMM2 with the PI3-Kinase inhibitor GDC-0941. The treatment reduced the signaling PI3Kα, AKT and mTOR in both, MSC and MM-cells. As stated MM-D-MSC supported the growth of myeloma cells better than other MSC types. However, upon GDC-0941 treatment, the proliferation of MM-D-MSCs was significantly reduced compared to the other MSC-types. In addition, the inhibition of proliferation of myeloma cell lines MM1S and SKMM2 was much more pronounced when they were cocultured with MM-D-MSC (32 and 34 %, p=0.04) compared to the growth of myeloma cells in coculture with MSC types, either in remission or other malignancies. Conclusion: We here identified functionally distinct differences in MM-D-MSCs compared to MM-R-MSCs or CTR-MSCs. Our data further provides a deeper insight into the molecular signature of MM-MSCs, a predictive of patient prognosis and treatment outcome. Targeting MSCs as a crucial part of the MM-BM niche by using PI3K-inhibitors could contribute to novel therapeutic strategies to effectively block MM-MSC interaction improving overall patient survival. Disclosures Raab: Roche: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Khandanpour: BMS/Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; GSK: Honoraria.

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High-Throughput Immunofluorescence and Electron Tomography to Characterize Centrosomal Aberrations in Plasma Cell Neoplasia

Blood ◽

10.1182/blood-2019-130713 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3077-3077

Author(s):

Tobias Dittrich ◽

Martin Schorb ◽

Isabella Haberbosch ◽

Elena Bausch ◽

Mandy Börmel ◽

...

Keyword(s):

Board Of Directors ◽

Cancer Patients ◽

Genomic Instability ◽

Cell Lines ◽

Plasma Cell ◽

High Throughput ◽

Research Funding ◽

Electron Tomography ◽

Automated Analysis ◽

Advisory Committees

Introduction Genomic instability is the basic prerequisite for a Darwinian-type evolution of neoplasia and as such represents a fundamental hallmark of cancer. Centrosomal aberrations have been identified as potent drivers of genomic instability (Cosenza et al., Cell Reports 2017; Krämer et al., Leukemia 2003). The current standard to investigate centrosomal aberrations in cancer patients is immunofluorescence (IF) staining. Although this method is fast and easily scalable, its diagnostic significance is controversially discussed. Moreover, ultrastructural analysis of centrosomes in cancer patients is required to gain a mechanistical understanding of the relationship between genomic instability and centrosomal aberrations. To address this, we combined semi-automated analysis of immunofluorescence (IF) images with high-throughput electron tomography (ET) of different cell lines and subentities of primary plasma cell neoplasia, which serve as surrogate for clonal evolution. Methods CD138+ plasma cells were isolated from bone marrow aspirates of consenting patients with plasma cell neoplasia. Each sample was split to be subsequently processed for IF and ET. The IF workflow included (1) chemical fixation, (2) staining for nuclei, cells, centrin and pericentrin, (3) semi-automated acquisition of >1000 cells, (4) semi-automated analysis of IF data using the software Konstanz Information Miner (KNIME) (Berthold et al., GfKL 2007). The ET workflow included (1) chemical fixation (2) agarose embedding, (3) dehydration and epoxy resin embedding, (4) serial sectioning at 200 nm, (5) semi-automated screening for centrioles with transmission electron microscopy (TEM) (Schorb et al., Nature Methods 2019), (6) semi-automated acquisition of previously identified centriole regions with serial section ET. Results So far, four patients with relapsed refractory myeloma as well as two cell lines (U2OS-PLK4, RPMI.8226) have been screened with TEM. No centrosomal amplification was apparent by IF in any of these patients. Within 5598 cells, 205 centrosomes have been detected. A total of 659 electron tomograms were performed on 141 regions of interest that were distributed on average over five sections. One patient with highly refractory multiple myeloma (resistance to eight prior therapies) showed over-elongated and partially fragmented centrioles (Figure), similar to recently reported findings in tumor cell lines (Marteil et al., Nature Communications 2018). Six out of 10 mother centrioles in this patient were longer than 500 nm, which is supposed to be the physiological length. The dimensions (mean [range]) of mother (decorated with appendages) and daughter centrioles in this patient were: length 919 nm [406 nm - 2620 nm] and 422 nm [367 nm - 476 nm]; diameter 221 nm [99 nm - 470 nm] and 236 nm [178 nm - 450 nm]. Moreover, the mother centrioles showed multiple sets of appendages (mean [range]: 5.9 [2 - 13]), while one set of appendages would be physiological. This is an ongoing study and additional results are expected by the date of presentation. Conclusions We present a semi-automated methodological setup that combines high-throughput IF and cutting-edge ET to study centrosomal aberrations. To our knowledge, this is the first study that systematically analyzes the centrosomal phenotype of cancer patients at the ultrastructural level. Our preliminary IF results suggest that supernumerary centrosomes in plasma cell neoplasia might be less common than previously reported. Moreover, we for the first time describe and characterize over-elongated centrioles in myeloma patients, reminiscent of previous findings in tumor cell lines. With increasing numbers of patients, we will be also able to correlate results from IF and ET to address the current uncertainty with respect to IF screens for centrosomal aberrations. Better insight into centrosomal aberrations will likely increase our understanding on karyotype evolution in plasma cell neoplasia and possibly facilitate the development of novel targeted therapies. Figure Disclosures Goldschmidt: John-Hopkins University: Research Funding; John-Hopkins University: Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding; Janssen: Consultancy, Research Funding; Mundipharma: Research Funding; Chugai: Honoraria, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Schönland:Medac: Other: Travel Grant; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding.

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Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition

Blood ◽

10.1182/blood-2020-142592 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 1-2

Author(s):

Yao Yao ◽

Woojun D Park ◽

Eugenio Morelli ◽

Mehmet Kemal Samur ◽

Nicholas P Kwiatkowski ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Mononuclear Cells ◽

Luciferase Reporter ◽

G1 Arrest ◽

Normal Donor ◽

Private Company ◽

Advisory Committees

Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.

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Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.133.133 ◽

2010 ◽

Vol 116 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Patricia Maiso ◽

AbdelKareem Azab ◽

Yang Liu ◽

Yong Zhang ◽

Feda Azab ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Cell Lines ◽

Mechanism Of Action ◽

Plasma Cells ◽

Bone Marrow Microenvironment ◽

Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽

10.1182/blood.v116.21.2999.2999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2999-2999 ◽

Cited By ~ 1

Author(s):

Samantha Pozzi ◽

Diana Cirstea ◽

Loredana Santo ◽

Doris M Nabikejje ◽

Kishan Patel ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Preliminary Data ◽

Research Funding ◽

Preclinical Studies ◽

Dependent Manner ◽

Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

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