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• W2122734929 abstract "•Phenotypic screening identifies compounds that overcome stromal resistance in MM•Compound BRD9876 only inhibits microtubule-bound Eg5, which is high in MM cells•This unusual mode of action enables MM over hematopoietic progenitor selectivity•Thus, compounds identified here can discover “druggable” vulnerabilities within MM Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876’s mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets. Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876’s mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets. Multiple myeloma (MM) is an incurable malignancy that caused more than 80,000 deaths in 2012 worldwide (Ferlay et al., 2013Ferlay, J., Soerjomataram, I., Ervik, M., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D., and Bray, F. (2013). GLOBOCAN 2012: estimated cancer incidence, mortality and prevalence worldwide in 2012. http://globocan.iarc.fr.Google Scholar). Novel therapeutic approaches are desperately needed, but target-based drug development against this disease is challenging due to complex genomic alterations including multiple driver mutations even within the same individual (Lohr et al., 2014Lohr J.G. Stojanov P. Carter S.L. Cruz-Gordillo P. Lawrence M.S. Auclair D. Sougnez C. Knoechel B. Gould J. Saksena G. et al.Multiple Myeloma Research ConsortiumWidespread genetic heterogeneity in multiple myeloma: implications for targeted therapy.Cancer Cell. 2014; 25: 91-101Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar). An alternative phenotypic approach to therapeutic discovery is to define a cellular phenotype representative of the disease and to use small-molecule screening to discover simultaneously relevant therapeutic targets and lead compounds. The success of this general approach is shown by the observation that the majority of first-in-class drugs approved by the US Food and Drug Administration (FDA) between 1999 and 2008 came from phenotypic approaches despite the dominance of target-based drug discovery during that period (Swinney and Anthony, 2011Swinney D.C. Anthony J. How were new medicines discovered?.Nat. Rev. Drug Discov. 2011; 10: 507-519Crossref PubMed Scopus (1376) Google Scholar). In MM, novel therapeutic discovery has occurred largely using the phenotypic approach. Lactacystin, a natural product with phenotypic effects in cancer cells, was used to discover that the proteasome could be targeted selectively by engaging a catalytic β-subunit N-terminal threonine (Fenteany et al., 1995Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin.Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar). This knowledge accelerated the discovery and development of bortezomib from previously unselective peptide aldehydes (Adams et al., 1998Adams J. Behnke M. Chen S. Cruickshank A.A. Dick L.R. Grenier L. Klunder J.M. Ma Y.T. Plamondon L. Stein R.L. Potent and selective inhibitors of the proteasome: dipeptidyl boronic acids.Bioorg. Med. Chem. Lett. 1998; 8: 333-338Crossref PubMed Scopus (639) Google Scholar), and subsequently, bortezomib transformed the treatment of MM (Richardson et al., 2005Richardson P.G. Sonneveld P. Schuster M.W. Irwin D. Stadtmauer E.A. Facon T. Harousseau J.L. Ben-Yehuda D. Lonial S. Goldschmidt H. et al.Assessment of Proteasome Inhibition for Extending Remissions (APEX) InvestigatorsBortezomib or high-dose dexamethasone for relapsed multiple myeloma.N. Engl. J. Med. 2005; 352: 2487-2498Crossref PubMed Scopus (2222) Google Scholar). The development of thalidomide and its analogs against MM also followed phenotypic observations of thalidomide’s effects on angiogenesis and immune function (Bartlett et al., 2004Bartlett J.B. Dredge K. Dalgleish A.G. The evolution of thalidomide and its IMiD derivatives as anticancer agents.Nat. Rev. Cancer. 2004; 4: 314-322Crossref PubMed Scopus (711) Google Scholar). Based on these encouraging precedents, we undertook a phenotypic screening approach to systematically discover compounds that uncover new therapeutic strategies against MM. In 95% of MM cases, the malignant cells are restricted to the bone marrow, where interactions with the stromal niche are thought to be critical for their survival (Hideshima et al., 2007Hideshima T. Mitsiades C. Tonon G. Richardson P.G. Anderson K.C. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets.Nat. Rev. Cancer. 2007; 7: 585-598Crossref PubMed Scopus (754) Google Scholar) and for drug resistance to traditional chemotherapy drugs (Meads et al., 2008Meads M.B. Hazlehurst L.A. Dalton W.S. The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance.Clin. Cancer Res. 2008; 14: 2519-2526Crossref PubMed Scopus (439) Google Scholar). The ability to overcome resistance factors from bone marrow stromal cells (BMSCs) is now considered an obligate requirement for any novel therapeutic agent against MM (Dalton and Anderson, 2006Dalton W. Anderson K.C. Synopsis of a roundtable on validating novel therapeutics for multiple myeloma.Clin. Cancer Res. 2006; 12: 6603-6610Crossref PubMed Scopus (18) Google Scholar). Agents like bortezomib, thalidomide, and lenalidomide overcome BMSC resistance in vitro (Hideshima et al., 2000Hideshima T. Chauhan D. Shima Y. Raje N. Davies F.E. Tai Y.T. Treon S.P. Lin B. Schlossman R.L. Richardson P. et al.Thalidomide and its analogs overcome drug resistance of human multiple myeloma cells to conventional therapy.Blood. 2000; 96: 2943-2950Crossref PubMed Google Scholar, Hideshima et al., 2001Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliott P.J. Adams J. Anderson K.C. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells.Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar) and achieve complete, durable responses in vivo, resulting in greatly improved survival (Hideshima et al., 2007Hideshima T. Mitsiades C. Tonon G. Richardson P.G. Anderson K.C. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets.Nat. Rev. Cancer. 2007; 7: 585-598Crossref PubMed Scopus (754) Google Scholar). McMillin and colleagues used an assay in which labeled MM cell lines are co-cultured with BMSCs and determined that the anti-MM activities of a variety of FDA-approved and bioactive compounds are attenuated by BMSCs (McMillin et al., 2010McMillin D.W. Delmore J. Weisberg E. Negri J.M. Geer D.C. Klippel S. Mitsiades N. Schlossman R.L. Munshi N.C. Kung A.L. et al.Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity.Nat. Med. 2010; 16: 483-489Crossref PubMed Scopus (265) Google Scholar). We adapted this BMSC-MM co-culture assay in 384-well plates for high-throughput screening to identify compounds that overcome the stroma-induced drug-resistance phenotype in MM. Because hematological toxicity is common for anti-MM agents, we prioritized compounds with selectivity over human hematopoietic progenitors to identify those with improved toxicity profiles. One such compound, BRD9876, was discovered to be a distinctive kinesin-5 (Eg5; kinesin spindle protein; KIF11) inhibitor that specifically targets microtubule-bound Eg5, allowing greater selectivity over hematopoietic cells. An Eg5 inhibitor, ARRY-520, is showing promising, durable responses in MM (Shah et al., 2011Shah J.J. Zonder J. Cohen A. Orlowski R.Z. Alexanian R. Thomas S.K. Weber D. Kaufman J.L. Harvey R.D. Walker D. et al.ARRY-520 shows durable responses in patients with relapsed/refractory multiple myeloma in a phase 1 dose-escalation study.ASH Annual Meeting Abstracts. 2011; 118: 1860Google Scholar), but its use is limited by hematological toxicity. We report here a novel mechanism of Eg5 inhibition that could preserve anti-MM efficacy while mitigating potentially life-threatening hematological toxicity during MM treatment. To recapitulate the MM niche in vitro, we used primary human BMSCs isolated from hip replacement samples, removed for non-cancerous indications. We co-cultured primary MM cells with these BMSCs and stained them with the vital dye Calcein AM, utilizing intensity and shape differences to distinguish and quantify the two cell types in the MetaXpress software (Molecular Devices). Primary MM cells displayed round, bright staining (Figure 1A), whereas BMSCs alone appeared as dull, spindle-shaped objects. Co-culture with BMSCs maintained the viability of CD138(+) primary MM cells >5-fold better than culture media alone (Figure 1B), confirming previously observed stroma dependence of primary MM (Zlei et al., 2007Zlei M. Egert S. Wider D. Ihorst G. Wäsch R. Engelhardt M. Characterization of in vitro growth of multiple myeloma cells.Exp. Hematol. 2007; 35: 1550-1561Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). To recapitulate this behavior in a cell line amenable to high-throughput screening, the stroma-dependent MM cell line MOLP5 (Matsuo et al., 2000Matsuo Y. Drexler H.G. Nishizaki C. Harashima A. Fukuda S. Kozuka T. Sezaki T. Orita K. Human bone marrow stroma-dependent cell line MOLP-5 derived from a patient in leukaemic phase of multiple myeloma.Br. J. Haematol. 2000; 109: 54-63Crossref PubMed Scopus (15) Google Scholar), stably expressing GFP, was similarly grown alone or in co-culture with stroma in 384-well plates. A similar ∼7-fold increase in GFP(+) MOLP5 cells was observed with stromal co-culture (Figure 1B) using image-based quantification, making this an attractive physiologically relevant high-throughput assay to screen for small-molecule inhibitors of MM within its niche. To determine the impact of stroma on the activity of small molecules, we chose two other MM cell lines (MM.1S cells and IL6-dependent INA6 cells) that grow with or without BMSCs. MM.1S (MM1S) cells grow well alone but double their proliferation in the presence of stromal cells (Hideshima et al., 2001Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliott P.J. Adams J. Anderson K.C. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells.Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar; Figure 1C). The survival of INA6 cells is dependent on the presence of high concentrations of IL6 or on co-culture with BMSCs (Chatterjee et al., 2002Chatterjee M. Hönemann D. Lentzsch S. Bommert K. Sers C. Herrmann P. Mathas S. Dörken B. Bargou R.C. In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6/gp130/STAT3 pathway.Blood. 2002; 100: 3311-3318Crossref PubMed Scopus (82) Google Scholar). The effect of BMSC co-culture on small-molecule activity was confirmed in these cells. Dexamethasone inhibited the growth of MM1S grown alone, but not in the presence of stroma (Figure 1D). In contrast, bortezomib, which overcomes microenvironment resistance (Hideshima et al., 2001Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliott P.J. Adams J. Anderson K.C. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells.Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar), is equally active in the presence or absence of stroma, consistent with its ability to induce durable complete responses in vivo. Thus, small molecules that overcome stromal resistance can be identified using MM1S and INA6 cells with or without stromal co-culture. Assay optimization was achieved using vorinostat as a positive control. The image-based viability assay and the Cell-Titer-Glo assay (Promega; measures cellular ATP content) produced similar dose-response curves (Figure 1E) with vorinostat. The Z’ factor, a statistical test of assay robustness that incorporates SDs and differences of means of negative and positive controls (Zhang et al., 1999Zhang J.H. Chung T.D. Oldenburg K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J. Biomol. Screen. 1999; 4: 67-73Crossref PubMed Scopus (5403) Google Scholar), was consistently above 0.5 for the MOLP5-BMSC imaging assay, confirming its suitability for high-throughput screening. We chose the MOLP5-BMSC co-culture assay as the primary assay for screening small molecules to identify compounds that either overcome stromal resistance or are active against stroma-dependent MM. A library of 25,280 compounds comprising FDA-approved drugs, bioactive compounds, natural products, commercial vendor compounds, kinase-biased compounds, chromatin-biased compounds, and compounds synthesized at the Broad Institute using diversity-oriented synthesis (DOS) (Nielsen and Schreiber, 2008Nielsen T.E. Schreiber S.L. Towards the optimal screening collection: a synthesis strategy.Angew. Chem. Int. Ed. Engl. 2008; 47: 48-56Crossref PubMed Scopus (427) Google Scholar) were tested in duplicate in the primary assay and showed good reproducibility (Figure 2A). Compounds that inhibited MOLP5 growth with Z scores ≤−2 relative to DMSO controls in both replicates were considered “hits”. We developed a prioritization schema to identify compounds of high interest (Figure 2B). We tested hits on BMSCs growing alone to exclude compounds with non-specific toxicity. Then, to exclude compounds that are susceptible to stromal resistance (like dexamethasone), we tested hits at eight doses on MM1S and INA6 cells with or without BMSCs. Compounds with IC50 with BMSCs ≤ IC50 without BMSCs were validated by ordering them in powder form and then retesting in primary and secondary assays. We identified 60 compounds that reproducibly inhibited MM growth, did not demonstrate non-specific toxicity, and could overcome stromal resistance (Table S1). Because hematological toxicity is common for most MM inhibitors, we then tested compounds on human CD34+ hematopoietic progenitors growing in serum-free liquid culture media, an assay predictive for in vivo hematological toxicity (Olaharski et al., 2009Olaharski A.J. Uppal H. Cooper M. Platz S. Zabka T.S. Kolaja K.L. In vitro to in vivo concordance of a high throughput assay of bone marrow toxicity across a diverse set of drug candidates.Toxicol. Lett. 2009; 188: 98-103Crossref PubMed Scopus (23) Google Scholar). Only a few compounds demonstrated greater inhibition of MM growth than hematopoietic growth (Table S1), and of these, three compounds were tested on primary MM cells co-cultured with BMSCs. We identified compounds with unique selectivity patterns. A sulfonamide BRD9647 (Figure 2C) displayed selective inhibition of stroma-dependent MOLP5 and primary MM cells, but not stroma-independent cell lines like MM1S (Figure 2D). This compound suppressed the viability of three out of six primary MM samples (Figures S1A and S1B) at greater than or equal to two concentrations ≤10 μM (p < 0.05 by t test), but the maximal effect did not exceed 50% in any sample, suggesting primarily cytostatic effects. A chromanone BRD2588 (Figure S1C) displayed enhanced activity in the presence of stroma (the opposite of dexamethasone). In contrast to vorinostat (Figure S1D), a hydrazone-containing hydroxamic acid BRD2318 displayed selectivity for IL6-dependent INA6 (Figure S1E). We focused our attention on a naphthalene bis-nitrile BRD9876 (Figure 2E) that inhibited MM1S growth equally with or without BMSCs (like bortezomib) with a modest (∼4-fold) but significant selectivity over normal CD34+-derived hematopoietic cells (MM1S IC50: 2.2 ± 0.5 μM; CD34 IC50: 9.2 ± 2 μM; Figure 2F). In contrast, no MM over CD34 selectivity was observed for most tested compounds such as doxorubicin (Figure 2F), which causes significant hematological toxicity in vivo in MM patients (Alberts and Salmon, 1975Alberts D.S. Salmon S.E. Adriamycin (NSC-123127) in the treatment of alkylator-resistant multiple myeloma: a pilot study.Cancer Chemother. Rep. 1975; 59: 345-350PubMed Google Scholar). BRD9876 was able to overcome, in MM1S cells, stromal resistance of BMSCs from MM bone marrow aspirates (Figure 2G), but only minimal effects were observed with BRD9876 against primary MM cells (Figure S2A). This was initially concerning, but we observed that primary MM cells do not proliferate in vitro, even in the presence of stroma in contrast to cell lines like MM1S (Figure S2B). Consistent with the lack of proliferation, primary MM cells were insensitive to the anti-mitotic Eg5 inhibitor ARRY-520 (Figure S2C), which is efficacious against MM in vivo, although they were sensitive to bortezomib that induces apoptosis (Figure S2D). These data suggested that BRD9876 may be specific for cycling MM cells. Because BRD9876 was able to inhibit diverse MM cell lines, overcome stromal resistance, and display selectivity over hematopoietic progenitors, we undertook studies to identify its mechanism of action. We first synthesized or purchased structural analogs of BRD9876 with the goal of immobilizing the compound on beads for affinity purification of protein-binding partners. However, all structural analogs of BRD9876 (Figure S3) were either less active against MM1S cells or lost selectivity between MM1S and CD34 blood cells. We therefore switched our attention to understanding the functional effects of BRD9876 on MM1S cells. Gene-expression analysis of MM1S cells treated for 6 hr with BRD9876 revealed sparse changes—only ten genes changed in expression >2-fold. Network analysis of genes with >1.5-fold changes revealed no significant relationship between genes (Figure S4A). We turned to analyzing individual genes, focusing on the top downregulated gene, ID1 (Figure 3A). We queried the Connectivity Map database (Lamb et al., 2006Lamb J. Crawford E.D. Peck D. Modell J.W. Blat I.C. Wrobel M.J. Lerner J. Brunet J.P. Subramanian A. Ross K.N. et al.The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease.Science. 2006; 313: 1929-1935Crossref PubMed Scopus (3601) Google Scholar), which contains over 7,000 gene-expression profiles of bioactive compounds, for compounds that downregulate ID1 expression. The top ten compounds that downregulate ID1 included the anti-mitotic paclitaxel (Figure S4B). This finding, in addition to literature reports of ID1 expression being associated with abnormal mitoses (Man et al., 2008Man C. Rosa J. Yip Y.L. Cheung A.L. Kwong Y.L. Doxsey S.J. Tsao S.W. Id1 overexpression induces tetraploidization and multiple abnormal mitotic phenotypes by modulating aurora A.Mol. Biol. Cell. 2008; 19: 2389-2401Crossref PubMed Scopus (13) Google Scholar), suggested possible anti-mitotic effects of BRD9876. Cell-cycle analysis of MM1S treated with 10 μM BRD9876 revealed rapid arrest of cells at the G2/M phase, starting as early as 2 hr of treatment (Figure 3B). In contrast, CD34 hematopoietic cells treated with BRD9876 showed markedly less G2/M arrest (Figure 3C), albeit these cultures contained fewer cycling cells. To determine whether treated MM1S cells arrested at G2 or entered mitosis, we stained the mitotic spindle protein α-tubulin. Not only were a significant number of cells arrested in mitosis, but they uniformly exhibited an aberrant mono-astral mitotic spindle (Figure 3D). Mono-astral spindles are a characteristic feature of inhibitors of mitotic kinesin-5 (Eg5), first demonstrated with the Eg5 inhibitor monastrol (Mayer et al., 1999Mayer T.U. Kapoor T.M. Haggarty S.J. King R.W. Schreiber S.L. Mitchison T.J. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.Science. 1999; 286: 971-974Crossref PubMed Scopus (1627) Google Scholar) but later observed with polo-like kinase or aurora kinase inhibitors (Tillement et al., 2009Tillement V. Remy M.H. Raynaud-Messina B. Mazzolini L. Haren L. Merdes A. Spindle assembly defects leading to the formation of a monopolar mitotic apparatus.Biol. Cell. 2009; 101: 1-11Crossref PubMed Scopus (27) Google Scholar). Therefore, we tested Eg5 activity in a microtubule-gliding assay in which recombinant full-length Eg5 drives motility of rhodamine-labeled microtubules (Mayer et al., 1999Mayer T.U. Kapoor T.M. Haggarty S.J. King R.W. Schreiber S.L. Mitchison T.J. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.Science. 1999; 286: 971-974Crossref PubMed Scopus (1627) Google Scholar). In the presence of 1 μM BRD9876, Eg5-mediated microtubule gliding was completely arrested (Figure 3E). Inhibition of Eg5 activity was further confirmed in a microtubule-stimulated ATPase assay of the recombinant Eg5 motor domain that demonstrated that BRD9876 was an ATP non-competitive inhibitor of Eg5 (Figures 3F and S5A). Eg5 inhibitors such as ARRY-520 have demonstrated promising durable responses in MM (Shah et al., 2011Shah J.J. Zonder J. Cohen A. Orlowski R.Z. Alexanian R. Thomas S.K. Weber D. Kaufman J.L. Harvey R.D. Walker D. et al.ARRY-520 shows durable responses in patients with relapsed/refractory multiple myeloma in a phase 1 dose-escalation study.ASH Annual Meeting Abstracts. 2011; 118: 1860Google Scholar), prompting a phase-III registration trial (Owens, 2013Owens B. Kinesin inhibitor marches toward first-in-class pivotal trial.Nat. Med. 2013; 19: 1550Crossref PubMed Scopus (19) Google Scholar). Current clinical Eg5 inhibitors such as ispinesib and ARRY-520 bind the monastrol-binding loop L5 region on Eg5 (Talapatra et al., 2012Talapatra S.K. Schüttelkopf A.W. Kozielski F. The structure of the ternary Eg5-ADP-ispinesib complex.Acta Crystallogr. D Biol. Crystallogr. 2012; 68: 1311-1319Crossref PubMed Scopus (41) Google Scholar, Woessner et al., 2009Woessner R. Tunquist B. Lemieux C. Chlipala E. Jackinsky S. Dewolf Jr., W. Voegtli W. Cox A. Rana S. Lee P. Walker D. ARRY-520, a novel KSP inhibitor with potent activity in hematological and taxane-resistant tumor models.Anticancer Res. 2009; 29: 4373-4380PubMed Google Scholar) and exhibit severe hematological toxicity (Burris et al., 2011Burris 3rd, H.A. Jones S.F. Williams D.D. Kathman S.J. Hodge J.P. Pandite L. Ho P.T. Boerner S.A. Lorusso P. A phase I study of ispinesib, a kinesin spindle protein inhibitor, administered weekly for three consecutive weeks of a 28-day cycle in patients with solid tumors.Invest. New Drugs. 2011; 29: 467-472Crossref PubMed Scopus (49) Google Scholar, Shah et al., 2011Shah J.J. Zonder J. Cohen A. Orlowski R.Z. Alexanian R. Thomas S.K. Weber D. Kaufman J.L. Harvey R.D. Walker D. et al.ARRY-520 shows durable responses in patients with relapsed/refractory multiple myeloma in a phase 1 dose-escalation study.ASH Annual Meeting Abstracts. 2011; 118: 1860Google Scholar) requiring myeloid growth factor support. Because BRD9876 exhibited selectivity between MM and hematopoietic cells, we reasoned that BRD9876 may have a different mode of Eg5 inhibition. To explore this, MM1S cells were raised to be resistant to BRD9876 by serial exposure to increasing compound concentrations. Consistent with our reasoning, an MM1S subline, MM1S-BRD9876R, had no cross-resistance to ispinesib (Figure 4A). Sequencing Eg5 cDNA from MM1S-BRD9876R revealed a previously unreported heterozygous mutation (Figure 4B) that alters the amino acid residue at position 104 from tyrosine (Y) to cysteine (C), suggesting a dominant drug-resistance allele. Intriguingly, this mutation is distant from the known binding site of existing Eg5 inhibitors (Figure 4C), suggesting a distinct allosteric binding site for BRD9876. Resistance to existing Eg5 inhibitors like ispinesib and monastrol is mediated by a loop L5 mutation at position 130 aspartate (D130; Figure 4C; Luo et al., 2007Luo L. Parrish C.A. Nevins N. McNulty D.E. Chaudhari A.M. Carson J.D. Sudakin V. Shaw A.N. Lehr R. Zhao H. et al.ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism.Nat. Chem. Biol. 2007; 3: 722-726Crossref PubMed Scopus (91) Google Scholar). To confirm the unique resistance mechanism of BRD9876, we generated recombinant Eg5 motor domain constructs, containing the wild-type (WT) sequence, Y104C mutation, or D130V mutation. Enzymatic microtubule-stimulated ATPase activities were identical for all three constructs (Figure S5B). The Y104C mutant was completely resistant to BRD9876 (Figure 4D) while showing no alteration to ispinesib activity. In contrast, the D130V mutant conferred ∼10-fold resistance to ispinesib and ∼10-fold sensitization to BRD9876. To test whether the Eg5 mutations were sufficient to confer resistance in cells, we introduced lentiviral vectors containing C-terminal GFP-tagged Eg5 open reading frame (ORF) with the WT sequence or Y104C or D130V mutations into adherent SW48 cells, chosen because of a high efficiency of lentiviral transduction. Cells expressing the Y104C mutant construct were resistant to BRD9876, consistent with the dominant drug-resistant nature of this allele, whereas the D130V mutation conferred enhanced sensitivity (Figures 4E). In contrast, the D130V, but not the Y104C, construct imparted resistance to ispinesib. Similar results were obtained using doxycycline-inducible lentiviral constructs in MM1S cells (Figure S5C). A recent report described a distinct Eg5 allosteric binding site between the α4 and α6 helices using a benzimidazole compound BI8 (Ulaganathan et al., 2013Ulaganathan V. Talapatra S.K. Rath O. Pannifer A. Hackney D.D. Kozielski F. Structural insights into a unique inhibitor binding pocket in kinesin spindle protein.J. Am. Chem. Soc. 2013; 135: 2263-2272Crossref PubMed Scopus (40) Google Scholar; Figure S5D). BI8 also bound, with weaker affinity, a site partially overlapping with the ispinesib L5 site. The crystal structure demonstrates that the Y104 residue participates in BI8 binding, forming a face-to-face stacking interaction with the trifluoromethylbenzyl group (Figure S5E). Indeed, biochemical ATPase assays confirmed that the Y104C mutation conferred resistance to BI8 activity (Figure S5F). In contrast to BRD9876, neither the Y104C or D130V mutants imparted resistance to BI8 in cells expressing those constructs (Figure S5G), suggesting that BI8 occupies both allosteric sites at the higher concentrations required for cellular growth inhibition. These studies confirm that the Y104 residue is a bona fide member of the α4/α6 allosteric site and that BRD9876 binds exclusively to this site. The Eg5 motor domain has basal ATPase activity in the absence of microtubules, but it is ∼100-fold less efficient (Luo et al., 2004Luo L. Carson J.D. Dhanak D. Jackson J.R. Huang P.S. Lee Y. Sakowicz R. Copeland R.A. Mechanism of inhibition of human KSP by monastrol: insights from kinetic analysis and the effect of ionic strength on KSP inhibition.Biochemistry. 2004; 43: 15258-15266Crossref PubMed Scopus (52) Google Scholar). Surprisingly, we found that BRD9876 was completely ineffective at inhibiting the basal ATPase activity of Eg5, in contrast to loop-L5-binding monastrol or α4/α6-binding BI8 (Figure 5A), which showed greater activity against basal Eg5 ATPase activity. To determine whether BRD9876 is selective for microtubule-bound Eg5 within cells, we altered the levels of microtubule-bound protein. Association of Eg5 with microtubules is regulated by CDK1-mediated phosphorylation of Eg5 threonine 927 (Blangy et al., 1995Blangy A. Lane H.A. d’Hérin P. Harper M. Kress M. Nigg E.A. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.Cell. 1995; 83: 1159-1169Abstract Full Text PDF PubMed Scopus (788) Google Scholar). CDK1 activity in turn is negatively regulated by the tyrosine kinase WEE1 and treatment wi" @default.
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• W2122734929 date "2015-02-01" @default.
• W2122734929 modified "2023-09-25" @default.
• W2122734929 title "Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors" @default.
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• W2122734929 doi "https://doi.org/10.1016/j.celrep.2015.01.017" @default.
• W2122734929 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4524791" @default.
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