Supplementary Figures 1-8 Supplementary Tables 1-2 Supplementary Materials and Methods

Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.

Abstract Several lines of evidence link the canonical oncogene BCL6 to stress response. Here we demonstrate that BCL6 evolved in vertebrates as a component of the HSF1-driven stress response, which has been co-opted by the immune system to support germinal center formation and may have been decisive in the convergent evolution of humoral immunity in jawless and jawed vertebrates. We find that the highly conserved BTB corepressor binding site of BCL6 mediates stress adaptation across vertebrates. We demonstrate that pan-cancer cells hijack this stress tolerance mechanism to aberrantly express BCL6. Targeting the BCL6 BTB domain in cancer cells induces apoptosis and increases susceptibility to repeated doses of cytotoxic therapy. The chemosensitization effect upon BCL6 BTB inhibition is dependent on the derepression of TOX, implicating modulation of DNA repair as a downstream mechanism. Collectively, these data suggest a form of adaptive nononcogene addiction rooted in the natural selection of BCL6 during vertebrate evolution. Significance: We demonstrate that HSF1 drives BCL6 expression to enable stress tolerance in vertebrates. We identify an HSF1–BCL6–TOX stress axis that is required by cancer cells to tolerate exposure to cytotoxic agents and points toward BCL6-targeted therapy as a way to more effectively kill a wide variety of solid tumors. This article is highlighted in the In This Issue feature, p. 565

Despite the proven clinical antineoplastic activity of histone deacetylase inhibitors (HDACI), their effect has been reported to be lower than expected in B-cell lymphomas. Traditionally considered as "epigenetic drugs", HDACI modify the acetylation status of an extensive proteome, acting as general lysine deacetylase inhibitors (KDACI), and thus potentially impacting various branches of cellular metabolism. Here, we demonstrate through metabolomic profiling of patient plasma and cell lines that the KDACI panobinostat alters lipid metabolism and downstream survival signaling in diffuse large B-cell lymphomas (DLBCL). Specifically, panobinostat induces metabolic adaptations resulting in newly acquired dependency on the choline pathway and activation of PI3K signaling. This metabolic reprogramming decreased the antineoplastic effect of panobinostat. Conversely, inhibition of these metabolic adaptations resulted in superior anti-lymphoma effect as demonstrated by the combination of panobinostat with a choline pathway inhibitor. In conclusion, our study demonstrates the power of metabolomics in identifying unknown effects of KDACI, and emphasizes the need for a better understanding of these drugs in order to achieve successful clinical implementation.

The primary components of the stromal lymphoma microenvironment (LME) are the cancer-associated fibroblasts (CAF) and the extracellular matrix (ECM) they produce. CAFs are derived from healthy fibroblasts that have been hijacked and transcriptionally reprogrammed by cancer cells into a novel biological entity to promote tumorigenesis. While the key effectors of these programs are largely unknown, recent data indicates that in a variety of solid tumors and T-acute lymphoblastic leukemias an important transcriptional programmer of the microenvironment is the Heat Shock Factor 1 (HSF1). Hence, we sought to determine the contribution of HSF1 within the stromal LME to the acquisition of lymphomagenic features in diffuse large B-cell lymphoma (DLBCL). We first analyzed the activity of HSF1 in 80 DLBCL patients by RNA-sequencing. Patients were segregated into having "high" and "low" HSF1 activity based on the expression of the canonical stress response target genes. We found distinct patterns of stromal-associated genes between DLBCL having "low" vs. "high" HSF1 activity. Furthermore, ChIP-sequencing data of lymphoma cells and CAFs showed the emergence of distinct transcriptional programs that are differentially orchestrated by HSF1. To delve deeper into the mechanism of these effects, we co-cultured lymphoma cells with fibroblasts having either HSF1 WT or HSF1 null in 3D collagen-containing lymphoma organoids. We found that only organoids harboring HSF1 WT fibroblasts were supportive of DLBCL cell proliferation. Moreover, DLBCL cells responsive and non-responsive to HSF1 downregulation in a cell-autonomous manner, failed to proliferate when cultured in HSF1-deficient LME organoids. To further elucidate this mechanism, we implanted a murine HSF1-positive DLBCL cell line (A20) into HSF1 WT (n=13) and HSF1 null (n=11) syngeneic mice and monitored lymphoma development for up to 14 days. We observed a continuous growth of HSF1-positive murine DLBCL in HSF1 WT mice; however, DLBCL in HSF1 null mice underwent spontaneous collapse after day 7, leading to almost complete tumor eradication. We further investigated the lymphoma microarchitecture, ECM biomechanics and composition in relation to the cellular components in serial tumor sections at several time points. At day 7, when no significant difference in tumor sizes exists between HSF1 WTs and nulls, we found significant differences in tissue stiffness (p=0.017), collagen microarchitectureby fiber alignment (p<0.001) and elastic fiber area (p<0.001). ECM proteomics by HPLC-MS/MS of the ECM-enriched fractions of these murine DLBCLs at days 5, 7 and 13 followed by network analysis demonstrated highly dynamic changes in ECM composition. Specifically, comparing the "matrisomes" of HSF1 WT and null tumors at day 7, we identified differentially regulated ECM modules that facilitate collagen functionalization and abundance of small leucine-rich proteoglycans; suggesting a failure of the HSF1 null stroma in efficiently remodeling the ECM. We identified dynamic changes in specific matrisome components with known roles in tumor proliferation and immune activity. These biochemical and biophysical changeswere in turn capable of modulating the cellular composition of DLBCL tumors. By day 7, the LME in HSF1 null was less supportive of lymphoma growth as measured by lower cellularity (p=4.5e-18) and proliferation of lymphoma cells by Ki67 (p=7.6e-6); and increased apoptosis by TUNEL (p=5.6e-5). In regards to CD31 positivity based on endothelial cells and vascularization, we observed no significant differences between HSF1 WT and HSF1 null tumors, however, there were significant changes in the repertoire of immune infiltrating cells. Specifically, HSF1 null LME harbored higher fractions of effector cd3+ T-cells (p=3.0e-6) and mac2+macrophages (p=9.4e-11); and a decrease in the suppressive cells cd3+foxp3+Tregs (p=6.6e-19) and cd11b+ gr1+myeloid-derived suppressor cells (p=0.0467). Taken together, our data in DLBCL specimens and cell lines, lymphoma-fibroblasts organoids, and a murine DLBCL model, all demonstrate that HSF1 activity in DLBCL drives an ECM program that confers microarchitectural and biophysical properties to the LME that are supportive of lymphoma cell proliferation and infiltration by immune suppressive populations. Cerchietti: Celgene: Research Funding; Weill Cornell Medicine: Employment.

To identify drugs with preferential activity against phenotypically defined subtypes of DLBCL, we carried out a high throughput screening of 100,000 drug-like compounds in two cell lines with and without tonic BCR activation. After 3 rounds of progressive viability screening we identified a small molecule, named Li5, which decreased viability of BCR-DLBCLs. In an extended DLBCL panel including cell lines with tonic or chronic (OCI-Ly1, OCI-Ly7, Farage, OCI-Ly10), and no (Toledo, Karpas422, Pfeiffer) BCR activation, we confirmed that Li5 is over 40 times more selective against DLBCL with tonic or chronic BCR activity (P= 0.001). To determine whether this differential effect is maintained in animal models, we formulated Li5 and evaluated its toxicity in C57BL/6 mice. Li5 was administered up to 200 mg/kg without evidence of toxicity in biochemical and histological assays. To asses the selective anti-lymphoma effect, we xenografted two BCR-DLBCL cell lines (OCI-Ly1 and OCI-Ly7) and a non-BCR-DLBCL cell line (Toledo) into SCID mice. Once tumors developed, we administered 10 mg/kg/day of Li5 or vehicle. Li5 significantly decreased lymphoma size in OCI-Ly7 and OCI-Ly1 mice (P < 0.005 and P= 0.005, respectively, vs. vehicle) but not in Toledo mice (P= n.s., vs. vehicle), indicating the compound holds its selectivity in vivo.

Peripheral T-cell lymphomas (PTCL) are aggressive diseases with poor response to chemotherapy and dismal survival. Identification of effective strategies to target PTCL biology represents an urgent need. Here we report that PTCL are sensitive to transcription-targeting drugs, and, in particular, to THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7). The STAT-signalling pathway is highly vulnerable to THZ1 even in PTCL cells that carry the activating STAT3 mutation Y640F. In mutant cells, CDK7 inhibition decreases STAT3 chromatin binding and expression of highly transcribed target genes like MYC, PIM1, MCL1, CD30, IL2RA, CDC25A and IL4R . In surviving cells, THZ1 decreases the expression of STAT-regulated anti-apoptotic BH3 family members MCL1 and BCL-XL sensitizing PTCL cells to BH3 mimetic drugs. Accordingly, the combination of THZ1 and the BH3 mimetic obatoclax improves lymphoma growth control in a primary PTCL ex vivo culture and in two STAT3-mutant PTCL xenografts, delineating a potential targeted agent-based therapeutic option for these patients.

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

Background: Refractory and/or relapsed diffuse large B cell lymphoma (RR-DLBCL) patients are incurable with conventional chemotherapy due to the aggressiveness and the chemorefractory state of these tumors. DNA hypermethylation and histone deacetylation are two major epigenetic modifications by which aggressive DLBCL maintain their oncogenic state. We have previously reported that DNA methyltransferase inhibitors (DNMTI) affect RR-DLBCL growth and improve chemosensitivity. Here, we hypothesized that the combination of DNMTI with histone deacetylase inhibitor (HDI) would be an active and feasible therapeutic strategy in RR-DLBCL. Thus, we evaluated the anti-lymphoma activity of the HDI vorinostat (VST) in combination with the DNMTI azacitidine (AZA) or decitabine (DAC) in pre-clinical models of RR-DLBCL, and we determined the feasibility of the combination by conducting a phase Ib trial in RR-DLBCL patients.Results: Concurrent combination of DNMTI and HDI resulted in synergistic anti-lymphoma effect toward RR-DLBCL cells in vitro and in vivo, with no significant toxicity increase. In a phase Ib trial, a total of 18 patients with a median of three prior therapies were treated with four different dose levels of AZA and VST. The most common toxicities were hematological, followed by gastrointestinal and metabolic. The clinical benefit was low as only one subject had a partial response and three subjects had stable disease. Interestingly, two of the seven patients that received additional chemotherapy post-study achieved a complete response and three others had a significant clinical benefit. These observations suggested that the combination might have a delayed chemosensitization effect that we were able to confirm by using in vitro and in vivo models. These studies also demonstrated that the addition of VST does not improve the chemosensitizing effect of DAC alone.Conclusions: Our data supports the strategy of epigenetic priming by employing DNMTI in RR-DLBCL patients in order to overcome resistance and improve their outcomes.

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3μM) and very weak activity in osteosarcoma (IC50 >10μM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent.

Treatment failure in acute myeloid leukemia (AML) is frequently due to the persistence of a cell population resistant to chemotherapy through different mechanisms, in which drug efflux via ATP-binding cassette (ABC) proteins, specifically P-glycoprotein, is one of the most recognized. However, disappointing results from clinical trials employing inhibitors for these transporters have demonstrated the need to adopt different strategies. We hypothesized that microtubule targeting compounds presenting high affinity or covalent binding could overcome the effect of ABC transporters. We therefore evaluated the activity of the high-affinity paclitaxel analog CTX-40 as well as the covalent binder zampanolide (ZMP) in AML cells. Both molecules were active in chemosensitive as well as in chemoresistant cell lines overexpressing P-glycoprotein. Moreover, ZMP or CTX-40 in combination with daunorubicin showed synergistic killing without increased in vitro hematopoietic toxicity. In a primary AML sample, we further demonstrated that ZMP and CTX-40 are active in progenitor and differentiated leukemia cell populations. In sum, our data indicate that high affinity and covalent-binding anti-microtubule agents are active in AML cells otherwise chemotherapy resistant.

Abstract Ureidomustine (BO-1055) is a water-soluble N- mustard derivative that has antitumor activity against a number of human tumors including prostate, colon cancers and gliomas. In this study, we investigated its pre-clinical activity in sarcoma models. We tested the activity of BO-1055 on early passages from Ewing sarcoma patient samples and patient-derived xenografts, preclinical models of rhabdomyosarcoma, desmoplastic small round cell tumor (DSRCT) and osteosarcoma. We used cell proliferation, viability, clonogenicity and cell death assays, and immunoblotting for differential expression of proteins in cells that are sensitive and resistant to BO-1055. We studied the toxicity of BO-1055 on cardiac myocytes, hematopoietic stem and progenitor cells (HSPCs), epithelial, mesenchymal and endothelial cells, and performed animal toxicity experiments in mice. Efficacy of BO-1055 was tested in NSG mice bearing A673 xenografts (Ewing sarcoma) treated at doses of 10mg/kg, 20mg/kg and 30mg/kg, Q2D×5, via i.v. injection after the tumors grew to 250-500 mm3. We also investigated the efficacy of this agent in NSG mice bearing A204 xenografts and Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. The antitumor effects of BO-1055 in combination with standard chemotherapeutic agents were also evaluated. Ureidomustine exhibited significant cytotoxicity against the in vitro growth of Ewing sarcoma and rhabdomyosarcoma with sub μM IC50 values and was more cytotoxic than conventional DNA damaging agents. BO-1055 had less cytotoxicity against a panel of normal HSPCs. BO-1055 had moderate activity (IC50 = 2-4 μM) on DSRCT cells and had only weak activity (IC50>10μM) against osteosarcoma cell lines. Notably, this agent has no cross-resistance to Taxol and Vinblastine. Growth arrest in G2/M phase was noted at 24h and maximal apoptosis and cell death at 48h and 72h after exposure of A673 cells. Apoptosis was due to the induction of caspase 3 and 7 activity in a dose- and time- dependent manner. Cardiotoxicity was evaluated by binding of BO-1055 to hERG, which was significantly less than that of Astemizole indicating that BO-1055 has less propensity for QTc prolongation. BO-1055 significantly suppressed Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. Remarkably, this agent induced complete suppression of tumors in nude mice bearing A204 xenografts, without significant weight loss. In two-drug combination studies, BO-1055 exhibited synergism with etoposide, SN-38 (active metabolite of irinotecan), doxorubicin and PU-H71 (HSP-90 inhibitor). Because of its broad therapeutic window, lack of cross-resistance, better safety profile and synergistic cytotoxicity with standard drugs, BO-1055 has high potential for clinical application for treatment of Ewing sarcoma and rhabdomyosarcoma patients. Citation Format: Srikanth R. Ambati, Shieh JaeHung, Benet Pera, Elissa W.P. Wong, Eloisi Caldas Lopes, Elizabeth Peguero, Tsann-Long Su, Malcolm A.S. Moore. Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2015-1625

Abstract DNA crosslinking agents continue to be an important part of chemotherapy for pediatric sarcomas. We tested potent analogues of phenyl N-mustard-benzene conjugates in several tumor models. In this work, we compared the efficacy of a novel water-soluble DNA alkylating agent, Ureidomustin (BO-1055) in Ewing sarcoma, rhabdomyosarcoma, osteosarcoma, desmoplastic small round cell tumor (DSRCT) cell lines and early cultures derived from primary patient samples. We tested its toxicity in normal human cells in vitro including the proliferation of mesenchymal stromal cells, bone marrow-derived endothelial cells and purified cord blood CD34+ cells in colony-forming assay (for hematopoietic progenitor cells) and in cobblestone area forming assay (for hematopoietic stem cells). We performed cell viability, cytotoxicity and caspase activation assays in a dose-dependent and time-dependent manner. We also performed apoptosis, cell cycle analyses using flow cytometry, immunoblot analysis and in vivo experiments in nude mice using Ewing sarcoma (A673) cell line transduced with GFP/luciferase for bioimaging. We evaluated cardiotoxicity in the HL-1 cardiomyocyte line in vitro and noted a 227 fold less cytotoxicity using BO-1055 compared to Doxorubicin. Based on MTD testing in nude mice, we used 30mg/kg q2d for 5 doses after tumors reached 75-100mm3 and were detected on luciferase bioimaging. Our results indicated that BO-1055 has a significant therapeutic window in its activity (mean IC50±SD μM) for rhabdomyosarcoma (0.16±0.11), Ewing sarcoma (0.48±0.29) and DSRCT (3.07±0.94) but not for osteosarcoma (IC50>10μM). The mean IC50 in benign cells was >10μM. Treatment with BO-1055 resulted in G2 arrest and activation of caspases 3 and 7 in a dose dependent manner. A673 xenografts were significantly inhibited by BO-1055 compared to the control mice injected with vehicle, based on mean fluorescence intensity and size of the tumors. Our results suggest that BO-1055 has potent activity against Ewing sarcoma and rhabdomyosarcoma and presents a strong rationale for further development as a therapeutic agent. Citation Format: Srikanth R. Ambati, Elissa WP Wong, Benet Pera, Elizabeth Peguero, Eloisi Caldas Lopes, Jin-Jer Chen, Jae-Hung Shieh, Tsann-Long Su, Malcolm AS Moore. Pre-clinical evaluation of a novel DNA crosslinking agent, Ureidomustin (BO-1055) in pediatric sarcomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3980. doi:10.1158/1538-7445.AM2014-3980

We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by P-gp overexpression, by mutations in the beta-tubulin binding site or by overexpression of the highly dynamic beta III-tubulin isotype.We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7-S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on P-gp and beta III overexpressing cells and on mutant cells.The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power. (C) 2014 Elsevier Ltd. All rights reserved.

Peripheral T-cell lymphomas (PTCL) are clinically aggressive diseases with poor response to available (largely B-cell lymphoma–tailored) chemotherapy regimens and dismal survival.

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.

Ten novel taxanes bearing modifications at the C2 and C13 positions of the baccatin core have been synthesized and their binding affinities for mammalian tubulin have been experimentally measured. The design strategy was guided by (i) calculation of interaction energy maps with carbon, nitrogen and oxygen probes within the taxane-binding site of β-tubulin, and (ii) the prospective use of a structure-based QSAR (COMBINE) model derived from an earlier series comprising 47 congeneric taxanes. The tubulin-binding affinity displayed by one of the new compounds (CTX63) proved to be higher than that of docetaxel, and an updated COMBINE model provided a good correlation between the experimental binding free energies and a set of weighted residue-based ligand-receptor interaction energies for 54 out of the 57 compounds studied. The remaining three outliers from the original training series have in common a large unfavourable entropic contribution to the binding free energy that we attribute to taxane preorganization in aqueous solution in a conformation different from that compatible with tubulin binding. Support for this proposal was obtained from solution NMR experiments and molecular dynamics simulations in explicit water. Our results shed additional light on the determinants of tubulin-binding affinity for this important class of antitumour agents and pave the way for further rational structural modifications.

Poor outcomes in patients with acute myeloid leukemia (AML) are related to the high proportion of patients with chemorefractory disease or relapse after an initial response due to the development of resistance. This treatment failure is frequently due to the persistence of a cell population that is inherently resistant to classical chemotherapeutic agents through different mechanisms, in which an increased efflux of these agents via transmembrane proteins of the ATP-binding cassette (ABC) family is one of the best recognized. Classically, the main approach to overcome the transport effect has involved the co-administration of competitive inhibitors of these transporters. However, the large number of failed clinical trials involving these inhibitors has demonstrated the necessity to adopt different strategies.

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg(2+)-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.