Felix Bachmann

The Novel Microtubule-Destabilizing Drug BAL27862 Binds to the Colchicine Site of Tubulin with Distinct Effects on Microtubule Organization.

Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin-BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC50 of 1.4 μM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244±30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin-BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin-BAL27862 and tubulin-colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization.

The Novel Tubulin-Binding Checkpoint Activator BAL101553 Inhibits EB1-Dependent Migration and Invasion and Promotes Differentiation of Glioblastoma Stem-like Cells.

Glioblastoma patients have limited treatment options. Cancer stem-like cells (CSLC) contribute to glioblastoma invasiveness and repopulation; hence, they represent promising targets for novel therapies. BAL101553 is a prodrug of BAL27862, a novel microtubule-destabilizing agent inhibiting tumor cell proliferation through activation of the spindle assembly checkpoint, which is currently in phase I/II clinical development. Broad anticancer activity has been demonstrated against human cancer models, including tumors refractory to conventional treatments. We have shown that overexpression of microtubule + end-binding 1-protein (EB1) correlates with glioblastoma progression and poor survival. Here, we show that BAL27862 inhibits the growth of two glioblastoma CSLCs. As EB1 is overexpressed in the CSLC line GBM6, which displays a high tumorigenicity and infiltrative pattern of migration in vivo, we investigated drug activity on GBM6 according to EB1 expression. BAL27862 inhibited migration and colony formation at subcytotoxic concentrations in EB1-expressing control cells (GBM6-sh0) but only at cytotoxic concentrations in EB1-downregulated (GBM-shE1) cells. Three administrations of BAL101553 were sufficient to provoke an EB1-dependent survival benefit in tumor-bearing mice. Patterns of invasion and quantification of tumor cells in brain demonstrated that GBM6-sh0 cells were more invasive than GBM6-shEB1 cells, and that the antiproliferative and anti-invasive effects of BAL101553 were more potent in mice bearing control tumors than in EB1-downregulated tumors. This was associated with inhibition of stem cell properties in the GBM6-sh0 model. Finally, BAL27862 triggered astrocytic differentiation of GBM6 in an EB1-dependent manner. These results support the potential of BAL101553 for glioblastoma treatment, with EB1 expression as a predictive biomarker of response. Mol Cancer Ther; 15(11); 2740–9. ©2016 AACR.

Abstract 1977: BAL27862: A unique microtubule-targeted drug that suppresses microtubule dynamics, severs microtubules, and overcomes Bcl-2- and tubulin subtype-related drug resistance

Microtubule-targeted drugs (MTDs) are widely used to treat human malignancies such as ovarian and breast cancers. However, a major limitation of their efficacy is the development of resistance modulated by P-gp over-expression or modification of the microtubular target and/or the expression/function of apoptotic pathway proteins such as Bcl-2 [1]. BAL27862 is a novel, orally bioavailable synthetic MTD that triggers apoptosis in cancer cell lines. BAL27862 induces a G2/M arrest, destabilizing microtubules (MT) to produce a unique MT phenotype distinct from that observed with vinca alkaloids (e.g. vinblastine), colchicine and taxanes. Moreover, BAL27862 retains its anti-proliferative activity against multi-drug-resistant, P-gp over-expressing tumor cells [2]. To investigate the mechanism of action of BAL27862, we analyzed its effects on MT dynamics in living breast cancer cells (SKBr3 cells). As measurements were not possible using equipotent cytotoxic concentrations of the MT destabilizer vinblastine, it was compared to the MT stabilizer paclitaxel. BAL27862 suppressed MT dynamic instability, supressing MT catastrophes and growth rate and increasing the mean duration of pauses. In contrast, paclitaxel suppressed the MT shortening rate and did not affect the MT growth rate. At equipotent cytotoxic concentrations, the suppression of MT dynamics by BAL27862 was 2-fold lower than paclitaxel. Strikingly, BAL27862 displayed a unique MT severing activity. We then determined the anti-proliferative potency of BAL27862 in paclitaxel- and Vinca-alkaloid-resistant ovarian cancer cells (A2780-TC1 cells) in comparison to the chemosensitive parental line (A2780-wt cells). A2780-TC1 cell resistance is mediated by P-gp over-expression, Bcl-2 down-regulation and modifications in tubulin subtype composition [1]. Importantly, BAL27862 remained fully active against A2780-TC1 cells. Indeed, when comparing EC50, the resistance factors observed were 1.14 (NS) and 147 (p In conclusion, BAL27862 shares properties with other MTDs such as suppression of MT dynamics, but displays unique features such as MT severing. In ovarian tumor cells, activity appears independent of Bcl-2 and tubulin modifications. These features make BAL27862 a promising compound for clinical development. 1. Esteve MA et al, Mol Cancer Ther, 2006 2. Lane H. et al, EORTC-NCI-AACR 2008, Abstract 444 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1977.

Abstract 3789: BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity

Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule (MT)-depolymerizing agent with a broad in vitro anti-proliferative activity against human tumor lines refractory to standard MT-targeting agents. The prodrug has anti-cancer activity in diverse tumor models when given orally or iv; alone or in combination therapy. In this study, the role of the ‘Spindle Assembly Checkpoint’ (SAC) in the mechanism of action of BAL27862 was evaluated. Methods: To evaluate BAL27862-resistance mechanisms, tumor lines were selected in vitro by increasing drug levels. Anti-proliferative activity was analyzed using Crystal Violet assay. siRNA transfection for 24 h was followed by drug treatment (50 nM) for 48 h. Mitotic index was evaluated after 18 h treatment by p-Histone H3 staining (pHH) or flow cytometry (FC). SAC complex formation was assessed by BubR1 immunoprecipitation (IP) and immunoblotting for SAC components. Results: The SAC ensures cell cycle arrest upon spindle disruption, promoting tumor cell death after prolonged mitotic arrest. SAC formation involves phosphorylation and assembly of a multiprotein complex, of which BubR1 and Mad2 are essential components. Using siRNA approaches, BubR1 was shown to be required for BAL27862 anti-proliferative activity in 5 BAL27862 sensitive tumor lines (IC50s: 8-20 nM). A similar analysis of Mad2 in 3 lines resulted in almost identical attenuation of the effect of BAL27862. For example, non-targeting control siRNA (NTC)-treated H460 lung cancer cells entered a cell death program following BAL27862 treatment (∼45% cell death vs. starting cell number); a response attenuated by BubR1 and Mad2 down regulation (71% and 62% proliferation resp. vs. DMSO controls). The accumulation of mitotic arrested (pHH positive) cells associated with BAL27862 treatment was also absent after BubR1 or Mad2 siRNA transfection. To investigate whether BAL27862 (50 nM, 18 h) induces the formation of the SAC, BubR1 IPs from 3 sensitive tumor lines (IC50: HeLa cervical: 20 nM; A549 lung: 15 nM; SKOV3 ovarian: 12 nM) were analyzed for SAC components. In all cases, complex formation was observed, consistent with a concomitant mitotic accumulation as measured by FC. Strikingly, in variants of the same lines selected for BAL27862 resistance (resistance factors: 3.2-9.4) no SAC formation or mitotic accumulation was observed under the same conditions. Moreover, BAL27862-induced protein mobility shifts, consistent with activating phosphorylation of SAC components, were also absent in the resistant variants. Conclusions: The association of SAC activation with BAL101553sensitivity suggests prediction of cancer response may lie in its tumor checkpoint control function. BAL101553 is currently in Phase 2a clinical evaluation in advanced cancer patients. Exploratory biomarkers are being evaluated to aid selection of patients most likely to respond. Citation Format: Felix Bachmann, Karin Burger, Heidi Lane. BAL101553 (prodrug of BAL27862): the spindle assembly checkpoint is required for anticancer activity. [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 3789. doi:10.1158/1538-7445.AM2015-3789

BAL27862: A Unique Microtubule Destabilizer Active Against Chemorefractory Breast Cancers.

Background: BAL27862, a novel, synthetic, small molecule, is a potent inhibitor of tubulin polymerization that induces cancer cell death. BAL27862 elicits a unique microtubule (MT) phenotype, distinct from paclitaxel, vinblastine and colchicine, has broad in vitro anti-proliferative activity against a diverse range of human tumor lines (low nM IC50s) and induces significant antitumor responses in a range of animal models of human cancer when administered orally (p.o.) or intravenously (i.v.). In this study, BAL27862 activity in a panel of experimental breast cancer models was assessed.Materials and Methods: Anti-proliferative activity was analyzed using a monolayer (crystal violet) or soft agar (clonogenic) assay. Effects on MT phenotypes were assessed by immunofluorescence for α-tubulin. Efficacy was assessed in mouse xenograft models bearing chemosensitive and multidrug resistant human breast tumors.Results: The unique BAL27862-associated MT phenotype in interphase cells consisted of a partially collapsed MT network without peripheral MTs. In dividing cells, tiny MT asters were found scattered within the nuclear region. Potent anti-proliferative activity was demonstrated against 8 breast cancer cell lines (crystal violet assay IC50 range: 6.5 – 22 nM for the SKBR3, MCF7, BT474, T47D, BT549, MDA-MB231, MDA-MB453 and MDA-MB468 lines), although one line (HCC1937) appeared relatively insensitive (IC50: >1000 nM). Interestingly, two breast cancer lines were sensitive to BAL27862 treatment in a clonogenic assay (IC50/IC70: MAXF 401 = 13/18 nM; MAXF MX1 = 22/46 nM), despite one being relatively resistant to paclitaxel treatment (MAXF 401 = 11/48 nM; MAXF MX1 = 127/>3500 nM). Moreover, using monolayer assays, BAL27862 activity was retained against five tumor lines overexpressing the Pgp efflux pump (including MT-3/ADR mammary adenocarcinoma cells), which were up to several thousand-fold resistant to paclitaxel and vinblastine. BAL27862 showed little activity against human stem cells or peripheral blood mononucleocytes.When administered p.o or i.v. to mice at well tolerated doses, BAL27862 treatment elicited statistically significant antitumor activity (p≤0.05) in three chemosensitive human breast tumor xenograft models (including MAXF 401, MaCa 4049 and MT-3); resulting in a final %T/C (ratio of mean tumor volume of treated and control group x 100) equivalent to that observed with comparator cytotoxics using MTD schedules (e.g. final %T/C in MT-3 model: 36% BAL27862, 35% paclitaxel, 50% doxorubicin). Strikingly, significant antitumor activity was maintained in the Pgp-overexpressing MT-3/ADR xenograft model, where paclitaxel and doxorubicin were ineffective (final %T/C: 37% BAL27862, 112% paclitaxel, 108% doxorubicin).Conclusions: BAL27862 is a new tubulin-interacting agent with an apparently novel mechanism of action. A potent antitumor activity in experimental models of breast cancer, including chemorefractory models, strongly support further development of BAL27862 as a novel breast cancer treatment modality with a possibility for both i.v. and p.o. administration. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2093.

Abstract 4781: The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts

Microtubule-targeting agents (MTA) have been employed in the treatment of many cancers for decades. BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizing agent that induces tumor cell death by activating the ‘spindle assembly checkpoint’. Given intravenously or orally, the drug penetrates the brain and has anti-cancer activity in diverse tumor models refractory to standard MTA or radiotherapy (RT). In this study, BAL101553 was evaluated in orthotopic xenografts from 16 GBM PDX models; 7 of 16 lines demonstrated significant (p Citation Format: Ann C. Mladek, Jenny L. Pokorny, Heidi Lane, Felix Bachmann, Mark A. Schroeder, Katrina K. Bakken, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4781.

Abstract 1347: BAL101553: An optimized prodrug of the microtubule destabilizer BAL27862 with superior antitumor activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: [BAL27862][1] is a novel small molecule, inducing apoptosis in cancer cells through microtubule destabilization. A series of amino acid-derived [BAL27862][1] prodrugs was evaluated for solubility and in vivo conversion into drug. The Lys-prodrug BAL101553 was further compared with [BAL27862][1] in animal models of human cancer. Methods: Kinetic solubility of compounds was determined by diluting DMSO stock solutions with aqueous buffer. Pharmacokinetics and prodrug conversion were evaluated in mice. In vivo efficacy was analyzed in colon carcinoma SW480 and patient-derived mammary MaCa4151 xenografts. MTD dosing was used in all cases, adapted for each mouse strain. Results: All amino acid-derived prodrugs showed significantly increased aqueous solubility compared to [BAL27862][1], most pronounced at pH 3. At pH 5 and 6.5, the dibasic Lys-prodrug BAL101553 proved to be the most soluble compound (>200 μM). In vivo conversion rates differed significantly between the prodrugs. Highest exposure to the parent drug was obtained with the Lys, Ala and Gly derivatives, whereas the exposure achieved with other amino acid prodrugs (e.g. Phe, Asn, Ser, Trp) was more than twofold lower. Combining high solubility, good conversion and oral bioavailability, BAL101553 was selected for further evaluation. In tumor models, [BAL27862][1] was rapidly distributed into SW480 tumors after i.v. administration of drug or BAL101553. Strikingly, [BAL27862][1] was retained in tumor ∼1.5 times longer after BAL101553 (T1/2: 8.3h) vs. [BAL27862][1] (T1/2: 5.4h) administration. In all models, a higher MTD was reached with the prodrug, related to ∼60% prodrug conversion in vivo. In MaCa4151 xenografts, twice-weekly i.v. administration of BAL101553 (17 mg/kg [BAL27862][1] equivalents [BE]) elicited superior antitumor effects (final T/C=30%; p<0.05 vs. controls) as compared to [BAL27862][1] (10 mg/kg; T/C=66%). Once-weekly dosing of BAL101553 (14 mg/kg BE) and [BAL27862][1] (8 mg/kg) in the SW480 model resulted in final T/Cs of 34% and 49% (both p<0.001), resp. Strikingly, fractionation of the same total weekly dose did not significantly affect outcome for either BAL101553 (3x per day once-weekly: T/C=40%; 3x per week: T/C=26%; both p<0.001) or [BAL27862][1] (3x per day once-weekly: T/C=54%; 3x per week: T/C=54%; p=0.001 and p=0.002, resp.), suggesting antitumor response is related to AUC. Again, indications of superior antitumor responses were observed with all prodrug schedules in this model. Conclusions: BAL101553 has been identified as a highly soluble prodrug of [BAL27862][1], which can be administered p.o. or i.v. in the absence of solubilizing excipients known to be associated with adverse side-effects. Its administration facilitates higher tumor exposure to the active agent, with more profound responses in some tumor models. These data, together with a flexible dosing potential, support profiling of BAL101553 in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2011-1347 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=BAL27862&atom=%2Fcanres%2F71%2F8_Supplement%2F1347.atom

Abstract 4419: BAL101553: A highly soluble prodrug of the potent microtubule destabilizer BAL27862

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: [BAL27862][1] is a synthetic small molecule, inducing apoptosis in cancer cells through a unique, destabilizing effect on microtubules (MT). [BAL27862][1] can be administered intravenously (i.v.) and orally, and exhibits broad antitumor activity against a range of human tumor histotypes, including models refractory to conventional agents such as taxanes and vinca alkaloids. To improve water solubility, allowing a well tolerated formulation, a novel prodrug (BAL101553) has been developed. Materials and Methods: Solubility was determined by HPLC-quantitation of compound concentration in saturated solutions after filtration and appropriate dilution. Antiproliferative activity in vitro was analyzed by Crystal Violet assay. Effects on MTs were assessed by immunoblotting (IB) or immunofluorescence (IF) for α-tubulin. For pharmacokinetic (PK) studies, colon carcinoma SW480 xenografted mice were treated i.v. with [BAL27862][1] or BAL101553 weekly for 4 weeks. Compound levels were determined in plasma, brain and tumors. Efficacy was assessed in a NSCLC A549 mouse xenograft model using weekly MTD doses. Results: [BAL27862][1] is insoluble in water ( 100 mg/mL. Using tumor lines in vitro, BAL101553 elicited antiproliferative IC50s approx. double those of the parent drug (e.g. A549: 34.5 nM and 18.5 nM, respectively), consistent with its conversion to active drug in cell culture. Importantly, BAL101553s antiproliferative activity was associated with efficient MT depolymerization, characterized by the formation of multiple, tiny MT asters in dividing cells. The latter is a phenomenon unique to [BAL27862][1] and is not observed with conventional MT-targeted agents like taxanes and vinca alkaloids. In vivo, BAL101553 was converted to active [BAL27862][1]. In mice, after i.v. administration of [BAL27862][1] or BAL101553, brain and tumor concentrations equal to plasma levels were detected 5 min and 30 min post-administration, respectively. There was no brain or tumor accumulation over time, as drug concentrations paralleled those in plasma. Notably, a higher maximum tolerated dose was reached with BAL101553 as compared to [BAL27862][1]. In terms of exposure, this translated to a 20% lower Cmax and a 10% higher AUC of active drug with BAL101553. When administered i.v. to A549 xenograft-bearing mice, the prodrug was well tolerated, eliciting significant antitumor effects (p≤0.05), equivalent to the parent drug (final %T/C: 44% [BAL27862][1] [10 mg/kg], 40% BAL101553 [28 mg/kg]). Conclusions: BAL101553 is a prodrug of [BAL27862][1] with high water solubility, allowing i.v. administration in the absence of solubilizing excipients known to be associated with adverse side-effects. The in vitro profile, favorable PK characteristics and efficacy in animal tumor models support further evaluation of BAL101553 for the treatment of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4419. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=BAL27862&atom=%2Fcanres%2F70%2F8_Supplement%2F4419.atom

Abstract 4412: In vitro activity of the novel tubulin active agent BAL27862 in MDR1(+) and MDR1(−) human breast and ovarian cancer variants selected for resistance to taxanes

BAL27862 is a synthetic small molecule that induces apoptosis due to the inhibition of tubulin polymerization via a potentially new mechanism of action. The activity of this novel compound was evaluated in human breast and ovarian cancer cell line variants selected for resistance by continuous exposure to taxanes. BAL27862 retained activity against MDR1 (ABCB1)/P-glycoprotein-mediated resistance variants, unlike vinca alkaloids and taxanes. Activity was also observed in the taxane-resistant ovarian 1A9/TxTP50 tumor cell line, which overexpresses TUBB3 and was approximately 35x resistant to taxanes. Moreover, BAL27862 retained activity in ovarian cancer cells with acquired mutations in the class I (M40) β-tubulin isotype (1A9/PTX10, Phe270->Val; 1A9/PTX22, Ala364->Thr) which confer taxane resistance. The MCF-7/TxTP50 breast tumor variant was 9x resistant to taxanes, and 5x resistant to epothilone B, yet retained sensitivity to BAL27862, the vinca alkaloids (vinblastine and vincristine), colchicine and anthracyclines. These cells have significantly reduced levels of the taxane-binding target, the class I β-tubulin isotype, and reduced levels of total α- and β-tubulin relative to wild-type cells, alterations which may be responsible for a 34% reduction in bound BODIPY-paclitaxel as detected by FACS analysis. Evidence of resistance to BAL27862 (3x) was only detected in the ovarian OVCAR-3/TxTP5 variant, which was also resistant to taxanes (8x), the vinca alkaloids (4x) and colchicine (2x) and ixabepilone (2x). Finally, silencing TUBB3 using a specific siRNA sensitized cells to taxanes but did not affect BAL27862 activity in both wild-type MCF-7 and OVCAR-3 cells relative to appropriate controls, suggesting that TUBB3 status may not influence BAL27862 response. Thus, BAL27862 is a new agent with a distinct in vitro activity profile against a broad panel of human breast and ovarian tumor cell lines resistant to standard tubulin-interacting agents. These data strongly support further development of BAL27862 as a novel anticancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4412.

The novel microtubule targeting agent BAL101553 in combination with radiotherapy in treatment-refractory tumor models

BACKGROUND AND PURPOSE Resistance to microtubule targeting agents (MTA) represents a major drawback in successful cancer therapy with MTAs. Here we investigated the combined treatment modality of the novel MTA BAL101553 in combination with radiotherapy in paclitaxel and epothilone-resistant tumor models. MATERIAL AND METHODS Multiple regimens of BAL101553, or its active moiety BAL27862 for in vitro experiments, were probed in combination with radiotherapy in P-glycoprotein-overexpressing, human colon adenocarcinoma cells (SW480) and in tubulin-mutated human NSCLC cells (A549EpoB40) and tumors thereof. RESULTS BAL27862 reduced the proliferative activity of SW480 and A549EpoB40 tumor cells with similar potency as in A549 wildtype cells. Combined treatment of BAL27862 with ionizing radiation in vitro resulted in an additive reduction of clonogenicity. Moreover, treatment of paclitaxel- and epothilone-resistant tumors with fractionated irradiation and different regimens of BAL101553 (a single i.v. bolus vs. oral daily) suppressed tumor growth and resulted in an extended additive tumor growth delay with strong reduction of tumor proliferation and mean tumor vessel density. CONCLUSIONS BAL101553 is a promising alternative in taxane- and epothilone-refractory tumors as part of a combined treatment modality with ionizing radiation. Its potent antitumor effect is not only tumor cell-directed but also targets the tumor microenvironment.