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Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In vitro, BAL0891 has a combined prolonged effect on TTK and a transient effect on PLK1, leading to rapid disruption of the SAC that potentiates aberrant mitotic progression of tumor cells. In this work, efficacy of BAL0891 was investigated in mouse models of human triple negative breast cancer (TNBC) including evaluation of dose-dependency, drug exposure, target occupancy and a screen of activity across a panel of PDX models. Methods: The MDA-MB-231 cell line was grown sc in nude mice and treated with BAL0891, administered IV weekly (QW) or twice-weekly (2QW). Thirteen sc TNBC PDX models were screened for BAL0891 response using 2QW administration. Efficacy was quantified as deltaT/C (treated/control tumors). Plasma and tumor were analyzed for drug levels or TTK target occupancy by LC-MS/MS. The latter used a biotinylated TTK-specific probe and streptavidin-mediated isolation of unoccupied TTK, trypsin digestion and quantification of TTK-representative peptides. Results: BAL0891 efficacy was tested in the TNBC xenograft model MDA-MB-231 with QW or 2QW IV dosing schedules. All treatments were well tolerated, with no drug-related animal deaths. With MTD dosing, tumor regressions were observed, while different MTD fractions for both QW and 2QW schedules showed dose-dependent anti-tumor activity. The weekly MTD group was followed for an additional 20 days after treatment cessation on day 100. Strikingly, 3 of 8 tumors continued to shrink resulting in 2 (25%) pathologically confirmed cures. Consistent with the potent efficacy of intermittent MTD dosing, and prolonged tumor drug exposure, tumor TTK was fully drug-occupied for ≥ 6 days after the last administration; target occupancy was also dose-and drug exposure-dependent. To further evaluate BAL0891 anti-cancer activity in TNBC, a screen in 13 TNBC PDX models was conducted. Seven models exhibited deltaT/C < 50%, with regressions observed in 3. Of these, 2 models showed persistent regressions ≥ 70% vs. baseline. Interestingly, evaluation of TTK target occupancy in selected models showed high target occupancy independent of tumor response, indicating target dependency rather than drug availability is important for anti-cancer activity. Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor with potent anti-cancer activity in TNBC models. Intermittent IV administration is well tolerated and associated with prolonged tumor drug exposure, prolonged TTK inhibition and notable anti-tumor efficacy. These data support further investigation of BAL0891 for the treatment of cancer patients (incl. TNBC). Citation Format: Heidi A. Lane, Felix Bachmann, Elisa Zanini, Paul McSheehy, Karine Litherland, Nicole Forster-Gross, Luc Bury, Diep Vu-Pham, Jos de Man, Wilhelmina E. van Riel, Guido JR Zaman, Rogier C. Buijsman, Laurenz Kellenberger. BAL0891: A novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division resulting in potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5645.

Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In this work, kinase inhibition by BAL0891 was linked to effects on SAC integrity and aberrant mitotic progression in tumor cells. Comparison with a TTK-specific inhibitor (CFI-402257, CFI) allowed further evaluation of the contribution of dual TTK/PLK1 inhibition to anti-cancer activity, associated with a promising anti-proliferative profile across diverse tumor cell lines. Methods: Kinase assays used a radiometric assay. Target residency was measured using surface plasmon resonance with recombinant kinase. Anti-proliferative activity was assessed with crystal violet or YO-PRO assay (5 days incubation), in-cell target inhibition by immunoblotting for phospho-TTK following drug wash-out. Effects on SAC integrity were followed by immunoprecipitation (IP) mitotic progression by flow cytometry/mitotic marker expression. Cells were blocked in mitosis using the microtubule-targeting agent nocodazole or the PLK1 inhibitor onvansertib. SAC KT accumulation was evaluated by immunofluorescence (IF) for co-localization of BubR1 with CENPC. Comparative studies with CFI used anti-proliferative IC50 concentrations. Results: In vitro kinase profiling showed that BAL0891 has low nM IC50s against TTK and PLK1, with prolonged TTK (>12 h) and transient PLK1 (4 min) target residency. Prolonged TTK inhibition (≥38 h) was also observed in HT29 tumor cells. Consistent with a dominant TTK-targeting activity, BAL0891 treatment of HT29 cells blocked in mitosis with nocodazole or the PLK1 inhibitor onvansertib led to aberrant mitotic release and accumulation of polyploid cells. This was preceded by SAC disruption as visualized by IP assays. Effects on the SAC and mitotic exit were evaluated in comparative studies with CFI; BAL0891 exhibited faster kinetics for both parameters suggesting a contribution of PLK1 inhibition. This was confirmed by directly evaluating acute effects on SAC integrity at the KT by IF. Specifically, 1 h BAL0891 treatment of mitotic HT29 cells resulted in a highly reproducible and significant reduction in KT-associated SAC (p5 uM). Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor. In tumor cells, prolonged effects on TTK and transient effects on PLK1 contribute to rapid SAC disruption and aberrant mitotic exit. This is associated with potent anti-proliferative activity in diverse tumor lines. Citation Format: Elisa Zanini, Nicole Forster-Gross, Felix Bachmann, Nicole Willemsen-Seegers, Jos de Man, Guido J. Zaman, Rogier C. Buijsman, Anna Groner, Mila Roceri, Karin Burger, Paul McSheehy, Laurenz Kellenberger, Heidi A. Lane. BAL0891: A novel, small molecule, dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5646.

Background: Avanbulin (BAL27862) is a microtubule-targeting agent (MTA) that promotes tumor cell death by modulating the spindle assembly checkpoint. Preclinical studies in glioblastoma (GBM) have shown that avanbulin is also active in human cancer cells resistant to other MTAs used in the clinic. Lisavanbulin (BAL101553) is a water-soluble, oral, lysine prodrug of avanbulin, currently in phase 1/2 for patients with recurrent GBM and, in combination with radiotherapy, for newly diagnosed GBM patients. Preclinical studies and clinical signals have also suggested an association between lisavanbulin activity and expression of microtubule plus-end binding protein (EB1). MTAs are active agents for lymphoma patients, as exemplified by the inclusion of vincristine in the R-CHOP regimen, standard treatment for diffuse large B cell lymphoma (DLBCL). Thus, our aim was to evaluate avanbulin, the active moiety of lisavanbulin, for its potential anti-lymphoma activity. Methods: Lymphoma cell lines derived from activated B cell like (ABC) and germinal center B cell type (GCB) DLBCL were exposed to increasing doses of avanbulin; cell proliferation was measured with MTT. Apoptosis induction was performed with annexin V staining on cells incubated in a live cell imaging system (Incucyte) treated with 5, 10, 20 or 40nM avanbulin. Images were taken every 4h for 72h. Cell cycle analysis was performed after 24, 48 and 72h of drug exposure at 20nM. Results: 26 DLBCL cell lines, treated for 72h with increasing avanbulin doses, showed high anti-proliferative activity of the compound, with a median IC50 = 11nM. No differences in terms of IC50 were observed comparing ABC (n=8) to GCB (n=18) DLBCLs, nor based on MYD88, TP53 and BCL2 status. Cell lines with MYC translocation were less sensitive than the ones without translocation. All lymphoma cell lines exhibited high EB1 RNA expression, with no correlation with sensitivity to avanbulin.Live cell imaging demonstrated a strong apoptosis induction in 15 out of 17 cell lines tested at 20 and 40nM. Among the 15 cell lines undergoing apoptosis, 8 cell lines already showed an apoptosis induction within 24h of drug exposure, 6 lines between 24 and 48h and 1 line after 48h exposure. These data confirmed the high activity of avanbulin already seen with the MTT assay.Cell cycle experiments performed at 24, 48 and 72h confirmed the strong cytotoxic effect of avanbulin in 4 DLBCL cell lines, including 2 ABC (OCILY3 and TMD8) and 2 GCB (SUDHL5 and SUDHL16) lines. Sub-G0 accumulation was already present at 24h in all 4 cell lines tested, with an increase over time. Especially at 24h, where we also observed a G2-M arrest in 3 out of 4 cell lines. Conclusions: Our data demonstrate the high cytotoxic anti-lymphoma activity of avanbulin, suggesting a potential activity of its prodrug lisavanbulin in lymphoma patients. Citation Format: Filippo Spriano, Luca Aresu, Luciano Cascione, Alberto J. Arribas, Nicole Forster-Gross, Felix Bachmann, Heidi Lane, Francesco Bertoni. Avanbulin, the active moiety of the tumor checkpoint controller lisavanbulin (BAL101553), has anti-lymphoma activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2575.

Background: BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizer inducing tumor cell death through the spindle assembly checkpoint. BAL101553 has anti-cancer activity in diverse tumor models refractory to standard therapies, and is currently undergoing Phase 1/2a evaluation in advanced cancer patients with IV (weekly) and oral (daily) dosing schedules. Here, the vascular disruption activity of BAL101553 was evaluated in the context of administration route and implications for combinations with VEGF-targeted therapies. Methods: MTD IV (day[d] 1) and oral (d1 & d1 - 4) dosing regimens were tested in H460 NSCLC xenografts. Tumors were analyzed by IHC for proliferation (Ki67) and vascularization (CD31, lectin perfusion). Drug combinations were performed in Pgp-overexpressing, colon carcinoma SW480 xenografts using BAL101553 IV 1xweek (21.3 mg/kg) or oral daily (15 mg/kg) and bevacizumab IP d1 & 4 (5 mg/kg). Results: H460 tumor-bearing mice received once weekly (IV 25 mg/kg; oral 60 mg/kg; high Cmax regimens) or daily (oral 25 mg/kg; low Cmax, high AUC) BAL101553 regimens which elicited equipotent anti-tumor effects (ΔT/Cs = 26-34%). Although all treatments elicited a similar anti-proliferative response (% reduction of mean Ki67 signal+/-SD=10+/-1.2; p Conclusions: BAL101553 targets tumor cell proliferation, tumor hypoxic adaptation and vascularization. The latter is Cmax-driven and can be attenuated by daily oral dosing, providing an alternative pharmacodynamic endpoint with implications for clinical treatment strategies. IV and orally administered BAL101553 both elicit superior anti-tumor responses in combination with anti-VEGF therapy. Citation Format: Ashish Sharma, Felix Bachmann, Angela Broggini-Tenzer, Matthias Guckenberger, Heidi Lane, Martin N. Pruschy. The novel tubulin-binding, tumor checkpoint controller BAL101553 has differential effects on tumor vascularization with IV and oral dosing and provides superior anti-tumor activity in combination with bevacizumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2017-LB-151

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.

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

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 microtubule-targeting agents (MTAs). The BAL27862 MT phenotype is distinct from standard MTAs (eg taxanes; vinca-alkaloids) and the prodrug has antitumor activity in diverse mouse tumor models when given orally or iv. In this study, activity was assessed in experimental breast cancer models alone and in combination with the ErbB2-targeted therapeutic antibody trastuzumab. Methods: Anti-proliferative activity was analyzed using monolayer or soft agar assays, MT phenotypes by immunofluorescence for α-tubulin and efficacy in mouse xenograft models of human breast tumors. Results: Potent anti-proliferative activity was observed in 8 of 9 breast cancer lines (IC50 range of sensitive lines: 8 - 25 nM) associated with the characteristic BAL27862 MT phenotype (tiny MT asters in G2/M arrested cells). Interestingly, 2 breast cancer lines were sensitive to BAL27862 in a clonogenic assay (IC50/IC70 - MAXF 401: 13/18 nM; MX1: 22/46 nM) despite one being relatively resistant to paclitaxel (MX1: 127/>3500 nM). Moreover, BAL27862 retained activity in 2 Pgp drug efflux pump-expressing breast cancer lines, 50 - 100-fold resistant to paclitaxel and vinblastine (MT-3/ADR, MCF7/TxT50), as well as in MCF7/TxTP50 cells ∼8-fold resistant to paclitaxel through non-Pgp-related mechanisms. When administered iv to mice at well tolerated doses, BAL101553 elicited statistically significant antitumor activity (p Conclusions: BAL101553 is currently undergoing Phase 1 clinical evaluation in advanced cancer patients with solid tumors. Potent antitumor activity alone and in combination with trastuzumab in preclinical chemo-refractory models of breast cancer supports clinical evaluation in breast cancer patients. Citation Format: Felix Bachmann, Karin Burger, George E. Duran, Branimir I. Sikic, Heidi A. Lane. BAL101553 (prodrug of BAL27862): A unique microtubule destabilizer active against drug refractory breast cancers alone and in combination with trastuzumab. [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 831. doi:10.1158/1538-7445.AM2014-831

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

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: The synthetic small molecule [BAL27862][1], active component of the prodrug BAL101553, destabilizes microtubules, potently inducing apoptosis in cancer cells. [BAL27862][1] elicits a broad antitumor activity and BAL101553/BAL27862 can be administered orally and intravenously. A unique microtubule phenotype suggests a novel mechanism of action (MoA), which is associated with activity in tumor models refractory to microtubule stabilizers (e.g. taxanes; epothilones) and destabilizers (e.g. Vinca-alkaloids such as vinblastine [VBL]). To further investigate the MoA and provide a platform for the identification of patient stratification biomarkers, [BAL27862][1]-resistant tumor models were developed. Methods: Drug-resistant tumor lines (Hela, H460, A549, SKOV3, Jurkat) were selected in vitro by incubation with increasing [BAL27862][1] concentrations. Proliferation was measured by crystal violet assay, apoptosis by flow cytometry. Tubulin polymerization was assessed by immunoblotting or electron microscopy (EM). Tubulin binding experiments used a spin column and [3H]-VBL and [14C]-BAL27862. Results: Selection for [BAL27862][1]-resistant tumor cells in vitro proved slow, requiring 8 – 12 months to culture lines with resistance factors (RF) ranging from 4.5 – 16. As an example, the EC50 for induction of apoptosis following [BAL27862][1] treatment in the parental (‘Par’) Jurkat line was 17 nM, whereas the resistant line (‘Res’) had an EC50 of 127 nM. Consistent with [BAL27862][1] not being a Pgp efflux pump substrate, Pgp up regulation could not account for resistance development in any line. Moreover, tubulin polymerization assays indicated a higher proportion of tubulin in the polymerized fraction in ‘Res’ lines, indicative rather of microtubule stabilization. Furthermore, the microtubule network in ‘Res’ lines was significantly less sensitive to the depolymerizing activity of [BAL27862][1] than in the ‘Par’ lines. The Jurkat ‘Res’ line was as sensitive as the ‘Par’ line to VBL treatment (EC50 for induction of apoptosis: 2 nM in both lines), whereas ‘Res’ H460 were equally resistant to [BAL27862][1] (RF: 5.5) and VBL (RF: 4.2). Both Jurkat and H460 ‘Res’ lines had a slightly increased sensitivity to paclitaxel treatment (2-3 fold). Drug cross-resistance in H460 could not be simply due to overlapping binding sites, as [BAL27862][1] and VBL bound independently to tubulin heterodimers. Furthermore, microtubule disassembly analysis by EM showed that [BAL27862][1] did not induce the small oligomeric structures associated with VBL treatment. Conclusion: Selection for [BAL27862][1] resistance in vitro requires prolonged culturing, is associated with more stable microtubules and may be mediated through diverse mechanisms. Results from work in progress to define the molecular changes associated with [BAL27862][1] resistance could facilitate patient stratification during clinical development of the prodrug BAL101553. 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 743. doi:10.1158/1538-7445.AM2011-743 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=BAL27862&atom=%2Fcanres%2F71%2F8_Supplement%2F743.atom

Background: BAL27862 is a synthetic small molecule, inducing apoptosis in cancer cells through a unique, destabilizing effect on microtubules (MT). BAL27862 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 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 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, BAL101553's 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 and is not observed with conventional MT-targeted agents like taxanes and vinca alkaloids. In vivo, BAL101553 was converted to active BAL27862. In mice, after i.v. administration of BAL27862 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. 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 [10 mg/kg], 40% BAL101553 [28 mg/kg]). Conclusions: BAL101553 is a prodrug of BAL27862 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.

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. Estève 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.

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.

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.