Plk1 Inhibitors Research Articles

Recent Progress in Development of Polo-Like Kinase 1 Inhibitors: Efforts So Far

Polo-like kinase 1(Plk1) plays an important role in the inhibition of cell proliferation and which is come under the family of serine/threonine-protein kinase. Which is a highly specific target for cancer therapy. In some clinical studies, Plk1 has been identified as a target for cancer. Currently, so many scientists are working on the development of the Plk1 inhibitors and so many scientists and researchers thinking about working on it. Recent strategy for Plk1 inhibition is the development of small molecule inhibitors which will inhibit the Plk1 through the ATP-binding site of the Plk. Now new generation Plk1 inhibitors being tested clinically which are targeting polo box domain. This review highlights the recent progress made in the development of Plk1 inhibitors as anticancer agents.

High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient’s outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.

Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.

Design, synthesis, and biological evaluation of novel 4,4-difluoro-1-methyl-N, 6-diphenyl-5, 6-dihydro-4H-pyrimido [4, 5-b] [1, 2, 4] triazolo [4, 3-d] [1, 4] diazepin-8-amine derivatives as potential BRD4 inhibitors.

Bromodomain-containing protein 4 (BRD4) plays an extremely important physiological role in cancer, and the BRD4 inhibitors can effectively inhibit the proliferation of tumor cells. By taking BI-2536 (PLK1 and BRD4 inhibitor) as the lead compound, sixteen novel BRD4 inhibitors with the 4,4-difluoro-1-methyl-N,6-diphenyl-5,6-dihydro-4H-pyrimido[4,5-b] [1,2,4] triazolo[4,3-d] [1,4] diazepine-8-amine structure were designed and synthetized. Among the target compounds, compound 15h exhibited outstanding inhibition for BRD4-BD1 (IC50 value of 0.42μM) in the BRD4-BD1 inhibitory activity assay. Additionally, cell growth inhibition assay demonstrated that compound 15h potently suppressed the proliferation of MV4-11 cells (IC50 value of 0.51μM). Besides, compound 15h induced apoptosis and G0/G1 cycle arrest in MV4-11 leukemia cells effectively, and downregulated the expression of c-Myc in a dose-dependent manner. In summary, the optimal compound 15h is expected to become the clinical therapeutic drug for further research.

P73.03 A Kinome CRISPR Screen in FGFR-Amplified Lung Cancer

Oncogenic alterations in fibroblast growth factor receptor (FGFR) are frequent, but therapeutic targeting of FGFR-driven cancers remains an unmet challenge in clinical oncology in that FGFR inhibitors as a single agent are inevitably limited by poor responsiveness and/or compensatory activation of various acquired resistance mechanisms. We performed kinome CRISPR/Cas9 screens to identify genetic determinants that limit efficacy of FGFR-targeted therapy in FGFR-mutant squamous lung carcinoma (SQLC). We identify several kinases including polo-like kinase I (PLK1) as key determinants of sensitivity to AZD4547, a selective FGFR inhibitor currently investigated in phase III clinical trials. We provided evidence showing that genetic and pharmacological inhibition of PLK1 synergistically enhances the effect of FGFR inhibitors in FGFR-amplified lung SQLC. CRISPR/Cas9 screening is a powerful tool to uncover genetic determinants underlying drug sensitivity in cancer.

Efficient biosynthesis of (S)-1-[2-(trifluoromethyl)phenyl]ethanol by a novel isolate Geotrichum silvicola ZJPH1811 in deep eutectic solvent/cyclodextrin-containing system

This study aimed to develop a biotransformation process for the production of (S)-1-[2-(trifluoromethyl)phenyl]ethanol, a key chiral intermediate of Plk1 inhibitor, and increase its productivity through medium engineering strategy. A fungus isolate Geotrichum silvicola ZJPH1811 was adopted as biocatalyst for 2ʹ-(trifluoromethyl)acetophenone reduction, and gave the best performance with > 99.2% product ee. To improve the yield, choline acetate/cysteine (ChAc/Cys) was introduced as co-solvent in reaction system, which accelerated mass transfer and protected cells from substrate inhibition. Moreover, a synergistic effect of methylated-β-cyclodextrin (MCD) and ChAc/Cys was found in the bioreduction, with further enhancement in substrate concentration and cell membrane permeability. Compared with buffer system, in the developed ChAc/Cys-MCD-containing system, substrate loading and product yield were increased by 6.7-fold and 2.4-fold respectively. This is the first report on (S)-1-[2-(trifluoromethyl)phenyl]ethanol production with G. silvicola, and provides valuable insight into the synergistic effect of DES and CDs in biocatalysis.

Dual Inhibition of γ-Tubulin and Plk1 Induces Mitotic Cell Death.

α/β-Tubulin inhibitors that alter microtubule (MT) dynamics are commonly used in cancer therapy, however, these inhibitors also cause severe side effects such as peripheral neuropathy. γ-Tubulin is a possible target as antitumor drugs with low side effects, but the antitumor effect of γ-tubulin inhibitors has not been reported yet. In this study, we verified the antitumor activity of gatastatin, a γ-tubulin specific inhibitor. The cytotoxicity of gatastatin was relatively weak compared with that of the conventional MT inhibitors, paclitaxel and vinblastine. To improve the cytotoxicity, we screened the chemicals that improve the effects of gatastatin and found that BI 2536, a Plk1 inhibitor, greatly increases the cytotoxicity of gatastatin. Co-treatment with gatastatin and BI 2536 arrested cell cycle progression at mitosis with abnormal spindles. Moreover, mitotic cell death induced by the combined treatment was suppressed by the Mps1 inhibitor, reversine. These findings suggest that co-treatment with Plk1 and γ-tubulin inhibitors causes spindle assembly checkpoint-dependent mitotic cell death by impairing centrosome functions. These results raise the possibility of Plk1 and γ-tubulin inhibitor co-treatment as a novel cancer chemotherapy.

A Regulatory Loop of FBXW7-MYC-PLK1 Controls Tumorigenesis of MYC-Driven Medulloblastoma.

Simple SummaryGroup 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a poor prognosis. FBXW7, a critical tumor suppressor in many types of cancer, regulates the proteasome-mediated degradation of oncoproteins including MYC. However, the role of FBXW7 in the tumorigenesis of group 3 MB has not been well studied. In this study, we show that FBXW7 is downregulated in group 3 MB patient samples, and FBXW7 stabilization is crucial for inhibiting c-MYC. We identified a FBXW7-MYC-PLK1 regulatory loop in MYC-driven MB, which provides a mechanism of using protein kinase inhibitors for translation in the future.Polo-like kinase 1 (PLK1) is highly expressed in group 3 medulloblastoma (MB), and it has been preclinically validated as a cancer therapeutic target in medulloblastoma. Here, we demonstrate that PLK1 inhibition with PCM-075 or BI6727 significantly reduces the growth of MB cells and causes a decrease of c-MYC mRNA and protein levels. We show that MYC activates PLK1 transcription, while the inhibition of PLK1 suppresses MB tumor development and causes a decrease in c-MYC protein level by suppressing FBXW7 auto poly-ubiquitination. FBXW7 physically interacts with PLK1 and c-MYC, facilitating their protein degradation by promoting ubiquitination. These results demonstrate a PLK1-FBXW7-MYC regulatory loop in MYC-driven medulloblastoma. Moreover, FBXW7 is significantly downregulated in group 3 patient samples. The overexpression of FBXW7 induced apoptosis and suppressed proliferation in vitro and in vivo, while constitutive phosphorylation mutation attenuated its tumor suppressor function. Altogether, these findings demonstrated that PLK1 inhibition stabilizes FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing medulloblastoma progression.

PLK1 Inhibition Induces Immunogenic Cell Death and Enhances Immunity against NSCLC.

PLK1 inhibitors were shown, in vitro and in vivo, to possess inhibitory activities against non-small cell lung cancer (NSCLC), and such inhibition has been proven by clinical trials. However, it remains unclear whether and how the immune microenvironment is associated with the action. In this study, we found that inhibiting PLK1 could alter the tumor immune microenvironment by increasing DC maturation, and enriching T cells infiltration. PLK1 inhibitors, serving as immunogenic cell death (ICD) inducers, indirectly activated DCs, instead of directly acting on DC cells, through the surface expression of costimulatory molecules on and enhanced phagocytosis by DCs. Furthermore, upon targeting PLK1, tumor cells that had undergone ICD were converted into an endogenous vaccine, which triggered the immune memory responses and protected the mice from tumor challenge. Collectively, these results suggested that the PLK1 inhibitor might function as an immune modulator in antitumor treatment.

PLK1 and NOTCH Positively Correlate in Melanoma and Their Combined Inhibition Results in Synergistic Modulations of Key Melanoma Pathways.

Melanoma is one of the most serious forms of skin cancer, and its increasing incidence coupled with nonlasting therapeutic options for metastatic disease highlights the need for additional novel approaches for its management. In this study, we determined the potential interactions between polo-like kinase 1 (PLK1, a serine/threonine kinase involved in mitotic regulation) and NOTCH1 (a type I transmembrane protein deciding cell fate during development) in melanoma. Employing an in-house human melanoma tissue microarray (TMA) containing multiple cases of melanomas and benign nevi, coupled with high-throughput, multispectral quantitative fluorescence imaging analysis, we found a positive correlation between PLK1 and NOTCH1 in melanoma. Furthermore, The Cancer Genome Atlas database analysis of patients with melanoma showed an association of higher mRNA levels of PLK1 and NOTCH1 with poor overall, as well as disease-free, survival. Next, utilizing small-molecule inhibitors of PLK1 and NOTCH (BI 6727 and MK-0752, respectively), we found a synergistic antiproliferative response of combined treatment in multiple human melanoma cells. To determine the molecular targets of the overall and synergistic responses of combined PLK1 and NOTCH inhibition, we conducted RNA-sequencing analysis employing a unique regression model with interaction terms. We identified the modulations of several key genes relevant to melanoma progression/metastasis, including MAPK, PI3K, and RAS, as well as some new genes such as Apobec3G, BTK, and FCER1G, which have not been well studied in melanoma. In conclusion, our study demonstrated a synergistic antiproliferative response of concomitant targeting of PLK1 and NOTCH in melanoma, unraveling a potential novel therapeutic approach for detailed preclinical/clinical evaluation.

The Polo-like kinase 1 inhibitor onvansertib represents a relevant treatment for head and neck squamous cell carcinoma resistant to cisplatin and radiotherapy.

Rationale: Head and neck squamous cell carcinoma (HNSCC) represent the 4th most aggressive cancer. 50% of patients relapse to the current treatments combining surgery, radiotherapy and cisplatin and die two years after the diagnosis. Elevated expression of the polo-like kinase 1 (Plk1) correlated to a poor prognosis in epidermoid carcinomas.Methods: The molecular links between Plk1 and resistance to cisplatin/radiotherapy were investigated in patients and cell lines resistant to cisplatin and/or to radiotherapy. The therapeutic relevance of the Plk1 inhibitor onvansertib, alone or combined with cisplatin/radiotherapy, was evaluated on the proliferation/migration on HNSCC cell lines, in experimental HNSCC in mice, in a zebrafish metastasis model and on patient-derived 3D tumor sections.Results: Plk1 expression correlated to a bad prognosis in HNSCC and increased after relapse on cisplatin/radiotherapy. Onvansertib induced mitotic arrest, chromosomic abnormalities and polyploidy leading to apoptosis of sensitive and resistant HNSCC cells at nanomolar concentrations without any effects on normal cells. Onvansertib inhibited the growth of experimental HNSCC in mice and metastatic dissemination in zebrafishes. Moreover, onvansertib combined to cisplatin and/or radiotherapy resulted in a synergic induction of tumor cell death. The efficacy of onvansertib alone and in combination with reference treatments was confirmed on 3D viable sections of HNSCC surgical specimens.Conclusions: Targeting Plk1 by onvansertib represents a new strategy for HNSCC patients at the diagnosis in combination with reference treatments, or alone as a second line treatment for HNCSCC patients experiencing relapses.

MBRS-57. IDENTIFICATION OF MYC-DEPENDENT THERAPEUTIC VULNERABILITIES FOR TARGETING GROUP 3 MEDULLOBLASTOMA

Group 3 medulloblastoma (MBGroup3) is a highly aggressive tumour characterised by MYC amplification and elevated expression (17% of MBGroup3). MYC amplification in MBGroup3 confers a dismal prognosis using standard therapies, and there is an urgent unmet need for novel therapeutic approaches. The identification and targeting of MYC’s biological dependencies thus represents a promising strategy to treat MYC-MBGroup3 tumours. Three independent isogenic MYC-regulable MBGroup3 human cell-based models, in which elevated MYC expression can be directly down-regulated by doxycycline-inducible shRNAs, were developed and used initially to establish MYC-dependent growth of each model. Our novel models were then used to investigate MYC-dependent drug sensitivity, by characterising responses to a panel of candidate cancer therapeutics and small molecule inhibitors, including a high-throughput compound screen of >500 established/clinically-relevant small molecule inhibitors. This approach identified several specific, consistently observed, druggable MYC-dependencies (e.g. cell cycle regulators, DNA-damage response controllers, mitotic control machinery) with potential for the development of treatments against MYC-MBGroup3 tumours. PLK1, CHK1 and AURK were identified as prime candidate targets with consistent MYC-dependent response profiles. Subsequent validation of each candidate, by genetic and pharmacological target inhibition, confirmed their MYC-dependent effects, associated with downregulation of MYC and established target-dependent pharmacodynamic biomarkers/pathways. Results were consistent across all of our MBGroup3 models. In summary, our novel models reveal druggable MYC-associated dependencies as a feature of MBGroup3. Our findings support the development of PLK1, CHK1 and AURK inhibition as therapeutic approaches against MYC-dependent MBGroup3. Future work is now essential to validate our findings in vivo, to support the design of future clinical trials.

Centrosomal Localization of RXRα Promotes PLK1 Activation and Mitotic Progression and Constitutes a Tumor Vulnerability.

Retinoid X receptor alpha (RXRα), a nuclear receptor of transcription factor, controls various physiological and pathological pathways including cellular growth, proliferation, differentiation, and apoptosis. Here, we report that RXRα is phosphorylated at its N-terminal A/B domain by cyclin-dependent kinase 1 (Cdk1) at the onset of mitosis, triggering its translocation to the centrosome, where phosphorylated-RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) through its N-terminal A/B domain by a unique mechanism. The interaction promotes PLK1 activation, centrosome maturation, and mitotic progression. Levels of p-RXRα are abnormally elevated in cancer cell lines, during carcinogenesis in animals, and in clinical tumor tissues. An RXRα ligand XS060, which specifically inhibits p-RXRα/PLK1 interaction but not RXRα heterodimerization, promotes mitotic arrest and catastrophe in a tumor-specific manner. These findings unravel a transcription-independent action of RXRα at the centrosome during mitosis and identify p-RXRα as a tumor-specific vulnerability for developing mitotic drugs with improved therapeutic index.

Mining Polo-Box domain of Polo-like kinase 1 as a new therapeutic target for cancer

Polo-like kinase 1 (Plk1) is widely regarded as one of the most promising targets for cancer therapy due to its essential role in cell division and tumor cell survival. At present, most Plk1 inhibitors have been developed based on kinase domain, some of which are in clinical trial. However, inhibitors targeting kinase domain face off-target effect and drug resistance owing to the conserved nature and the frequent mutations in the ATP-binding pocket. In addition to a highly conserved kinase domain, Plk1 also contains a unique Polo-Box domain (PBD), which is essential for Plk1's subcellular localization and mitotic functions. Inhibitors targeting Plk1 PBD show stronger selectivity and less drug resistance for cancer therapy. Therefore, Plk1 PBD is an attractive target for the development of anti-cancer agents. In this review, we will summarize the up-to date drug discovery for targeting Plk1 PBD, including the molecular structure and cellular functions of Plk1 PBD. Small-molecule inhibitors targeting Plk1 PBD not only provide an opportunity to specifically inhibit Plk1 activity for cancer treatment, but also unveil novel biological basis regarding the molecular recognition of Plk1 and its substrates.

Computational Exploration of Potential Polo-Like Kinase 1 Inhibitors as New Chemotherapeutic Agents

Background and Objective: Polo like kinase-1 (PLK-1) enzyme belongs to serine/threonine protein kinase family that is regarded as a principle mitotic controller of G2-M phase transition. The antimitotic therapies are a cornerstone for the treatment of metastatic as well as benign cancer. Therefore, PLK-1 has recently gained much interest in the field of targeting it by novel and effective inhibitors.
 Materials and Methods: The present study described the used of pharmacophore modelling based on the potent and selective clinical agent, Volasertib and followed by hybrid selection of a kinase inhibitors databank of 4800 diverse compounds by pharmacophore- and docking-based virtual screening.
 Results: The retrieved hits were filtered on the bases of their pharmacophore-fit scores, docking binding affinity scores, ADME-T profiles as well as ligand quality assessments. Among the five hit compounds that fulfil the criterion, three compounds, Z1991791422, Z56115729 and Z1991791176 were selected for binding dynamic analyses by molecular dynamic simulation. The Z1991791422 and Z56115729 compounds illustrated stable binding behaviours at the proposed binding site. Conclusion: Thus, these compounds might emerge as potent inhibitors of PLK1 and could be applied as seeds for designing better PLK1 inhibitors in near future.

Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance.

USP7 is a promising target for the development of cancer treatments because of its high expression and the critical functions of its substrates in carcinogenesis of several different carcinomas. Here, we demonstrated the effectiveness of targeting USP7 in advanced malignant cells showing high levels of USP7, especially in taxane-resistant cancer. USP7 knockdown effectively induced cell death in several cancer cells of lung, prostate, and cervix. Depletion of USP7 induced multiple spindle pole formation in mitosis, and, consequently, resulted in mitotic catastrophe. When USP7 was blocked in the paclitaxel-resistant lung cancer NCI-H460TXR cells, which has resistance to mitotic catastrophe, NCI-H460TXR cells underwent apoptosis effectively. Furthermore, combination treatment with the mitotic kinase PLK1 inhibitor volasertib and the USP7 inhibitor P22077 showed a strong synergism through down-regulation of MDR1/ABCB1 in paclitaxel-resistant lung cancer. Therefore, we suggest USP7 is a promising target for cancer therapy, and combination therapy with inhibitors of PLK1 and USP7 may be valuable for treating paclitaxel-resistant cancers, because of their strong synergism.

Polo-like kinase 1 is involved in apoptosis, invasion, and metastasis of pancreatic ductal adenocarcinoma.

BackgroundPolo-like kinase 1 (Plk1) is overexpressed in different types of carcinomas, and it is widely pursued as a novel target for treatment of neoplasms. However, the role of Plk1 in invasion and metastasis of pancreatic cancer has not been reported.MethodsIn order to evaluate whetherPlk1 can potentially serve as a therapeutic target in pancreatic cancer, we herein explore and discuss the relationship between the inhibition of Plk1 by RNA interference (RNAi) and invasion and metastasis of pancreatic cancer cells. For this, three pancreatic cancer cell lines (AsPC-1, BxPC-3, and PANC-1) were studied. Plk1-specific short hairpin RNAs (shPlk1) were introduced into these cell lines by viral transduction.ResultsWe found that Plk1 mRNA and protein levels were significantly reduced in the shPlk1 group. Moreover, knockdown of Plk1 expression by shPlk1 promoted apoptosis, while cell invasion and metastasis potentials were significantly reduced.ConclusionsPlk1 expression levels were closely correlated with proliferation, invasion, and metastasis of pancreatic cancer, and inhibition of Plk1 expression by RNAi could promote cell apoptosis and suppress metastasis and invasion of pancreatic cancer cells in vitro. Furthermore, Plk1 might constitute novel target for treatment of pancreatic cancer, with inhibition of Plk1 potentially providing a new strategy for the prevention of pancreatic cancer invasion and metastasis.

The Frequently Amplified Gene TRIM37 Mediates PLK4 Inhibitor Response

Inhibitors of the centrosome-duplicating protein PLK4 selectively target cells with high TRIM37 expression.

Brain delivery of Plk1 inhibitor via chimaeric polypeptide polymersomes for safe and superb treatment of orthotopic glioblastoma

The chemotherapy toward glioblastoma (GBM) is severely challenged by blood-brain barrier and dose-limiting toxicity. Herein, we adopt brain delivery of Plk1 inhibitor volasertib (Vol), which is highly specific and presents low off-target toxicity, as a new means to treat GBM, for which angiopep-2-docked chimaeric polypeptide polymersome (ANG-CPP) was designed and prepared from poly(ethylene glycol)-b-poly(L-tyrosine)-b-poly(L-aspartic acid) for loading Vol to its watery interior via electrostatic interactions. ANG-CPP loaded with 13.9 wt% Vol (ANG-CPP-Vol) exhibited a small size of about 76 nm, superb colloidal stability (against dilution, serum and long-term storage), and enzyme-triggered drug release behavior (about 73% of Vol released within 8 h with proteinase K). In sharp contrast to free Vol, ANG-CPP-Vol induced complete G2/M cell cycle arrest in U-87 MG GBM cells giving 7.8-times better anti-tumor activity, prolonged circulation time and largely increased GBM enrichment. ANG-CPP-Vol effectively suppressed the growth of orthotopic U-87 MG GBM and significantly boosted mice survival rate. Importantly, ANG-CPP-Vol showed further reduced toxicity over free Vol. This great safety and remarkable efficacy of ANG-CPP-Vol renders it a high potential for treating GBM.

Calibrated liposomal release of the anti-mitotic agent BI-2536 increases the targeting of mitotic tumor cells

Cancer drugs which are specifically targeted at mitosis have generally under-delivered as a class. One likely reason is that only a small percentage of cancer cells in a tumor are actually dividing at any moment. If this is the case, then prolonged bioavailability in the tumor should significantly increase the efficacy of antimitotic agents. Here, we show that if the Plk1 inhibitor BI 2536 is co-encapsulated in a liposome with a pair of anions, its release rate is dependent on both the identity and stoichiometry of the anions. We created a library of liposomes with varying release rates using this approach and found that liposomal drug release rates correlated inversely with in vitro cancer cell killing. Xenografted mice treated with a single dose of slow-releasing liposomal BI 2536 experienced tumor volume decreases lasting 12 days and complete responses in 20% of mice. Treatment with two doses a week apart increased the response rate to 75%. This approach, which we termed Paired Anion Calibrated Release (PACeR), has the potential to revive the clinical utility of antimitotic cancer drugs which have failed clinical trials.