Why Great Mitotic Inhibitors Make Poor Cancer Drugs
Authors: Victoria C. Yan, Hannah E. Butterfield, Anton H. Poral, Matthew J. Yan, Kristine L. Yang, Cong-Dat Pham, Florian L. Muller
Journal: Trends in cancer (2020)

A common misconception is that all cancers proliferate more rapidly compared with normal tissue. However, many non-malignant tissues have proliferation indices higher than those of certain cancers.Many successful DNA-damaging chemotherapies are prodrugs and achieve sufficient therapeutic windows due to selective bioactivation in cancer cells over normal cells.Despite strong preclinical promise, inhibitors of cell-essential mitotic kinases were relegated to the drug development graveyard due to severe off-target toxicities in rapidly proliferating normal tissues.The utility of mitotic kinase inhibitors may be realized by applying the prodrug strategy, used successfully in classical chemotherapies, to this class of inhibitors. Selectivity for cancer cells will then arise from proliferation rate and tumor-specific prodrug bioactivation. Chemotherapy is central to oncology, perceived to operate only on prolific cancerous tissue. Yet, many non-neoplastic tissues are more prolific compared with typical tumors. Chemotherapies achieve sufficient therapeutic windows to exert antineoplastic activity because they are prodrugs that are bioactivated in cancer-specific environments. The advent of precision medicine has obscured this concept, favoring the development of high-potency kinase inhibitors. Inhibitors of essential mitotic kinases exemplify this paradigm shift, but intolerable on-target toxicities in more prolific normal tissues have led to repeated failures in the clinic. Proliferation rates alone cannot be used to achieve cancer specificity. Here, we discuss integrating the cancer specificity of prodrugs from classical chemotherapeutics and the potency of mitotic kinase inhibitors to generate a class of high-precision cancer therapeutics. Chemotherapy is central to oncology, perceived to operate only on prolific cancerous tissue. Yet, many non-neoplastic tissues are more prolific compared with typical tumors. Chemotherapies achieve sufficient therapeutic windows to exert antineoplastic activity because they are prodrugs that are bioactivated in cancer-specific environments. The advent of precision medicine has obscured this concept, favoring the development of high-potency kinase inhibitors. Inhibitors of essential mitotic kinases exemplify this paradigm shift, but intolerable on-target toxicities in more prolific normal tissues have led to repeated failures in the clinic. Proliferation rates alone cannot be used to achieve cancer specificity. Here, we discuss integrating the cancer specificity of prodrugs from classical chemotherapeutics and the potency of mitotic kinase inhibitors to generate a class of high-precision cancer therapeutics. Chemotherapies inducing DNA damage remain the mainstay of cancer treatment. 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The best clinical response rates (objective, PR) were <20%.DrugStructureTarget (Ki)Furthest clinical stageDose-limiting toxicityBest clinical response rateRefsAlisertibAURKA (1.2 nM)Phase III (2012- 2015) NCT01482962Febrile neutropenia17% in aggressive B cell lymphoma[21.Manfredi M.G. et al.Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.Clin. Cancer Res. 2011; 17: 7614-7624Crossref PubMed Scopus (190) Google Scholar]IspinesibEg5/KIF11 (1.7 nM)Phase II (2004-2006) NCT00089973Febrile neutropenia, vomiting0% in malignant melanoma[22.Lad L. et al.Mechanism of inhibition of human KSP by ispinesib.Biochemistry. 2008; 47: 3576-3585Crossref PubMed Scopus (82) Google Scholar, 23.Carol H. et al.Initial testing (stage 1) of the kinesin spindle protein inhibitor ispinesib by the pediatric preclinical testing program.Pediatr. Blood Cancer. 2009; 53: 1255-1263Crossref PubMed Scopus (0) Google Scholar, 24.Lee C.W. et al.A phase II study of ispinesib (SB-715992) in patients with metastatic or recurrent malignant melanoma: a National Cancer Institute of Canada Clinical Trials Group trial.Invest. New Drugs. 2008; 26: 249-255Crossref PubMed Scopus (0) Google Scholar]DinaciclibCDK2 (1 nM) CDK5 (1 nM) CDK1 (3 nM) CDK9 (4 nM)Phase I (2016-2018) NCT02684617; NCT03484520Neutropenia, Pneumocystis11% PR in multiple myeloma[25.Parry D. et al.Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.Mol. Cancer Ther. 2010; 9: 2344-2353Crossref PubMed Scopus (272) Google Scholar,26.Kumar S.K. et al.Dinaciclib, a novel CDK inhibitor, demonstrates encouraging single-agent activity in patients with relapsed multiple myeloma.Blood. 2015; 125: 443-448Crossref PubMed Scopus (105) Google Scholar]GSK461364PLK1 (2.2 nM)Phase I (2007–2009) NCT00536835Neutropenia, thrombocytopenia, thrombic emboli, myelosuppression0% in solid tumors[27.Olmos D. et al.Phase I study of GSK461364, a specific and competitive Polo-like kinase 1 inhibitor, in patients with advanced solid malignancies.Clin. Cancer Res. 2011; 17: 3420-3430Crossref PubMed Scopus (0) Google Scholar,28.Gilmartin A.G. et al.Distinct concentration-dependent effects of the polo-like kinase 1-specific inhibitor GSK461364A, including differential effect on apoptosis.Cancer Res. 2009; 69: 6969-6977Crossref PubMed Scopus (88) Google Scholar]BAY1217389TTK (0.63 nM)Phase I (2015–2019) NCT02366949Hematological toxicity, neutropeniaN/A[29.Wengner A.M. et al.Novel Mps1 kinase inhibitors with potent antitumor activity.Mol. Cancer Ther. 2016; 15: 583-592Crossref PubMed Google Scholar]a An overview of structures, corresponding target affinities, and furthest progression in clinical trials for several potent mitotic kinase inhibitors is shown. Even with single-digit nanomolar Kis, major dose-limiting toxicities are still observed, stalling these trials. According to RECIST criteria, none of these included drugs achieved a complete response (CR) at toxicity-limiting doses. The best clinical response rates (objective, PR) were <20%. Open table in a new tab Table 2Examples of Clinically Impactful Chemotherapeutic Agents that Are Prodrugs and Rely on Cancer-Specific Biotransformation for Specific KillingaA representative panel of clinically useful conventional chemotherapies is shown. Combined treatment with one or more agents shown is responsible for curing a substantial number of patients with cancer. Prodrug moieties that undergo biological removal are indicated in red; those that undergo biological addition are indicated in magenta. Mechanisms outlined for each example are applicable for the broader class that it represents; for example, all nucleotide analogs require biotransformation to their monophosphate forms for bioactivity.Representative example (class)Structure of administered drug ()Active agent ()Mode of actionMechanism of selectivityRefsCyclophosphamide (nitrogen mustards)DNA damage through crosslinkingLoss of ALDH1A1 in dedifferentiated cancer cells, basic tumor intracellular[30.Russo J.E. Hilton J. Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells.Cancer Res. 1988; 48: 2963-2968PubMed Google Scholar]Temozolomide (triazenes)DNA damage through methylationMGMT silencing, cytosolic alkaline pH in GBM[31.Newlands E.S. et al.Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials.Cancer Treat. Rev. 1997; 23: 35-61Abstract Full Text PDF PubMed Scopus (604) Google Scholar]5-Fluorouracil (nucleotide analog)DNA, RNA damage, inhibition of thymidylate synthaseOverexpression of enzymes of thymidine phosphate synthesis[32.van Triest B. et al.Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines.Clin. Cancer Res. 1999; 5: 643-654PubMed Google Scholar]Irinotecan (topoisomerase inhibitors)Topoisomerase inhibition leads to DNA breakageButyrylcholine esterase activity, overexpressed in cancer cells[33.Morton C.L. et al.The anticancer prodrug CPT-11 is a potent inhibitor of acetylcholinesterase but is rapidly catalyzed to SN-38 by butyrylcholinesterase.Cancer Res. 1999; 59: 1458-1463PubMed Google Scholar]Cisplatin (platinum agents)DNA damage: crosslinkingAquation due to more alkaline intracellular environment and lower intracellular chloride[34.Davies M.S. et al.Slowing of cisplatin aquation in the presence of DNA but not in the presence of phosphate: improved understanding of sequence selectivity and the roles of monoaquated and diaquated species in the binding of cisplatin to DNA.Inorg. Chem. 2000; 39: 5603-5613Crossref PubMed Scopus (0) Google Scholar]Methotrexate (antifolates)Antifolate: inhibition of nucleotide synthesisIncreased import via overexpressed folate receptor and trapping by polyglutamylation in cancer[35.Rajagopalan P.T.R. et al.Interaction of dihydrofolate reductase with methotrexate: Ensemble and single-molecule kinetics.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13481-13486Crossref PubMed Scopus (0) Google Scholar]a A representative panel of clinically useful conventional chemotherapies is shown. Combined treatment with one or more agents shown is responsible for curing a substantial number of patients with cancer. Prodrug moieties that undergo biological removal are indicated in red; those that undergo biological addition are indicated in magenta. Mechanisms outlined for each example are applicable for the broader class that it represents; for example, all nucleotide analogs require biotransformation to their monophosphate forms for bioactivity. Open table in a new tab The development of mitotic kinase inhibitors came as an outgrowth of studies on the molecular mechanisms of the cell cycle during the 1990s [36.Piwnica-Worms H. Reversible phosphorylation and mitotic control.J. Lab. Clin. Med. 1996; 128: 350-354Abstract Full Text PDF PubMed Google Scholar, 37.Hartwell L. Kastan M. Cell cycle control and cancer.Science (80-. ). 1994; 266: 1821-1828Crossref PubMed Google Scholar, 38.Draetta G. et al.Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate.Nature. 1988; 336: 738-744Crossref PubMed Google Scholar], which promised the development of precise inhibitors of mitosis without the toxic sequelae of classical chemotherapeutics. Yet, for all the initial excitement, mitotic kinase inhibitors have now been relegated to the drug development graveyard because they suffered from a lack of specificity between cancer cells and prolific, non-malignant cells. Multiple clinical trials have shown that they lack a therapeutic window and are dose limited by toxicities to tissues, such as the bone marrow and gut (Table 1). However, given their high affinities and potencies, we contend that mitotic kinase inhibitors may find clinical utility if refashioned with cancer-specific prodrug moieties. A detailed survey of the literature indicates that most classic chemotherapy drugs owe their therapeutic windows to specific prodrug activation and deactivation pathways exclusive to cancer cells. Here, we describe the utility of mitotic kinase inhibitors, review the bioactivation mechanisms of various chemotherapeutics, and then synthesize these concepts to discuss the application of cancer-specific prodrugs to mitotic kinase inhibitors. 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Examples of such inhibitors and their corresponding targets include dinaciclib [25.Parry D. et al.Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.Mol. Cancer Ther. 2010; 9: 2344-2353Crossref PubMed Scopus (272) Google Scholar] (pan-CDK), ispinesib [22.Lad L. et al.Mechanism of inhibition of human KSP by ispinesib.Biochemistry. 2008; 47: 3576-3585Crossref PubMed Scopus (82) Google Scholar] (KIF11/Eg5), alisertib [21.Manfredi M.G. et al.Characterization of Alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.Clin. Cancer Res. 2011; 17: 7614-7624Crossref PubMed Scopus (190) Google Scholar] (AURAK), and tozasertib [47.Harrington E.A. et al.VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo.Nat. 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Knockout of Cdk7/8/9 in mice, while embryonic lethal, thus showing them to be essential, are not exclusively mitotic kinases and their expression is not restricted to cell cycling [46.Bar-Joseph Z. et al.Genome-wide transcriptional analysis of the human cell cycle identifies genes differentially regulated in normal and cancer cells.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 955-960Crossref PubMed Scopus (0) Google Scholar]. CDK1 is an ideal cancer drug target due to its dispensability in postmitotic tissues [61.Diril M.K. et al.Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 3826-3831Crossref PubMed Scopus (171) Google Scholar]. CRISPR and RNAi data via DepMap corroborate early molecular biology on CDK1, supporting its essentiality in all cancer cell lines profiled to-date. Other CDKs, such as CDK4/6, are only essential in some cell lines (Tables S1 and S2 in the supplemental information online), underscoring their promotive but nonessential roles in the cell cycle [60.Santamaría D. et al.Cdk1 is sufficient to drive the mammalian cell cycle.Nature. 2007; 448: 811-815Crossref PubMed Scopus (553) Google Scholar]. Unlike nonessential CDKs, CDK1 expression is strongly correlated with survival in many cancers in a manner similar to that of Ki67 and PCNA (Tables S1 and S2 in the supplemental information online) [62.Piao J. et al.High expression of CDK1 and BUB1 predicts poor prognosis of pancreatic ductal adenocarcinoma.Gene. 2019; 701: 15-22Crossref PubMed Scopus (10) Google Scholar, 63.Jayanthi V.S.P.K.S.A. et al.Grade-specific diagnostic and prognostic biomarkers in breast cancer.Genomics. 2020; 112: 388-396Crossref PubMed Scopus (1) Google Scholar, 64.Pabla S. et al.Proliferative potential and resistance to immune checkpoint blockade in lung cancer patients.J. Immunother. Cancer. 2019; 7: 27Crossref PubMed Scopus (7) Google Scholar]iii. Such properties coincide with the exclusivity of CDK1 expression in dividing cells, making it impossible for a highly specific CDK1 inhibitor to exhibit (on-target) adverse effects to nonproliferative tissue. Following the rigorous candidate validation modeled for CDK1, we contend that other cell cycle-essential proteins would have similar proliferative-cell dependencies to CDK1 (Figure 1, and Tables S1 and S2 in the supplemental information online), although the extensive bioinformatics data have yet to be corroborated with the full complement of biological validation, including mouse knockout phenotypes. The discrepancy between preclinical success and clinical failure of mitotic kinase inhibitors (Table 1) can be explained by two reasons: (i) interspecies differences between mice and humans; and (ii) the high proliferation index of critical non-neoplastic tissues versus tumors. Preclinical xen