inhibitors targeting mitosis: tales of how great drugs ...transport kinesins, which function in...

Inhibitors Targeting Mitosis: Tales of How Great Drugsagainst a Promising Target Were Brought Down by a FlawedRationale

Edina Komlodi-Pasztor1, Dan L. Sackett2, and Antonio Tito Fojo1

AbstractAlthough they have been advocated with an understandable enthusiasm, mitosis-specific agents such as

inhibitors of mitotic kinases and kinesin spindle protein have not been successful clinically. These drugs

were developed as agents thatwould build on the success ofmicrotubule-targeting agentswhile avoiding the

neurotoxicity that encumbers drugs such as taxanes and vinca alkaloids. The rationale for using mitosis-

specific agents was based on the thesis that the clinical efficacy of microtubule-targeting agents could be

ascribed to the induction of mitotic arrest. However, the latter concept, which has long been accepted as

dogma, is likely important only in cell culture and rapidly growing preclinical models, and irrelevant in

patient tumors, where interference with intracellular trafficking on microtubules is likely the principal

mechanism of action. Here we review the preclinical and clinical data for a diverse group of inhibitors that

target mitosis and identify the reasons why these highly specific, myelosuppressive compounds have failed

to deliver on their promise. Clin Cancer Res; 18(1); 51–63. �2012 AACR.

Introduction

On the premise that tumors harbor a (much) largerfraction of actively dividing cells than do normal tissuesand should therefore bemore vulnerable to agents aimed atcell division, researchers have developed drugs to targetnumerous components of this intricate process as che-motherapeutics. Chief among these are agents that targetthe processes of mitosis and the accompanying cytokinesis,with inhibitors of mitotic kinases being among the mostrecent entries (1). Although the clinical efficacy of agentsthat target the process of cell division [e.g., microtubule-targeting agents (MTA)] provided support for this rationale,accumulating clinical and preclinical evidence is encourag-ing a reassessment of how MTAs act (2). Our increasingunderstanding of the working of cells in general and cancercells in particular, together with greater knowledge aboutthe diversity of targets of our chemotherapeutic agents,suggests that a more complex approach than only targetingcell division may be warranted. Among the components of

cell division, mitosis, the process whereby a eukaryotic cellseparates its replicated chromosomes into identical sets in 2offspring, has long been viewed as an attractive target forchemotherapeutic agents. Together, mitosis and the accom-panying cytokinesis that divides the nucleus, cytoplasmicorganelles, and cellmembrane into 2 cells define themitoticor M-phase of the cell cycle. In most cells, this accounts forless (and often substantially less) than 10%of the cell cycle.Emerging knowledge is revealing the complexity of themitotic process and allowing the identification of a diversegroup of proteins whose activity is precisely orchestratedduring mitosis. Mitosis requires many other players inaddition to tubulin and microtubules (MT). Many of theseother proteins are primarily functional only during mitosisand are responsible for controlling different steps in theassembly and function of the complex machinery of themitotic spindle. Among these proteins, the Aurora kinases(AK), Polo-like kinases (PLK), and kinesin spindle protein(KSP) have emerged as targets for cancer therapeutics (3, 4).

Mitosis: The Role of MTs and Mitotic Kinases

As shown in Fig. 1, ordered arrays of MTs play essentialroles in both interphase (G1-, S-, and G2-phases) andmitosis. In animal cells, centrosomes act as MT-organizingcenters (i.e., the site of MT nucleation), the structures fromwhich MTs emerge (5). Centrosomal nucleation of polar-ized arrays of MTs is essential for mitosis and for cellularorganization during the large portion of the cell cycle that isnot mitosis. In interphase, the MT-organizing center orga-nizes the array of MTs that provides polarity to the cyto-plasm. This array is an essential structure for the cellulartrafficking of a myriad of proteins, including many

Authors' Affiliations: 1Medical Oncology Branch, Center for CancerResearch, National Cancer Institute, and 2Program in Physical Biology,Eunice Kennedy Shriver National Institute of Child Health and HumanDevelopment, NIH, Bethesda, Maryland

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Antonio T. Fojo, National Cancer Institute, 10Center Drive, MSC 1906, Bldg. 10 Rm. 12N226, Bethesda, MD 20892-1906. Phone: 301-402-1357; Fax: 301-402-1680; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-0999

�2012 American Association for Cancer Research.

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important oncoproteins. Polarized arrays of MTs growoutward from near the nucleus in growing epithelial cells,and fromapical to basal in polarized cells (6). Their polarityis recognized by motor proteins of the dynein and kinesinsuperfamilies, allowing for directed movement of cargo onMTs (7). Dyneins move toward the minus (nuclear) end ofMTs, whereas kinesinsmostlymove to the plus (peripheral)end of MTs. In cells undergoing cell division, a majorfunction of the centrosome is to organize the 2 opposingarrays that form the mitotic spindle apparatus, which isrequired for separation of chromosomes during celldivision.

MTs provide the structure and machinery for chromo-some segregationduringmitosis, a process that also requiresan array of other proteins, including essential serine/thre-onine kinases. Figure 2 depicts the principal localization ofthe mitosis-associated proteins that have been targets ofdrugs in clinical trials (8). Figure 3 shows the proteins thatmodulate the activity of the mitotic kinases and thosethrough which the effects of these mitosis-associated pro-teins aremediated. Although themitotic kinases often act inconcert, they also perform unique functions without over-lap. During S-phase, Aurora kinase A (AKA) localizes to thecentrosome. It then translocates to themitotic poles and theadjacent spindle MTs as the cell progresses through pro-phase, metaphase, and anaphase, eventually locating in themidbody during telophase and cytokinesis (9). Duringtranslocation from the centrosome, the level of AKA

increases. This indicates that expression is largely restrictedto mitosis and that agents designed to target AKA would beinactive in cells in other phases of the cell cycle (this point isdiscussed further below). Aurora kinase B (AKB) localizes tobundles of specialized MTs termed K-fibers and helps con-nect the kinetochore, a protein structure that assembles onthe centromere of chromosomes, to spindle fibers duringprometaphase and metaphase. It then relocalizes to themidzone or central spindle at the metaphase–anaphasetransition, influencing chromosome separation and cyto-kinesis. Like AKB, Aurora kinase C (AKC) is a chromosomepassenger protein that localizes first to the inner centro-mere, then to the central spindle, andfinally to themidbodyofmitotic cells as the cell cycle progresses (10). Aswith AKA,the mRNA and protein expressions of AKB and AKC aremaximally elevated during the G2/M-phase of the cell cycleand decrease rapidly as the cells enter G1 (10).

PLK1–4 localize to the centrosomeduring interphase andprophase, move to the spindle poles after prophase, andrelocate to the midbody for telophase and cytokinesis (11).

Like the mitotic kinases, KSP is mitosis-specific and thushas attracted attention as a target for anticancer agentstargeting mitosis (12). KSP is a homotetrameric kinesinmotor protein and a member of the kinesin superfamily ofproteins that binds and hydrolyzes ATP, coupling the ener-gy released from hydrolysis with force production thatallows for unidirectional movement along MTs. Unliketransport kinesins, which function in cytosolic movement

Figure 1. Organization of MTsduring the cell cycle and theexpression of proteins involvedin mitosis. During the G1-, S-,and G2-phases, MTs areassembled in parallel, polarizedarrays with their plus (þ) endpointing outward from the cellcenter. The minus (�) end of thearrays is nucleated (anchoredand initiated) by the centrosome(also known as the MT-organizing center). Enteringmitosis, the centrosomes moveto the 2 opposite sides of thecell, forming the mitotic spindleapparatus that separateschromosomes during celldivision. Theexpression levels ofspecialized proteins in mitosis,such as AKA (red), AKB (yellow),and PLK (green), increase ascells traverse the G2 portion ofthe cell cycle and reach theirmaximum level during mitosis.

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Clin Cancer Res; 18(1) January 1, 2012 Clinical Cancer Research52



and the localization of organelles and vesicles, mitotickinesins such as KSP participate in the assembly, mainte-nance, and elongation of the mitotic spindle, chromosomealignment and segregation, and MT depolymerization.KSP’s antiparallel MT-sliding function is required for sep-

aration of the 2 centrosomes in early mitosis, an essentialevent for bipolar spindle formation. Consequently, inhibi-tion of KSP results in mitotic arrest because the failure ofcentrosomal separation results in anMT arraywith only onepole.

Figure 2. Localization of AKA,AKB, and PLK during mitosis.During mitosis, AKA (red)translocates from the centrosometo the mitotic poles and theadjacent spindle MTs, AKB(yellow) localizes to MTs near thekinetochores, and PLK (green)moves from the centrosome to thespindle poles. By telophase andcytokinesis, all 3 kinases relocateto the midbody.

Tales of How Great Drugs Were Brought Down by a Flawed Rationale

www.aacrjournals.org Clin Cancer Res; 18(1) January 1, 2012 53



Numerous studies have shown that AK, PLK, and KSP areexpressed primarily in theM-phase of the cell cycle and, to alesser extent, in G2 (8). During G1-, G0-, and S-phases,expression either does not occur or occurs only at very lowlevels. Consequently, drugs aimed at these proteins willonly find their target and have an effect on cells that arerapidly cycling (dividing) and likely to be in or passingthrough the G2- or M-phase while the drug is present. Thishighly restricted, cycle-specific expression likely explainswhy these agents have failed to affect tumor growth inpatients.

What happens when a cell is exposed to an inhibitorthat targets mitosis?

Inhibitors of mitotic proteins all cause disruption ofnormal mitotic function, as do MTAs, which additionallydisrupt interphase functions. The mechanism wherebymitosis is disrupted differs among these agents. MTAs andinhibitors of KSP and PLK interfere with proper assembly ofthemitotic spindle and lead to arrest inmid-mitosis (4, 13).Inhibitors of AKA cause transient arrest followed by mitoticexit with misaligned chromosomes, and inhibitors of AKBcause premature mitotic exit with major defects in chromo-some attachment (13). Although the mechanistic detailsdiffer, all of these compounds, including MTAs, disrupt thenormal process of mitosis, but of course, they only do sowhen mitosis occurs. None of these agents produces orpromotes the initiation of mitosis, and hence will onlycause damage to cells when mitosis happens to occur. Ifmitosis occurs rarely, agents whose action is restricted to

mitosis will rarely cause damage. However, MTs are presentthroughout the cell cycle, andMTarrays aremost vulnerableduring mitosis due to the increased rate of MT turnover.Hence, MTAs potently target mitosis when it occurs, butthey can also target interphase cells.

Duration of mitotic arrestStudies involving a number of cell types and a variety of

methods, including the use of mitosis inhibitors, showedthat mitotic arrest can only be sustained for �1–2 days inhuman cells with constant drug exposure, and less than thatin rodent cells (14). Although 1–2 days is a significantduration, given that the normal duration of mitosis is only1–2 hours inmany different cell types and tissues, it is by nomeans indefinite (15). Mitotic arrest causes many differenttypes of stress. For example, condensed chromosomes can-not be transcribed, which makes it impossible to replenishneeded transcripts—a state that cannot be sustained. Con-sequently, this period is limited by mitotic slippage, espe-cially in normal cells, or cell death, especially in cancer cells(14). A further complication is that cells display significantintra- and interline variability in the duration of mitoticarrest, and great variation among different drugs and themode of exit from arrest (16). The latter presumably hap-pens when the slow degradation of cyclin B1, which con-tinues during arrest, is reduced below the level that isrequired to maintain the mitotic state. At this point somecells die; some exit mitosis (termed mitotic slippage),remain tetraploid, and possibly die later; and some showother fates (reviewed in ref. 17). Whatever the details, the

Figure 3. Binding partners ofAKA, AKB, and PLK duringmitosis. After phosphorylationby Lats2, Ajuba interacts withAKA. AKA phosphorylatesBRCA1 (101), Cdc25b (102),CENP-A (103), p53 (104), Eg5(105), TACC3 (106), and Tpx2(107). PLK phosphorylatesAPC/C (108), cyclin B (109),NudC (110), and cohesinsubunit SCC1 (111). AKBphosphorylates INCENP (112),Dam1 (113), CENP-A (114),histone 3 (115), Ncd80 (116),myosin II (117), Nlp (118), desmin(119), andseptin (120), andbindsto survivin (121) and vimentin(122). APC/C, anaphase-promoting complex/cyclosome;CENPA, centromere protein A;Eg5, kinesin-like protein; H3,histone 3; Nlp, ninein-likeprotein; NudC, nucleardistribution gene C.

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data show that mitotic arrest is not maintained for anyextendedperiod. Furthermore, it is unclear howany of theseresults would change with pulsed or varying drug concen-trations. Very few data are available regarding mitotic dura-tion in patients. In one study, mitotic counts that wereobtained before and after treatment with an MTA (pacli-taxel) showed great variability from patient to patient in theextent and duration of mitotic arrest (18).

Doubling times of human tumorsCancer is often mistakenly thought of as a mass of

abnormal cells growing rapidly in an uncontrolledmanner.This is misleading for at least 2 reasons. First, the data showthat cell division is a very precisely regulated process thatfollows similar pathways in both normal and cancer cellsand involves highly specialized proteins. Second, onemightthink that frequent cell division is a hallmark of tumor cells.This misconception is encouraged by the rapid rate of celldivision of cancer cells observed in vitro and in xenograftmodels. Although it can be challenging to measure tumordoubling times in humans, studies have shown that tumorsdonot double as rapidly as onemight think. As summarizedin Table 1, data obtained by a variety of radiologic imagingmodalities show median doubling times for many humantumors of >100 days, which is much longer than thedoubling times observed in preclinical animal models(2). This means that at any one time, only a very smallpercentage of tumor cells are undergoing mitosis. Indeed,the meanmitotic index in a variety of tumor types has beenshown to be <1% (19, 20). Additionally, the mean labelingindex measured by radiolabeled imaging [e.g., tritiatedthymidine ([3H]-TdR)] was only a few percent of the tumor(21). Together, these data show that in contrast to in vitroand xenograft models, human tumors have very long dou-bling times. This makes such tumors indifferent to drugsthat target cell proliferation or proteins whose expression ishighly restricted to one phase of the cell cycle.

Preclinical in vivo dataTable 2 and Fig. 4 show preclinical in vivo data obtained

for mitotic kinase and KSP inhibitors, and they provideinsight into why this class of agents has not been successfulclinically. Also shown in Table 2 are the doubling timesof the preclinical models used in various studies (see

also Table 1). As noted above, this rate of doubling ismarkedly faster than that found in any solid tumor inhumans, and this difference may be significant for targetgenes/proteins whose expression is highly restricted duringthe cell cycle, such as occurs with mitosis-specific proteins.Furthermore, in most models the agents were administeredfrequently (often daily and even twice daily). Frequentadministration is essential for agents that target proteinswhose expression is restricted to a very short part of the cellcycle (22). The importance of frequent drug administrationis underscored by the results depicted in Fig. 4, which showa decline in activity when drug administration was changedfrom twice per week to once per week. Finally, we note, assummarized in Table 2, that complete responses wereachieved inonly 2 of the 53models evaluated, underscoringthe fact that despite a frequent schedule of administration torapidly growing tumors, complete tumor disappearancecould not be achieved. From these data, it is clear that inthe models with rapid doubling times, cells that are not inG2/M-phase are refractory to therapy and can repopulate therapidly dividing tumormass unless frequent administrationis used (23).

Clinical dataFew (if any) agents have shown as much activity as

paclitaxel did during its development, in terms of bothbreadth and magnitude. A review in 1995 of early evalua-tions of the drug’s activity showed single-agent responserates of 17% to 62%, 20% to 48%, and 21% to 41% inbreast, ovarian, and lung cancers, respectively (24). Activitywas subsequently reported in other cancers, includingKaposi sarcoma and esophageal, urothelial, and head andneck cancers. In our own tabulation of 29 studies of single-agent paclitaxel in these seven cancers, we recordedresponse rates ranging from 7% to 71%, with a median of37% and an overall response rate of 28%, in a group of2,271 patients (data not shown; see references in ref. 25).With this as background, we summarize in Table 3 andSupplementary Tables S1A and S1B the clinical results of 46studies conductedwith20different agents targeting theAKs,PLK, and KSP. The range and median values for all patientgroups are presented at the bottomof each table. The overallresponse rate of 1.6% for all studies shows unequivocallythat these agents lack activity against solid tumors [787

Table 1. Tumor doubling time in days: a comparison of preclinical in vivo models and patient data

Preclinical models Patient data

Mean Median Reference Mean Median Reference

Breast cancer 5.6 4.5 (34) 152 137 (35–46)Colon cancer 3.4 3.4 (34) 391 334 (47–51)Lung cancer 4.4 3.8 (34) 114 100 (52–60)Prostate cancer 3.4 3.4 (34) 219 126 (61)Melanoma 5.4 5.7 (34) 147 78 (62–67)





Table 2. Mitotic kinase inhibitors in xenograft models

Drug Cell lineTumororigin

In vivoDT,daysa Hostb

Treatmentschedule(dose peradministration)

Tumorreduction(comparedwith) Reference

AK inhibitorsMK-0457/VX-680 HL-60 AML 3.3 Nude i.p., bid � 13d (75 mg/kg) 98% (C) (68)

MIA PaCa-2 Pancreas — Nude i.p., bid (50 mg/kg) 22% (ITS)HCT-116 Colon 2.6 Nude rats i.v., 3d/w (1 mg/kg/h) 56% (ITS)

AZD1152(barasertib)

SW-620 Colon 2.4 AT s.c., 48-h infusion(150 mg/kg/d)

87% (C) (69)

Colo 205 Colon 4.3 AT s.c., 48-h infusion(150 mg/kg/d)

94% (C)

HCT116 Colon 2.6 AT s.c., 48-h infusion(150 mg/kg/d)

74% (C)

A549 Lung 8.4 AT s.c., 48-h infusion(150 mg/kg/d)

69% (C)

Calu-6 Lung — AT s.c., 48-h infusion(150 mg/kg/d)

55% (C)

HL-60 AML 3.3 AT s.c., 48-h infusion(150 mg/kg/d)

>100% (C)

PHA739358(danusertib)

Huh-7 Liver — NOD/SCID i.p., bid (30 mg/kg/d) 75% (C) (70)HepG2 Liver — NOD/SCID i.p., bid (30 mg/kg/d) 88% (C)

MLN8054 HCT-116 Colon 2.6 Nude p.o., bid � 21d (30 mg/kg) 81% (C) (71)MLN8237 MM1.S MM — SCID p.o., bid � 21d (40mg/kg) 80% (C) (72)R763 MIA PaCa-2 Pancreas — SCID p.o. 3d/w � 4w (15 mg/kg) 25% (C) (73)AT9283 HCT-116 Colon 2.6 AT nu/nu q9d � 3 27% (C) (74)SNS-314 A2780 Ovarian nu/nu i.p., qw � 3w 57% (C) (75)

nu/nu i.p., biw � 3w (170 mg/kg) 54% (C)A375 Melanoma — nu/nu i.p., qw � 3w (170 mg/kg) 35% (C)

nu/nu i.p., biw � 3w (170 mg/kg) 65% (C)H1299 Lung — nu/nu i.p., qw � 3w (170 mg/kg) 18% (C)

nu/nu i.p., biw � 3w (170 mg/kg) 69% (C)MDA-MB-231 Breast 4.4 nu/nu i.p., qw � 3w (170 mg/kg) 49% (C)

nu/nu i.p., biw � 3w (170 mg/kg) 74% (C)PC-3 Prostate 2.4 nu/nu i.p., qw � 3w (170 mg/kg) 56% (C)

nu/nu i.p., biw � 3w (170 mg/kg) 68% (C)Calu-6 Lung — nu/nu i.p., qw � 3w (170 mg/kg) 35% (C)

nu/nu i.p., biw � 3w (170 mg/kg) 91% (C)ENMD-2076 HCT-116 Colon 2.6 Nude or SCID p.o., bid (200 mg/kg) 89% (C) (76)

HT-29 Colon 5.1 Nude or SCID p.o., qd (100 mg/kg) 62% (C)CT-26 Mouse colon — Nude or SCID p.o., qd (100 mg/kg) 21% (C)A375 Melanoma — Nude or SCID p.o., qd (151 mg/kg) 81% (C)MDA-MB-231 Breast 4.4 Nude or SCID p.o., qd (100 mg/kg) 94% (C)H929 MM — Nude or SCID p.o., qd (150 mg/kg) 88% (C)OPM-2 MM — Nude or SCID p.o., qd (75 mg/kg) 56% (C)MV4 AML — Nude or SCID p.o., bid (50 mg/kg) 99% (C)HL-60 AML 3.3 Nude or SCID p.o., qd (150 mg/kg) 83% (C)

PLK inhibitorsBI2536 Caki-1 Kidney 2.1 nu/nu i.v., qd � 2d, 5d off

(50 mg/kg)Minor effect (77)

SN12C Kidney 5.6 nu/nu i.v., qd � 2d, 5d off(50 mg/kg)

Minor effect

(Continued on following page)

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(AKs, Table 3) þ 345 (PLK, Table S1A) þ 267 (KSP, TableS1B) ¼ 1,399 patients total; objective response rate: 22/1399¼ 1.6%]. Such a response rate might even be recordedwith a placebo (26, 27). Some may wonder whether lowbioavailability can explain why these drugs were inactive inpatients. We think this unlikely given that poor results wereseen even in cases where adequate biomarkers for mitoticarrest (mitotic index or histone H3 phosphorylation mostoften in skin biopsies) showed target engagement (28, 29).Moreover, wewould argue that the neutropenia observed inmany of the treated patients represents a biomarker orsurrogate that clearly proves the drug hit the target andinhibited mitosis (even if it was not in the tumor). Inaddition, we emphasize our belief that a stable disease rateof �20% can only be interpreted as a measure of theinherent biology of the tumors being treated. As reviewedabove,mitotic arrest canonly be sustained for�1–2days, soany assumption that this class of agents (or any other) canarrest cells in mitosis for any period that might be scoredclinically as stable disease is not scientifically defensible.Similarly, given the restricted expression of the targets in

mitosis, only a small percentage of cells would provevulnerable to inhibitors targetingmitosis. This would argueagainst a situation inwhich the fraction of killed cells equalsthe quantity that has been newly added to the tumor bydivision.

Not surprisingly, we note that in the subset of patientswith hematologic malignancies, the response rate washigher, but still low, at 8.2%. Given that hematologiccancers often have faster doubling times than solid tumors,one could envision a potential role for agents that targetmitosis in these malignancies. However, the neutropeniathat will invariably occur might limit treatment such thatboth the amount and frequency of drug administrationwould be inadequate.

Finally, although infrequent responseshavebeenobservedclinically, occasionally an off-target effect inhibiting a kinaseother than a mitotic kinase has resulted in activity. Forexample, activity has been reported with an AK inhibitor,tozasertib (MK-0457/VX680), in patients with imatinib-resistant chronic myeloid leukemia (CML) harboring theT315Imutation (30).However, these effects are independent

Table 2. Mitotic kinase inhibitors in xenograft models (Cont'd )

Drug Cell lineTumororigin

In vivoDT,daysa Hostb

Treatmentschedule(dose peradministration)

Tumorreduction(comparedwith) Reference

786–0 Kidney 6.7 nu/nu i.v., qd � 2d, 5d off(50 mg/kg)

No effect

nu/nu i.t., qd � 2d, 5d off(50 mg/kg)

No effect

nu/nu i.t., bid � 2d, 5d off(100 mg/kg)

Significantregression

A498 Kidney 3.4 nu/nu i.v., qd � 2 d, 5d off(50 mg/kg)

No effect

nu/nu i.t., bid � 2d, 5d off(100 mg/kg)

Significantregression

HMN-214 PC-3 Prostate 2.4 Nude p.o., qd � 28d(20 mg/kg)

79% (C) (78)

WiDr Colon — Nude p.o., qd � 28d(20 mg/kg)

73% (C)

A549 Lung 8.4 Nude p.o., qd � 28d(20 mg/kg)

75% (C)

KSP inhibitorsIspinesib(SB-715992)

MCF-7 Breast 4.5 nu/nu i.p., q4d � 3 (10 mg/kg) 92% (C) (79)

HCC-1954 Breast — nu/nu i.p., q4d � 3 (10 mg/kg) 95% (C)KLP4 Breast — nu/nu i.p., q4d � 3 (10 mg/kg) 94% (C)BT-474 Breast NA SCID i.p., q4d � 3 (8 mg/kg) 61% (C)MDA-MB-468 Breast — SCID i.p., q4d � 3 (8 mg/kg) 100% (C)

Abbreviations: AT, athymic; bid, twice per day; biw, twice per week; C, control; d, day; DT, doubling time; i.p., intraperitoneal; i.t.,intratumoral; ITS, initial tumor size; MM, multiple myeloma; NOD, nonobese diabetic; nu, nude; p.o., orally; SCID, severe combinedimmunodeficient; qw, once per week; qd, once per day; w, week.aPlowman et al. (34).bMouse, unless otherwise indicated.





of the cell cycle anddonot contradict the reasonsgivenhereinto explain why these drugs have failed to benefit the over-whelming majority of patients with solid tumors.

Conclusions

Mitotic kinase inhibitors were developed as nonneuro-toxic alternatives to MTAs; however, the rationale behindthe development of these agents, i.e., the thesis that MTAskill cells inhuman tumors onlyby inhibitingmitosis,meantthat if unrecognized mechanisms proved to be important,such agents would fail when they reached the clinic. In an invitro or a xenograft model with a doubling time of a fewdays, a large fraction of cells will prove vulnerable to atherapy that targets a protein that is crucial for mitosis.Therefore, it is not surprising that in a patient with a tumordoubling time of�30–60 days and an S-phase fraction of atmost a few percent, only a small, insignificant fraction ofcells will be vulnerable to a drug aimed at a target that isexpressed only transiently in most tumor (and normal)cells. Inhibitors that target mitosis could prove effective inrapidly growing (i.e., rapidly dividing) leukemias and lym-phomas (i.e., Burkitt lymphoma), and their value in suchmalignancies should be further explored. However, theirlack of activity against a majority of cancers highlightsseveral aspects of cancer drug development in the 21stcentury and informs our understanding of the mechanismsof drug action, as summarized below.

First, the dose-limiting toxicity of mitotic kinase inhibi-tors—reversible neutropenia—stands as a testament to theprowess of pharmaceutical drug developers, especially thechemists who synthesized these agents, and the biologists

who identified and characterized the targets and developedthe models used in their validation. Although the majorityof human tumors do not divide rapidly enough to besusceptible to these mitotic poisons, the same cannot besaid of vulnerable marrow elements. The doubling time ofgranulocyte precursors is very short [17 hours for myelo-blasts, 63 hours for promyelocytes, and 55 hours for mye-locytes (31)]. Thus, reversible neutropenia would beexpected of an agent targeting a mitotic kinase or KSP,because at any one time �25% of marrow neutrophils areundergoing mitosis. Indeed, we would argue that the neu-tropenia observed nearly uniformly in clinical trials withagents that inhibit mitosis can be viewed as a biomarker orsurrogate of activity.

Second, although this pharmaceutical prowess has pos-itive attributes, it led to theoverdevelopment of drugs aimedat targets that have not yet been validated (e.g., AKs, PLKs,and KSP). To date, clinical data have been reported for atleast 20 different mitosis-specific poisons, and there arelikely more yet to be reported. This has involved a consid-erable financial outlay and patient recruitment effort with-out a return on the investment (the response rate to aninhibitor targeting mitosis in clinical trials involving 1,399patients with solid tumors was 1.6%; see Fig. 4 and Sup-plementary Tables S1A and S1B). Although a certainamount of redundancy represents attempts to design abetter agent, the clinical evidence and the nearly uniformmyelosuppression suggest that these drugs were remarkablysimilar and potent.

Third, the results indicate yet again the deficiencies ofthe preclinical models used in drug development.Although these preclinical models are far from ideal (butat some level valuable), it is clear that the rapid doublingtimes in such models compared with human tumorsallow for an accelerated drug-development timeline.However, this precludes their use when the doublingtime is a critical factor in a drug’s activity. The rapiddoubling times of the preclinical models explains whyagents targeting mitosis proved active in these models butwere ineffective against patient tumors.

Fourth, the results highlight the increasing use/abuse of"stable disease" as a measure of drug efficacy. As discussedabove, scoring stable disease as a measure of activity cannotbe defended scientifically for this class of compounds,arguing against its use as a measure of drug activity. Asnoted above, studies have shown that inhibitors targetingmitosis cannot be expected to stabilize tumor growth byarresting cells in mitosis for prolonged periods of time.Indeed, one can persuasively argue that these studies havecollectively defined �20% as the stable disease rate dueto the inherent tumor biology of patients harboringadvanced solid tumors who enroll in similar phase I/IItrials, because in effect, these patients received a drugthat had no disease-stabilizing activity. We note that thisestimate of 20% as the stable disease rate due to inherenttumor biology is not excessive, because stable disease ratesof 55% to 67% have been obtained with placebos in renalcell and hepatocellular carcinomas (26, 27). Finally, the

© 2012 American Association for Cancer Research

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100

Figure 4. Frequent administration of SNS-314 leads to greater antitumoreffect. SNS-314, a selective inhibitor of AKA, AKB, and AKC, wasadministered once or twice a week in mouse xenograft models. Acomparison with the control tumor size shows that the twice-weeklyschedule resulted in significantly greater tumor reduction than the once-weekly treatment schedule. Data from Arbitrario et al. (75).

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Table 3. AK inhibitors in clinical trials

Drug (company) Disease TreatmentPatients,N

Cyclesmedian(range) Response DLT/AE Reference

MK-0457/VX-680(Vertex/Merck)

CML/ALL 5-d CIV q2–3 wk 3 ND 1 PHR 1 CHR #BM (30)ST 5-d CIV q28d 16 2 (1–6) 3 SD #ANC, F/N, allergic reaction (80)ST 24-h CIV q21d 27 Total: 8 1 SD #ANC,N/V/diarrhea, fatigue (81)

AZD1152/barasertib(AstraZeneca)

AML 7-d CIV q21d 16 Total: 28 2 CR, 1 PR,6 SD

#ANC, F/N, #WBC, #Plt,fatigue, pneumonia,ARDS, sepsis

(82)

PHA 739358/danusertib(Nerviano)

ST 6-h CIV 42 ND 7 SD #ANC, HTN, fatigue,anorexia, N/diarrhea

(83)

ST 24-h CIV q14d �G-CSF

56 3 (1–20) 1 PR; 18 SD,4 PSD

F/N, fatigue, anorexia, N/V/D, mucositis, "LFTs, #Kþ,HTN, fever

(84)

ST 3-wk CIV q28d 50 2 (1–28) 14 SD #ANC, #WBC, #Hgb N/diarrhea anorexia, fatigue

(85)

MLN8054(Millenium)

ST p.o. d1–5 þ d8–12q28d or p.o. QIDd1–14 q28d

43 1 (1–10) 3 SD #MS, "LFTs, mucositis (86)

ST p.o. � 7d q21d; p.o.� 14d q28d; p.o.�21d q35d

61 2 (1–14) 9 SD #MS, N/V, confusion,cognitive disorder,hallucination, fatigue

(87)

MLN8237(Millenium)

ST p.o. � 7d q21d; p.o.� 14d q28d; p.o.�21d q35d

65 ND 1 PR, 8 SD #ANC, #Plt, sepsis,mucositis, N/D, fatigue,alopecia

(28)

NHM p.o. bid � 7d q21d 17 2 (1–8) ND F/N, #ANC, fatigue (88)R763 (Merck/Serono)

ST p.o. d1, 8 q21d; p.o.d1–3 q21d

15 ND 2 PD ND (89)

AT9283 (Astex) Leuk 72h CIV q3wk 29 ND 2 PHR; 1 PCR #ANC, #BM, "LFTs;alopecia

(90)

ST 72h CIV q3wk 22 2 (2–7) 3 SD F/N (91)ST 72h CIV q3wk 33 2 (1–12) 1 PR, 4 SD F/N, GI, fatigue (92)

SNS-314 (Sunesis) ST 3-h CIV d1, 8and 15 q28d

32 2 6 SD N/V, fatigue, constipation,pain

(93)

SU6668 (Sugen/Pfizer)

ST p.o. bid, 28d 35 ND 4 SD #Plt, pericarditis, pleuritis,fatigue

(94)

ST p.o. bid, 28d 19 ND 3 SD GI, fatigue, pleuritis, SOB,pericardial effusion

(95)

ENMD-2076(EntreMed)

ST p.o. qd, 28d 14 3 (< 1–9) 4 SD HTN, fatigue, proteinuria,diarrhea

(96)

ST p.o. qd � 21d,q28d

67 3 (1–24) 2 PR, 49 SD #ANC, HTN, N/V/D, fatigue,"LFTs

(97)

OvCa p.o. 64 2 3 PR, 27 SD #BM, fatigue, HTN,mucositis, "LFTs, HFS,thromboembolic event,subarachnoidhemorrhage, #LVfunction, PRES

(98)

BI 811283(BoehringerIngelheim)

ST 24h CIV q21d 57 Mean ¼ 3 33% SD #ANC, F/N, #WBC (99)

ST 24h CIV q2wk 52 Mean ¼ 3 29% SD #ANC, #WBC (100)Tumor type No. of patients No. of treatments:

range/medianNo. of responders(percentage of all)

(Continued on following page)





disappointing clinical results ratify the paradigm thatMTAs do not kill cancer cells in humans primarily byinhibiting mitosis, even though both MTAs and inhibitorsthat target mitosis are lethal to bone marrow elements inthis way. We previously proposed the inhibition of traffick-ing on MTs as the principal mechanism of action of MTAs(2).One such example forwhich clear evidencenowexists isthe androgen receptor, which plays a crucial role in thegrowth of prostate cancer (32, 33). The evidence that MTdisruption affects androgen-receptor trafficking helpsexplain the activity of both docetaxel and cabazitaxel in adisease that is often so indolent that questions about the

need for therapy remain unresolved. Therefore, when study-ing cells in which an MTA or a combination of MTAs isactive, one should seek to determine which of the criticalproteins that traffic on MTs have been affected by thetherapy in question.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Received July 28, 2011; revised October 19, 2011; accepted November 7,2011; published online January 3, 2012.

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Table 3. AK inhibitors in clinical trials (Cont'd )

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Hematologic malignancies 48 ND Partial/tumor response:8 (16.7%)

Stable disease:6 (12.5%)

Abbreviations:#ANC,decreasedabsoluteneutrophil count (neutropenia); ARDS, acute respiratory distresssyndrome; #BM,decreasedbonemarrow (pancytopenia); CHR, complete hematologic response; CIV, continuous i.v. infusion; CML/ALL, T315I abl-mutated CMLor (Ph)þ ALL; cycles, median number of cycles administered; DLT/AE, dose-limiting toxicity/adverse event; F/N, febrile neutropenia;G-CSF, granulocyte colony-stimulating factor; GI, gastrointestinal disturbance; HFS, hand-foot syndrome; #Hgb, anemia; #Kþ,hypokalemia; HTN, hypertension; Leuk, refractory leukemia; "LFTs, elevated liver function tests; #MS, reduced mental status/somnolence; NHM, nonhematologic malignancies; N/V, nausea/vomiting; OvCa, platinum-resistant ovarian cancer; PCR, partialcytogenetic response; PHR, partial hematologic response; #Plt, thrombocytopenia; p.o., orally; PR, partial response; PRES, reversibleposterior leukoencephalopathy syndrome; SD, stable disease; SOB, shortness of breath; ST, advanced solid tumors or advanced andrefractory solid tumors or refractory solid tumors; #WBC, decreased white blood cell count (leukopenia).

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